HK1241412A1 - Aminion derived adherent cells - Google Patents

Abstract

Provided herein are novel angiogenic cells from amnion, referred to as amnion derived adherent cells, and populations of, and compositions comprising, such cells. Further provided herein are methods of obtaining such cells and methods of using the cells in the treatment of individuals.

Description

Amnion derived adherent cells

The present application is a division of the invention application having an application date of 2009 from 11/19, application No. 200980154477.2, and a name of "amnion-derived adherent cells".

This application claims the benefit of U.S. provisional patent application No. 61/116,248, filed on 11/19/2009, the disclosure of which is incorporated by reference in its entirety.

1. Field of the invention

The present invention provides novel angiogenic cells from amnion, and cell populations thereof, referred to herein as "amnion derived adherent cells" (AMDACs). Amnion derived adherent cells differ from the placental stem cells previously described, including tissue culture plastic adherent placental stem cells.

2. Background of the invention

Cell compositions, e.g., stem cell compositions, have become attractive therapeutic approaches to a variety of physiological deficiencies, e.g., for bone marrow replacement therapy. There is a need for other cell populations, for example, stem or progenitor cells with angiogenic potential and/or properties.

3. Summary of the invention

In one aspect, the invention provides an isolated amnion derived adherent cell, also referred to herein as AMDAC, wherein the cell is attached to tissue culture plastic, and wherein the cell is OCT-4^–(OCT-4 NEGATIVE, OCT-4 is also known as POU5F1 or octamer BINDING PROTEIN 4), or by reverse transcriptase polymerase chain reaction (RT-PCR) assay, e.g., by 30 cycles of RT-PCR assay, in comparison to a suitable control cell line, e.g., an embryonic carcinoma-derived stem cell line (e.g., NTERA-2, e.g., from American type culture Collection, ATCC accession number CRL-1973), which is OCT-4^–. In a specific embodiment, the cells are OCT-4 as determined by RT-PCR^–And VEGFR1/Flt-1 as determined by immunolocalization⁺(vascular endothelial growth factor receptor 1) and/or VEGFR2/KDR⁺(vascular endothelial growth factor receptor 2, also known as kinase insert domain receptor). In another specific embodiment, the cells are OCT-4 as determined by RT-PCR^–And CD49f as determined by immunolocalization⁺(integrin- α 6⁺). In a specific embodiment, the cell is OCT-4 as determined by RT-PCR^–And HLA-G by RT-PCR^–. In another specific embodiment, the cell is OCT-4 as determined by RT-PCR^–And CD90 as determined by immunolocalization⁺、CD105⁺Or CD117^–. In a more specific embodiment, the OCT-4^–The cell is CD90⁺、CD105⁺And CD117^–. In another specific embodiment, the cells are OCT-4 as determined by, for example, 30 cycles of RT-PCR^–And no SOX2 is expressed.

In another embodiment, the OCT-4^–The cells were immunolocalized and determined to be CD29⁺、CD73⁺、ABC-p⁺And CD38^–One or more of (a).

In another specific embodimentThe OCT-4^–The cells were CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺(angiogenin receptor), TEM-7⁺(tumor endothelial cell marker 7), CD31^–、CD34^–、CD45^–、CD133^–、CD143^–(angiotensin I converting enzyme, ACE), CD146^–(melanoma cell adhesion molecule), CXCR4^–(chemokine (C-X-C motif) receptor 4). In a more specific embodiment, the cell is immunolocalized and determined to be CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And CXCR4^–. In another more specific embodiment, the amnion derived adherent cells provided herein are OCT-4 as determined by RT-PCR^–(ii) a Determined by immunolocalization as VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺(ii) a And CD31 as determined by immunolocalization^–、CD34^–、CD45^–、CD133^–And/or Tie-2^–One or more or all of them. In a specific embodiment, the amnion derived adherent cells or amnion derived adherent cell population are as described, for example, in>Under 20 cycles of PCR amplification, at least 2log less OCT-4mRNA was expressed than NTERA-2 cells or NTERA-2 cell populations with equivalent cell numbers. In another specific embodiment, the OCT-4^–Whether the cells are VE-cadherin as determined by immunolocalization^–(CD144^–). In another specific embodiment, the OCT-4^–Cells were also subjected to immunolocalization assay for CD105⁺And CD200⁺Is positive. In another specific embodiment, the OCT-4^–Cells do not express CD34 after 4-21 days of exposure to 1-100ng/mL VEGF (vascular endothelial growth factor) as determined by immunolocalization.

In another aspect, the inventionThere is provided an isolated amnion derived adherent cell, wherein said cell is attached to a tissue culture plastic, and wherein said cell is OCT-4 as determined by RT-PCR^–And SOX-2^–(ii) a And CD90 as determined by flow cytometry⁺、CD105⁺And CD117^–. In one specific embodiment, OCT-4^–，SOX-2^–Determination of whether cells are HLA-G by flow cytometry^–Or CD271^–. In a more specific embodiment, the cell is OCT-4 as determined by RT-PCR^–And SOX-2^–(ii) a And CD90 as determined by flow cytometry⁺、CD105⁺、CD117^–、CD271^–And HLA-G^–。

In another aspect, the invention provides an isolated amnion derived adherent cell, wherein the cell is attached to tissue culture plastic, and wherein the cell is for CD309 (also known as VEGFR 2/KDR)⁺) Is positive.

In another aspect, the invention provides an isolated amnion derived adherent cell, wherein the cell is attached to a tissue culture plastic, and wherein the cell is OCT-4 as determined by RT-PCR^–And VEGFR2/KDR as determined by immunolocalization⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺Or VE-cadherin^–One or more of (a). In a specific embodiment, the cell is cultured, for example, by>The RT-PCR assay for 20 cycles was OCT-4^-And measured by immunolocalization as VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺And VE-cadherin^–. In another specific embodiment, the cells do not express CD34 after 4-21 days of exposure to 1-100ng/mL VEGF as determined by immunolocalization.

In another embodiment, the amnion derived adherent cells are OCT-4^–、CD49f⁺、HLA-G^–、CD90⁺、CD105⁺And CD117^–. At one endIn a more specific embodiment, the cell is CD9 as determined by immunolocalization or flow cytometry⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–(melanoma cell adhesion molecule), or CXCR4^–One or more of (a). In a more specific embodiment, the cell is immunolocalized and determined to be CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And CXCR4^–. In another specific embodiment, the cells are also VEGFR1/Flt-1 as determined by immunolocalization⁺And/or VEGFR2/KDR⁺(ii) a And CD31 as determined by immunolocalization^–、CD34^–、CD45^–、CD133^–And/or Tie-2^–One or more of (a). In another specific embodiment, the cells are also VEGFR1/Flt-1 as determined by immunolocalization⁺、VEGFR2/KDR⁺、CD31^–、CD34^–、CD45^–、CD133^–And Tie-2^–。

In another embodiment, the invention provides an isolated amnion derived adherent cell, wherein the cell does not express mRNA of FGF4, IFNG, CXCL10, ANGPT4, ANGPTL3, FGA, LEP, PRL, PROK1, TNMD, FLT3, XLKD1, CDH5, LECT1, PLG, TERT, SOX2, NANOG, MMP-13, DLX5, or BGLAP as determined by, for example, 30 cycles of RT-PCR. In another embodiment, the invention provides an isolated amnion derived adherent cell, wherein the cell is incapable of constitutively expressing one or more of invariant chain, HLA-DR-DP-DQ, CD6, CD271, as determined by flow cytometry.

The invention further provides an isolated population of cellsComprising an amnion derived adherent cell. In a specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are amnion derived adherent cells. In one embodiment, the cell is OCT-4 as determined by RT-PCR^–And VEGFR1/Flt-1 as determined by immunolocalization⁺And/or VEGFR2/KDR⁺And wherein the isolated population of cells is not amniotic membrane. In another embodiment, the invention provides an isolated cell population comprising an amnion derived adherent cell that has been OCT-4 as determined by RT-PCR^–And HLA-G^–And VEGFR1/Flt-1 as determined by immunolocalization⁺Or VEGFR2/KDR⁺(ii) a And wherein the isolated population of cells is not amniotic membrane. In a specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are the amnion derived adherent cells. In another embodiment, the invention provides an isolated cell population comprising an amnion derived adherent cell, wherein said cell is attached to a tissue culture plastic, wherein said cell is OCT-4 as determined by RT-PCR^–Measured by immunolocalization as VEGFR1/Flt-1⁺And VEGFR2/KDR⁺Wherein said cell is further CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–Or CXCR4^–Or via, for example>20 cycles of RT-PCR assay were HLA-G^–And wherein the isolated population of cells is not amniotic membrane. In a specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are the amnion derived adherent cells. In another embodiment, the invention provides an isolated cell population comprising an amnion derived adherent cell, wherein said cell is attached to a tissue culture plastic, wherein said cell is OCT-4 as determined by RT-PCR^–Measured by immunolocalization as VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺Wherein the cells do not express CD34 after 4-21 days of exposure to 1-100ng/mL of VEGF as determined by immunolocalization, and wherein the isolated population of cells is not amniotic membrane. In a specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are the amnion derived adherent cells. In another embodiment, any of the cell populations comprising amnion derived adherent cells described above are cultured on a substrate, e.g., MATRIGEL, in the presence of an angiogenic factor, e.g., Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), or basic fibroblast growth factor (bFGF)^TMIn the above, a bud or tubular structure is formed.

In another embodiment, the invention provides a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion derived adherent cells that are OCT-4 as determined by RT-PCR^–And positive for VEGFR2/KDR, CD9, CD54, CD105, or CD 200. In a specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion derived adherent cells as determined by OCT-4 by RT-PCR^–And measured by immunolocalization as VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺And CD200⁺. In a more specific embodiment, the amnion derived adherent cells do not express CD34 as determined by immunolocalization after 4-21 days of exposure to 1-100ng/mL VEGF. In a specific embodiment, the amnion derived adherent cells are attached to a tissue culture plastic. In another specific embodiment, the population of cells is cultured on a substrate, e.g., MATRIGEL, in the presence of an angiogenic factor, such as Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF)^TMIn the above, a bud or tubular structure is formed.

In another embodiment, the invention provides a population of cells, e.g., human cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion derived adherent cells, it expresses one or more of ACTA, ADAMTS, AMAT, ANG, ANGPT, ANGPTL, BAI, CD200, CEACAM, CHGA, COL15A, COL18A, COL4A, CSF, CTGF, CXCL, DNMT3, ECGF, EDG, EDIL, ENPP, EPHB, FBLN, F, FGF, FIGF, FLT, FN, FST, FOXC, GRN, HGF, HEY, HSPG, IFNB, IL12, ITGA, GAITV, ITGB, MDK, MMP, MYOZ, NRP, PDGFB, PDGFRB, PEVEGFR, PGK, PROX, PTN, SEMA3, SERPINI, SERPINB, SERPINF, TIMP, TGFAB, TGTFFA, TGFAF, THGFRP, TNGFRP, VEGF/VEGFR.

In another embodiment, the invention provides a population of cells, e.g., amnion derived adherent cell populations; or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion-derived adherent cells that express CD49d, connexin-43, HLA-ABC, β 2-microglobulin, CD349, CD318, PDL1, CD106, galectin-1, ADAM17 precursor (disintegrin and metalloprotease domain 17) (TNF- α convertase), angiotensinogen precursor, filamin a (α -filamin) (filamin 1) (endothelial actin-binding protein) (ABP-280) (non-muscle filamin), α -actinin 1(α -actinin cytoskeletal subtype) (non-muscle α -actinin 1) (F-actinin), low density lipoprotein receptor-related protein 2 precursor (Megalin) (330) (gp330), glycoprotein (gp330), or a variant thereof, One or more or all of type I and type II macrophage depleting receptors (macrophage acetylated LDL receptors I and II), activin receptor type II precursor B (ACTR-IIB), Wnt-9 protein, glial fibrillary acidic protein, astrocytes (GFAP), myosin-binding protein C, cardiac-type (cardiac MyBP-C) (C-protein, myocardial subtype), or myosin heavy chain, non-muscle type a (cellular myosin heavy chain, type a) (non-muscle myosin heavy chain-a) (NMMHC-a).

In another aspect, the invention provides a population of cells, e.g., amnion derived adherent cells; or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of the cells in the isolated population of cells are amnion derived adherent cells that secrete one or more or all of VEGF, HGF, IL-8, MCP-3, FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2, uPAR, or galectin-1 in, e.g., the medium in which the cells are grown.

In another embodiment, the invention provides a population of cells, e.g., amnion derived adherent cell populations; or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion-derived adherent cells that express angiogenic micrornas (mirnas) at a level greater than bone marrow-derived mesenchymal stem cells, wherein the mirnas comprise one or more or all of miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, and/or miR-296. In another embodiment, the invention provides a population of cells, e.g., amnion derived adherent cell populations; or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion-derived adherent cells that express angiogenic micrornas (mirnas) at a level that is lower than bone marrow-derived mesenchymal stem cells, wherein the mirnas comprise one or more or all of miR-20a, miR-20b, miR-221, miR-222, miR-15b, and/or miR-16. In certain embodiments, the AMDACs, or population of AMDACs, express one or more or all of the angiogenic mirnas: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, miR-296, miR-221, miR-222, miR-15b, and/or miR-16.

In another specific embodimentThe invention provides an amniotic membrane derived angiogenic cell, or a population of amniotic membrane derived angiogenic cells, that is exposed to hypoxic conditions (e.g., less than about 5% O)₂) Under relatively normoxic conditions (e.g., about 20% or about 21% O)₂) The increased levels express CD202b, IL-8 and/or VEGF.

In another specific embodiment, the isolated population of amnion derived adherent cells further comprises a second cell type. In specific embodiments, the AMDACs comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or at least 98% of the cells in the population. In a specific embodiment, the second cell type is contained in or isolated from placental blood, umbilical cord blood, coarse bone marrow, or other tissue. In a more specific embodiment, the second cell type is an embryonic stem cell, a blood cell, a stem cell isolated from peripheral blood, a stem cell isolated from placental perfusate, a stem cell isolated from placental tissue, a stem cell isolated from umbilical cord blood, an umbilical cord stem cell, a bone marrow-derived mesenchymal stem cell, a hematopoietic stem cell, a somatic stem cell, a chondrocyte, a fibroblast, a muscle cell, an endothelial progenitor cell, a pericyte, a muscle cell, a cardioblast, a myoblast, an angioblast, or a cardiac myoblast. In another specific embodiment, the second cell type is a hematopoietic stem or progenitor cell, e.g., CD34⁺A cell. In another more specific embodiment, said second cell type comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 98% of the cells in said population.

In another specific embodiment, any of the above cells are being or have been cultured to proliferate. In another specific embodiment, any of the above cells are from the cell culture that has been passaged at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, or more. In another specific embodiment, any of the above cells is from a culture that doubles at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or at least 50 times in culture, or more.

In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising any one of the amnion derived adherent cells, or a cell population comprising amnion derived adherent cells, provided herein. In a specific embodiment, the composition is a matrix or scaffold, e.g., a natural tissue matrix or scaffold, e.g., a permanent or degradable decellularized tissue matrix or scaffold; or a synthetic matrix or scaffold. In a more specific embodiment, the matrix or scaffold is in the shape of a tube or other organ-like three-dimensional form. In another more specific embodiment, the matrix is a decellularized tissue matrix. In another specific embodiment, a composition comprises one or more isolated amnion derived adherent cells, or a population of cells comprising amnion derived adherent cells, provided herein, in a physiologically acceptable solution, e.g., saline solution, culture medium, and the like.

In another aspect, the invention provides a method of treating an individual having a disease or disorder of the circulatory system, comprising administering to the individual one or more amnion derived adherent cells described herein in an amount and for a time sufficient to substantially ameliorate one or more symptoms of the disease or disorder. In another embodiment, the invention provides a method of treating an individual having a disease or disorder of the circulatory system, comprising administering amnion derived adherent cells to the individual, the amount and duration of administration is sufficient to significantly improve one or more cardiac function indicators as compared to the individual prior to administration of the amnion derived adherent cells, wherein the cardiac function index is thoracic Cardiac Output (CO), Cardiac Index (CI), Pulmonary Artery Wedge Pressure (PAWP), Cardiac Index (CI),% fractional shortening (% FS), Ejection Fraction (EF), Left Ventricular Ejection Fraction (LVEF), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), contractility (dP/dt), decreased atrial or ventricular function, increased pump efficiency, decreased rate of pump efficiency loss, decreased hemodynamic function loss, or decreased complications associated with cardiomyopathy.

In a specific embodiment, the disease or disorder is myocardial infarction. In another specific embodiment, the disease or disorder is cardiomyopathy. In other specific embodiments, the disease or disorder is aneurysm, angina, aortic stenosis, aortic inflammation, arrhythmia, arteriosclerosis, arteritis, asymmetric ventricular septal hypertrophy (ASH), atherosclerosis, atrial fibrillation and flutter, bacterial endocarditis, Barlow syndrome (mitral valve prolapse), bradycardia, vasculitis disease (thromboangiitis obliterans), cardiac hypertrophy, myocarditis, carotid artery disease, aortic stenosis, congenital heart defects, congestive heart failure, coronary artery disease, eisenmenenger syndrome, embolism, endocarditis, erythromelalgia, fibromyalgia, fibromuscular dysplasia, cardiac conduction block, heart murmur, hypertension, hypotension, idiopathic infantile arterial calcification, kawasaki disease (mucocutaneous lymph node syndrome, mucocutaneous lymphadenopathy, pediatric polyarteritis), Metabolic syndrome, microvascular angina, myocarditis, Paroxysmal Atrial Tachycardia (PAT), periarteritis nodosa (polyarteritis, polyarteritis nodosa), pericarditis, peripheral vascular disease, critical limb ischemia, phlebitis, pulmonary stenosis (pulmonary stenosis), Raynaud's disease, renal artery stenosis, renal hypertension, rheumatic heart disease, diabetic vasculopathy, septal defects, asymptomatic myocardial ischemia, syndrome X, tachycardia, takayasu's arteritis, Falltetrad syndrome, large vessel transposition, tricuspid valve occlusion, arterial stem, valvular heart disease, varicose ulcer, varicose, vasculitis, ventricular septal defects, Wolff-Parkinson-Wharton syndrome, endocardial defects, acute rheumatic fever, acute rheumatic pericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis, chronic rheumatic heart disease, myocardial infarction, myocardial, Mitral valve disease, mitral valve stenosis, rheumatic mitral insufficiency, aortic valve disease, other diseases of the endocardial structure, ischemic heart disease (acute, subacute), angina pectoris, acute pulmonary heart disease, pulmonary embolism, chronic pulmonary heart disease, postspinal scoliotic heart disease, myocarditis, endocarditis, fibrosis of the endocardium and myocardium, hyperplasia of the fibroelastocardia endocardium, atrioventricular block, arrhythmia, cardiac degeneration, cerebrovascular disease, arterial, arteriolar and capillary disease, or venous and lymphatic disease.

In other specific embodiments, the disease or disorder is an occlusion and stenosis of a pre-cerebral artery, or an occlusion of a cerebral artery. In one aspect, the invention provides a method of treating an individual with impaired blood flow in and around the brain, for example, symptoms or neurological dysfunction resulting from impaired blood flow in or around the brain or Central Nervous System (CNS) of the individual, comprising administering to the individual a therapeutically effective amount of AMDACs. In certain embodiments, the disruption of blood flow results in hypoxic or hypoxic damage to the brain or CNS of the individual.

In other specific embodiments, the disease or disorder is peripheral arterial occlusion and stenosis. In one aspect, the invention provides a method of treating an individual with internal or peripheral blood flow disruption in a limb, e.g., a symptom or vascular defect arising from internal or peripheral blood flow disruption in the peripheral vascular system of the individual, comprising administering to the individual a therapeutically effective amount of an AMDAC. In certain embodiments, the disruption of blood flow results in hypoxic or hypoxic injury to a limb and or limb of the individual.

In another aspect, the invention provides a method of treating an individual having a wound or mental injury comprising administering to the individual one or more amnion derived adherent cells described herein in an amount and for a time sufficient to substantially ameliorate the wound or mental injury.

In another specific method of treatment embodiment, said cells are administered to said individual by injection. In a more specific embodiment, the injection is into an ischemic region of an individual's heart. In another particular method of treatment embodiment, the cells are administered to the individual intravenously. In another particular method of treatment embodiment, the cells, or population of cells comprising the cells, are administered to the individual by implanting in the individual a matrix or scaffold comprising amnion derived adherent cells as described above.

The isolated amnion derived adherent cells and cell populations provided herein are not isolated placental stem cells or cell populations such as described in U.S. Pat. No. 7,255,879 or U.S. patent application publication No. 2007/0275362. The isolated amnion-derived adherent cells provided herein are also not endothelial progenitor cells, amniotic epithelial cells, trophoblasts, embryonic germ cells, embryonic stem cells, cells obtained from the inner cell mass of an embryo, or cells obtained from the gonadal ridges of an embryo.

The term "about" as used herein means, for example, within 10% of the stated number or value.

The term "angiogenic" as used herein with respect to amnion-derived adherent cells described herein means that the cells are capable of forming a vessel or vessel-like bud, or the cells are capable of promoting angiogenesis (e.g., formation of a vessel or vessel-like structure) in another cell population, e.g., endothelial cells.

The term "angiogenesis" as used herein refers to the process of angiogenesis, which includes, but is not limited to, activation, migration, proliferation, matrix remodeling and cell stabilization of endothelial cells.

The term "stem cell" as used herein defines the functional attributes of any given cell population, which is capable of widespread, but not necessarily unlimited proliferation, and which facilitates the formation of a variety of tissues during embryonic development or tissue replacement and repair after birth.

The term "progenitor cell" as used herein defines the functional attributes of any given cell population that is capable of extensive, but not necessarily unlimited proliferation, and facilitates the formation of a wide variety of tissues, limited relative to the number of stem cells, during embryonic development or post-natal tissue replacement and repair.

The term "derived" as used herein means isolated or otherwise purified. For example, amnion-derived adherent cells are isolated from amnion. The term "derived" encompasses cells cultured in culture isolated directly from tissue such as amniotic membrane, as well as cells cultured or expanded from primary isolates.

As used herein, "immunolocalization" refers to the detection of compounds such as cell markers in, for example, flow cytometry, fluorescence activated cell sorting, magnetic cell sorting, in situ hybridization, immunohistochemistry, and the like, using an immune protein, e.g., an antibody or fragment thereof.

The term "isolated cell" as used herein means a cell that is substantially isolated from other cells or tissues, such as the amniotic membrane or placenta from which the cell is derived. A cell is considered "isolated" if at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with which the stem cell is naturally associated are removed from the cell, for example, during collection and/or culture of the cell.

The term "isolated population of cells" as used herein means a population of cells that is substantially isolated from other cells or tissues, such as the amniotic membrane or placenta from which the population of cells was derived. A population of cells is considered "isolated" if at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with which the population is naturally associated or from which the population is derived are removed from the population during, for example, collection and/or culture of amnion-derived adherent cells.

When a specific marker can be detected from the background, for example by immunolocalization or by flow cytometry or by RT-PCRCells used herein are considered "positive" for this particular marker. For example, a cell is described as positive for CD105 if an amount of CD105 that is significantly above background (relative to, e.g., an isotype control) can be detected on the cell. In the context of, for example, antibody-mediated detection, "positive" as a hint of the presence of a particular surface marker of a cell means that the marker can be detected with an antibody, e.g., a fluorescently labeled antibody specific for the marker; "Positive" also means that the cell carries the marker, the amount of which can produce a signal in, for example, a cytometer, which can be detected in the background. For example, the cell is "CD 105⁺", wherein the cells are detectably labeled with a CD 105-specific antibody and the signal from the antibody is detectable above a control (e.g., background). Conversely, in the same context, "negative" means that the cell surface marker is undetectable using its specific antibody against background. For example, the cell is "CD 34^–", wherein the cells are not detectable with an antibody label specific for CD 34. Unless otherwise indicated herein, differentiation antigen ("CD") markers are detected using antibodies. For example, if OCT-4mRNA can be detected using, for example, 30 cycles of RT-PCR, it can be determined that OCT-4 is present and the cell is OCT-4⁺。

4. Brief description of the drawings

FIG. 1 shows stem cell-related gene expression of amnion-derived adherent cells and NTERA-2 cells.

Figure 2 shows TEM-7 expression at the cell surface of amnion derived adherent cells (AMDACs).

Figure 3 shows that amnion derived adherent cells secrete selected angiogenic proteins.

Figure 4 shows the angiogenic effect of amnion derived adherent cell conditioned medium on human endothelial cell (HUVEC) luminal formation.

Figure 5 shows the angiogenic effect of amnion derived adherent cell conditioned medium on human endothelial cell migration.

Figure 6 shows the effect of amnion derived adherent cell conditioned medium on human endothelial cell proliferation.

FIG. 7 shows the uptake of acetylated LDL by HUVEC and amnion derived adherent cells.

Figure 8 shows luminal formation of HUVEC and amnion derived adherent cells.

FIG. 9 shows the secretion of amnion derived adherent cells VEGF and IL-8 under hypoxic and normoxic conditions.

FIG. 10 shows the oxygen concentration in normal oxygen (about 21% O)₂) And hypoxia (less than about 5% O)₂) Expression of the cell marker Tie2 under conditions. Y-axis: percentage of Tie2 positive cells as determined by flow cytometry.

Figure 11 shows cardiomyocyte differentiation potential of amnion-derived adherent cells, wherein AM represents amnion-derived adherent cells (AMDAC), HD represents untreated hanging drops, HD IND represents hanging drops exposed to induction conditions, and HD IND +5-AZA represents induction in the presence or absence of 5-azacytidine. CTRL represents untreated hanging-drop control.

FIG. 12 shows the positive effect of AMDAC on angiogenesis in a chick chorioallantoic angiogenesis model. Lot1, Lot 2, Lot 3: AMDACs from three different cell preparations. bFGF: basic fibroblast growth factor (positive control). MDAMB 231: angiogenic breast cancer cell lines (positive control). Y-axis: the degree of vascularization.

FIG. 13 shows the positive effect of AMDAC-conditioned medium on angiogenesis in a chick chorioallantoic membrane angiogenesis model. Lot1, Lot 2, Lot 3: AMDACs from three different cell preparations. bFGF: basic fibroblast growth factor (positive control). MDAMB 231: angiogenic breast cancer cell lines (positive control). Y-axis: the degree of vascularization.

Fig. 14A, 14B: co-culture of mesenchymal stem cells (BM-MSC) in astrocyte cultures, astrocytes and bone marrowActive oxygen produced by hydrogen peroxide from cocultures of astrocytes and AMDAC. 14A: AMDAC, Lot 1; 14B: AMDAC, Lot 2. HA (human astrocytes) alone, astrocytes + H₂O₂And astrocytes + BM-MSC + H₂O₂The conditions of (c) are the same for fig. 14A and 14B. RFU ROS activity: relative fluorescence units of active oxygen.

5. Detailed description of the invention

5.1 characterization of amnion-derived adherent cells

The present invention provides unique adherent angiogenic cells and populations of such cells, isolated from amniotic membrane, referred to herein as "amnion derived adherent cells" or AMDACs. Amnion derived adherent cells are somewhat similar in appearance to mesenchymal cells, having a substantially fibroid shape. The cells attach to the surface of a cell culture, e.g., tissue culture plastic.

AMDACs can display cellular markers that distinguish them from other amnion-or placenta-derived cells. For example, in one embodiment, the amnion derived adherent cells are OCT-4 as determined by RT-PCR^–(octamer binding protein 4). In another specific embodiment, OCT-4^–The amnion derived adherent cells were CD49f as determined by immunolocalization⁺. In another specific embodiment, the OCT-4^–The cells were HLA-G as determined by RT-PCR^–. In another specific embodiment, OCT-4^–The cells were measured by immunolocalization to be VEGFR1/Flt-1⁺(vascular endothelial growth factor receptor 1) and/or VEGFR2/KDR⁺(vascular endothelial growth factor receptor 2). In one specific embodiment, OCT-4^–Amnion derived adherent cells or OCT-4^–Amnion derived adherent cell populations, e.g. in>At 20 cycles of PCR-amplification, at least 2log of PCR-amplified OCT-4mRNA was expressed less than NTERA-2 cells or NTERA-2 cell populations with equivalent cell numbers and RNA amplification cycles. In another specific embodiment, the OCT-4^–The cell is CD90⁺、CD105⁺Or CD117^–. In a more specific embodiment, the OCT-4^–The cell is CD90⁺、CD105⁺And CD117^–. In a more specific embodiment, the cell is OCT-4^–Or HLA-G^–And also CD49f⁺、CD90⁺、CD105⁺And CD117^–. In a more specific embodiment, the cell is OCT-4^–，HLA-G^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–. In another specific embodiment, OCT-4^–Cells do not express SOX2 as determined by, for example, 30 cycles of RT-PCR. In a specific embodiment, the cell is OCT-4 as determined by immunolocalization or flow cytometry^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–And SOX2 as determined by RT-PCR over, for example, 30 cycles^–。

In another embodiment, the OCT-4^–The cells were CD29 by immunolocalization assay⁺、CD73⁺，ABC-p⁺And CD38^–One or more of (a).

In another specific embodiment, for example, OCT-4^–AMDAC can also be CD9 by immunolocalization determination⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、TEM-7⁺(tumor endothelial cell marker 7), CD31^–、CD34^–、CD45^–、CD133^–、CD143^–(angiotensin I converting enzyme, ACE), CD146^–(melanoma cell adhesion molecule), or CXCR4^–(chemokine (C-X-C motif) receptor 4) or HLA-G as determined by RT-PCR^–. In a more specific embodiment, the cell is immunolocalized and determined to be CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And CXCR4^–And HLA-G by RT-PCR^–. In one embodiment, the amnion derived adherent cells provided herein are CD31^–、CD34^–、CD45^–And/or CD133^–One or more of (a). In a specific embodiment, the amnion derived adherent cells are OCT-4 as determined by RT-PCR^–(ii) a Measured by immunolocalization to be VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺(ii) a And CD31^–、CD34^–、CD45^–And/or CD133^–One or more or all of them.

In another specific embodiment, said cell is also VE-cadherin as determined by immunolocalization^–. In another specific embodiment, the cells are additionally CD105 as measured by immunolocalization⁺And CD200⁺Is positive. In another specific embodiment, the cells do not express CD34 after 4-21 days of exposure to 1-100ng/mL VEGF as determined by immunolocalization. In a more specific embodiment, the cell does not express CD34 as determined by immunolocalization after exposure to 25-75ng/mL VEGF for 4-21 days, or 50ng/mL VEGF for 4-21 days. In a more specific embodiment, the cell does not express CD34 as determined by immunolocalization after 4-21 days exposure to 1, 2.5, 5, 10, 25, 50, 75, or 100ng/mL VEGF. In a more specific embodiment, the cells do not express CD34 as determined by immunolocalization after 7-14 days, e.g., 7 days, exposure to 1-100ng/mL VEGF.

In a specific embodiment, the amnion derived adherent cells are OCT-4 as determined by RT-PCR^–And VE-cadherin as determined by immunolocalization^–、VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺And/or CD200⁺One or more of (a). In a specific embodiment, the amnion-derived cells are OCT-4 as determined by RT-PCR^–And VE-cadherin as determined by immunolocalization^–、VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺And CD200⁺. In another specific embodiment, the cells do not express CD34 as determined by immunolocalization after, for example, exposure to 1-100ng/mL VEGF for 4-21 days.

In another embodiment, the amnion derived adherent cells are OCT-4^–、CD49f⁺、HLA-G^–、CD90⁺、CD105⁺And CD117^–. In a more specific embodiment, the cell is immunolocalized and determined to be CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–Or CXCR4^–One or more of (a). In a more specific embodiment, the cell is immunolocalized and determined to be CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And CXCR4^–. In another specific embodiment, the cells are also VEGFR1/Flt-1 as determined by immunolocalization⁺And/or VEGFR2/KDR⁺(ii) a And CD31 as determined by immunolocalization^–、CD34^–、CD45^–、CD133^–And/or Tie-2^–One or more of (a). In another specific embodiment, the cells are also VEGFR1/Flt-1 as determined by immunolocalization⁺、VEGFR2/KDR⁺、CD31^–、CD34^–、CD45^–、CD133^–And Tie-2^–。

In another embodiment, OCT-4^–The amnion derived adherent cells are CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD49f⁺、CD54⁺、CD90⁺、CD98⁺、CD105⁺、CD200⁺、Tie-2⁺、TEM-7⁺、VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺(CD309⁺) One or more or all of; or CD31 as determined by immunolocalization^–、CD34^–、CD38^–、CD45^–、CD117^–、CD133^–、CD143^–、CD144^–、CD146^–、CD271^–、CXCR4^–、HLA-G^–And/or VE-cadherin^–Or SOX2 by RT-PCR^–。

In certain embodiments, the isolated amnion derived adherent cells adherent to tissue culture plastic are CD49f⁺. In a specific embodiment, the CD49f⁺The cells were CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD54⁺、CD90⁺、CD98⁺、CD105⁺、CD200⁺、Tie-2⁺、TEM-7⁺、VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺(CD309⁺) One or more or all of; or CD31 as determined by immunolocalization^–、CD34^–、CD38^–、CD45^–、CD117^–、CD133^–、CD143^–、CD144^–、CD146^–、CD271^–、CXCR4^–、HLA-G^–、OCT-4^–And/or VE-cadherin^–Or SOX2 by RT-PCR^–。

In certain other embodiments, the isolated tissue culture plastic-adherent amnion-derived cells are HLA-G^–、CD90⁺And CD117^–. In a specific embodiment, the HLA-G^–、CD90⁺And CD117^–The cells were CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD49f⁺、CD54⁺、CD98⁺、CD105⁺、CD200⁺、Tie-2⁺、TEM-7⁺、VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺(CD309⁺) One or more or all of; or CD31 as determined by immunolocalization^–、CD34^–、CD38^–、CD45^–、CD133^–、CD143^–、CD144^–、CD146^–、CD271^–、CXCR4^–、OCT-4^–And/or VE-cadherin^–Or SOX2 by RT-PCR^–。

In another embodiment, the isolated amnion-derived adherent cells or amnion-derived angiogenic cell population do not constitutively express the following mRNA as measured by, for example, 30 cycles of RT-PCR under standard culture conditions: fibroblast growth factor 4(FGF4), interferon gamma (IFNG), chemokine (C-X-C motif) ligand 10(CXCL10), angiopoietin 4(ANGPT4), angiopoietin-like 3(ANGPTL3), fibrinogen alpha chain (FGA), Leptin (LEP), Prolactin (PRL), prodynein 1(PROK1), Tenascin (TNMD), FMS-like tyrosine kinase 3(FLT3 gene), the extracellular linking domain houses 1(XLKD1), cadherin 5, type 2 (CDH5), leukocyte-derived chemokine 1(LECT1), Plasminogen (PLG), telomerase reverse transcriptase (TERT), (sex-determining region Y) -cassette 2(SOX2), NANOG, matrix metalloproteinase 13(MMP-13), distalmless-less homeobox 5(DLX5), and/or bone gamma-carboxyglutamic acid (GLA) protein (BGLAP). In other embodiments, the isolated amnion-derived adherent cells or amnion-derived angiogenic cell population express mRNA from: (ARNT2), Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), glial cell derived neurotrophic factor (GDNF), neurotrophic factor 3(NT-3), NT-5, hypoxia-inducible factor 1 α (HIF1A), hypoxia-inducible protein 2(HIG2), heme oxidase (decyclization) 1(HMOX1), extracellular superoxide dismutase [ Cu-Zn ] (SOD3), Catalase (CAT), transforming growth factor β 1(TGFB1), transforming growth factor β 1 receptor (TGFB1R), and hepatocyte growth factor receptor (HGFR/c-met).

In another aspect, the invention provides an isolated population of cells comprising amnion derived adherent cells as described herein. The population of cells can be a homogeneous population, e.g., at least about 90%, 95%, 98%, or 99% of the population of cells are amnion-derived adherent cells. The cell population can be mixed, e.g., up to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the cells in the cell population are amnion derived adherent cells. However, the isolated cell population is not a tissue, i.e., is not an amniotic membrane.

In one embodiment, the invention provides an isolated cell population, e.g., a substantially homogeneous cell population, comprising AMDACs, wherein the AMDACs are attached to a tissue culture plastic, and wherein the AMDACs are OCT-4 as determined by RT-PCR^–. In a specific embodiment, the AMDAC is CD49f, as determined, for example, by immunolocalization or RT-PCR⁺Or HLA-G⁺. In another specific embodiment, the population of AMDACs is VEGFR1/Flt-1 as determined by immunolocalization⁺And/or VEGFR2/KDR⁺Wherein the isolated population of cells is not amniotic membrane (amnion/amniotic membrane). In a more specific embodiment, AMDAC is OCT-4 as determined by RT-PCR^–And/or HLA-G^–And VEGFR1/Flt-1 as determined by immunolocalization⁺And/or VEGFR2/KDR⁺. In a specific embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are the amnion derived adherent cells. In another specific embodiment, the AMDAC is CD90⁺、CD105⁺Or CD117^–. In a more specific embodiment, the AMDAC is CD90⁺、CD105⁺And CD117^–. In a more specific embodiment, the AMDAC is OCT-4^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–. In another specific embodiment, AMDACs do not express SOX2, as determined by, for example, 30 cycles of RT-PCR. In a more specific embodiment, the population contains AMDACs, and wherein said AMDACs are OCT-4 as determined by immunolocalization or flow cytometry^–、HLA-G^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–And for example SOX2 over 30 cycles of RT-PCR^–。

In another specific embodiment, said AMDACs in said cell population are CD90 as determined by immunolocalization or flow cytometry⁺、CD105⁺Or CD117^–. In a more specific embodiment, the AMDAC is CD90 as determined by immunolocalization or flow cytometry⁺、CD105⁺And CD117^–. In a more specific embodiment, AMDAC is OCT-4, as determined by RT-PCR^–Or HLA-G^–And is also CD49f as determined by immunolocalization or flow cytometry⁺、CD90⁺、CD105⁺And CD117^–. In a more specific embodiment, the AMDAC in the cell population is OCT-4^–、HLA-G^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–. In another specific embodiment, AMDACs do not express SOX2, as determined by, for example, 30 cycles of RT-PCR. In a more specific embodiment, the cell is OCT-4 as determined by immunolocalization or flow cytometry^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–And SOX2 as determined by RT-PCR over, for example, 30 cycles^–. In a more specific embodiment, the AMDAC is OCT-4^–Or HLA-G^–And additionally CD49f⁺、CD90⁺、CD105⁺And CD117^–. In a more specific embodiment, the AMDAC is OCT-4^–、HLA-G^–、CD49f⁺、CD90⁺、CD105⁺And CD117^–。

In another embodiment, the amnion derived adherent cells in the cell population are attached to tissue culture plastic that is OCT-4 as determined by RT-PCR^–And immunolocalization assay by VEGFR1/Flt-1⁺And/or VEGFR2/KDR⁺And are immunologically localizedMeasured also as CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–Or CXCR4^–Or HLA-G by RT-PCR^–And wherein the isolated population of cells is not amniotic membrane. In another embodiment, the invention provides an isolated cell population comprising an amnion derived adherent cell, wherein said cell is attached to a tissue culture plastic, wherein said cell is OCT-4 as determined by RT-PCR^–And VEGFR1/Flt-1 as determined by immunolocalization⁺And/or VEGFR2/KDR⁺Wherein the cells do not express CD34 after 4-21 days of exposure to 1-100ng/mL of VEGF as determined by immunolocalization, and wherein the isolated population of cells is not amniotic membrane. In a specific embodiment of any one of the above embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are the amnion derived adherent cells.

In another embodiment, any of the above cell populations comprising amnion derived adherent cells form a sprout or tubular structure when cultured in the presence of an extracellular matrix protein, such as collagen type I and IV, or an angiogenic factor, such as Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), or basic fibroblast growth factor (bFGF), such as placental collagen or, for example, MATRIGEL^TMIs cultured in or on the substrate for at least 4 days and up to 14 days.

Amnion-derived adherent cells and amnion-derived adherent cell populations exhibit the property of being capable of expressing proteins involved in angiogenesis-related or cardiomyogenesis-related genes. In certain embodiments, the invention provides a cell or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion derived adherent cells that express one or more or all of the following RNAs: ACTA2 (actin,. alpha.2, smooth muscle, aorta), ADAMTS1 (ADAM metallopeptidase with thrombin-sensitized protein 1-type motif), AMAT (angiostatin-binding protein), ANG (angiogenin), ANGPT1 (pro-angiogenic protein factor 1), ANGPT2, ANGPTL1 (pro-angiogenic protein factor-like 1), ANGPTL2, ANGPTL4, BAI1 (brain-specific angiogenesis inhibitor 1), CD44, CD200, CEACAM1 (embryonic antigen-associated cell adhesion molecule 1), CHGA (chromogranin A), COL15A1 (collagen, type XV, type. alpha.1), COL18A1 (collagen, type XVIII,. alpha.1), COL4A1 (collagen, type IV,. alpha.1), COL4A2 (collagen, type GF, type IV,. alpha.2), COL4A3 (collagen, type IV, alpha.3), connective tissue growth factor (CTC-derived factor 3985), and colony stimulating factor (CTC-derived factor 12), CXCL2, DNMT3B (DNA (cytosine-5-) -methyltransferase 3 β), ECGF1 (thymine phosphorylase), EDG1 (endothelial cell differentiation gene 1), EDIL3 (EGF-like repeat and discodermin I-like domain 3), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), EPHB2(EPH receptor B2), FBLN5(FIBULIN 5), F2 linkage (clotting factor II (thrombin)), FGF1 (acidic fibroblast growth factor), FGF2 (basic fibroblast growth factor), fiff (C-fos-induced growth factor (vascular endothelial growth factor D)), FLT4 (fms-related tyrosine kinase 4), FN1 (fibronectin 1), FST (follistatin), FOXC2 (wishbone C2(MFH-1, wishbone 1)), GRN (reticulon protein), HGF (fibroblast growth factor) ("GRN (mast cell growth factor) (" fln)), HEY1 (YRPW-associated hairlike/division enhancer motif 1), HSPG2 (heparan sulfate proteoglycan 2), IFNB1 (interferon, β 1, fibroblast), IL8 (interleukin 8), IL12A, ITGA4 (integrin, α 4; CD49d), ITGAV (integrin, α V), ITGB3 (integrin, β 3), MDK (heparin-binding cytokine), MMP2 (matrix metalloproteinase 2), MYOZ2(myozenin 2), NRP1 (neuropilin 1), NRP2, PDGFB (platelet-derived growth factor β), PDGFRA gene (platelet-derived growth factor receptor α), PDGFRB, PEFR 1 (platelet/endothelial cell adhesion molecule), PF4 (platelet factor 4), K1 (phosphoglycerate kinase 1), PROX1 (prosporo homeobox 1), SEN (SEMEN 3), PTMEN 5963 (pleiotrophin 2), PGMA peptidase inhibitor (PGMA 5963), ramus b (ovalbumin), member 5), serpin 1, serpin nf1, TIMP2 (tissue metalloproteinase 2 tissue inhibitor), TIMP3, TGFA (transforming growth factor, α), TGFB1, THBS1 (thrombin-sensitized protein 1), THBS2, TIE1 (immunoglobulin-like tyrosine kinase and EGF-like domain 1), TIE2/TEK, TNF (tumor necrosis factor), TNNI1 (troponin I, type 1), TNFSF15 (tumor necrosis factor (ligand) superfamily, member 15), VASH1 (angiostatin 1), VEGF (vascular endothelial growth factor), VEGFB, VEGFC, VEGFR1/FLT1 (vascular endothelial growth factor receptor 1), and/or VEGFR 2/KDR.

When human cells are used, all of the designated genes represent human sequences, and representative sequences can be found in the literature or in GenBank, as is well known to those skilled in the art. Probes for these sequences may be determined by publicly available sequences, or by commercial sources, e.g., specificityProbe orAngiogenesis arrays (Applied Biosystems, trade name 4378710).

Amnion-derived adherent cells and amnion-derived adherent cell populations exhibit the property of being capable of expressing angiogenesis-related proteins. In certain embodiments, the invention provides a cell or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion-derived adherent cells that express CD49d, connexin-43, HLA-ABC, β 2-microglobulin, CD349, CD318, PDL1, CD106, galectin-1, ADAM17 precursor (a disintegrin and metalloproteinase domain 17) (TNF- α convertase), angiotensinogen, filamin a (α -filamin) (filamin 1) (endothelial binding protein) (ABP-280) (non-muscle filamin), α -actinin 1(α -actinin cytoskeletal subtype) (non-muscle α -actinin 1) (F-actinin cross-linking protein), Low density lipoprotein receptor-associated protein 2 precursor (megalin) (glycoprotein 330) (gp330), macrophage scavenger receptors type I and II (macrophage acetylated LDL receptor type I and II), activin receptor type IIB precursor (ACTR-IIB), Wnt-9 protein, glial fibrillary acidic protein, astrocytes (GFAP), myosin-binding protein C, heart-type (cardiac MyBP-C) (C-protein, cardiac muscle subtype), and/or myosin heavy chain, non-muscle type a (cellular myosin heavy chain, type a) (non-muscle myosin heavy chain-a) (NMMHC-a).

The amnion-derived adherent cells provided herein further secrete angiogenesis-promoting proteins in cells such as endothelial cells and endothelial progenitor cells. In certain embodiments, the amnion derived adherent cells, amnion derived adherent cell population, or cell population comprising amnion derived adherent cells, e.g., wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated cell population are amnion derived adherent cells, secrete one or more or all of VEGF, HGF, IL-8, MCP-3, FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2, uPAR, galectin-1 into, e.g., a medium in which the cells or cell population are grown.

In another embodiment, any of the above cell populations comprising amnion derived adherent cells is capable of causing the formation of a bud or tube-like structure in a population of endothelial cells contacted with the amnion derived adherent cells. In a particular embodiment, for example in the presence of collagen such as types I and IV, and/or angiogenic factors such as Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), Platelet Derived Growth Factor (PDGF) or basic fibroblast growth factor (bFGF) extracellular matrix proteins, for example, in or on a matrix such as placental collagen or MATRIGEL^TMAfter at least 4 days and/or up to 14 days of culturing, the amnion-derived angiogenic cells are co-cultured with human endothelial cells to form a sprout or tube-like structure or to support the sprouting of endothelial cells.

In another embodiment, any of the above cell populations comprising amnion derived adherent cells are in the presence of extracellular matrix proteins such as collagen types I and IV, within or on a matrix such as placental collagen or MATRIGEL^TMWhen cultured, secretes angiogenic factors such as Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), or interleukin-8 (IL-8), and thereby can induce human endothelial cells to form bud or tube-like structures.

In another embodiment, the invention provides a population of cells, e.g., amnion derived adherent cell populations; or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion-derived adherent cells that express angiogenic micrornas (mirnas) at a level greater than bone marrow-derived mesenchymal stem cells, wherein the mirnas comprise one or more or all of miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, and/or miR-296. In another embodiment, the invention provides a population of cells, e.g., amnion derived adherent cell populations; or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the isolated population of cells are amnion-derived adherent cells that express one or more or all angiogenic micrornas (mirnas) at a level that is lower than bone marrow-derived mesenchymal stem cells, wherein the mirnas comprise one or more or all of miR-20a, miR-20b, miR-221, miR-222, miR-15b, and/or miR-16. In certain embodiments, the AMDAC or population of AMDACs expresses one or more or all of the angiogenic mirnas: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, (a member of an angiogenic miRNA cluster 17-92), miR-296, miR-221, miR-222, miR-15b, and/or miR-16.

In one embodiment, the invention provides an isolated amnion derived adherent cell, wherein the cell is attached to a tissue culture plastic, and wherein the cell is OCT as determined by RT-PCR-4^–And CD49f as determined by immunolocalization⁺、HLA-G^–、CD90⁺、CD105⁺And CD117^–And wherein the cells express one or more of CD, CD200, Tie-2, TEM-7, VEGFR/Flt-1, VEGFR/KDR (CD309), or VEGF/KDR (CD309), or do not express SOX as determined by RT-PCR, or express mRNA for ACTA, ADAMTS, AMAT, ANGG, ANGPT, ANGPTT, ANGPTL, ANGPTTL, BAI, CD200, CEACAM, CHGA, COL15A, COL18A, COL4A, COLCSF, CTGF, CXCL, NI 3, MT, ECL, ENCSF, COL18A, COL4A, COL GF, FGF, VEGF-7, miR-11, miR-7, miR-1, miR-7, VEGF-CD-or VEGFR (VEGFR, VEGFR-CD-7, or VEGFR (VEGFR) protein, or miR-CD-or RT (RT-CDMesenchymal stem cells of myeloid origin; (h) and (3) expressing miRNA: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, miR-296, miR-221, miR-222, miR-15b, or miR-16; and/or (i) to 21% O₂The following expression of CD202b, IL-8 or VEGF when cultured in less than about 5% O₂Express increased levels of CD202b, IL-8, or VEGF. In a specific embodiment, the isolated amnion derived adherent cells are OCT-4 as determined by RT-PCR^–And CD49f as determined by immunolocalization⁺、HLA-G^–、CD90⁺、CD105⁺And CD117^–And (a) a protein which expresses CD, CD200, Tie-2, TEM-7, VEGFR/Flt-1, and/or VEGFR/KDR (CD309) as determined by immunolocalization, (b) a protein which does not express CD, CD133, CD143, CD144, CD146, CD271, CXCR, HLA-G, and/or VEGFR-cadherin as determined by immunolocalization, or (C) a protein which expresses mRNA selected from ACTA, ADAMTS, AMAT, ANGG, ANGPT, ANGPTT, ANGPTTL, BAI, CD200, ACACCEM, CHGA, COL15A, COL18A, COL4A, COLCSF, CTGF, CXCL, CXGF, DNN 3, ECGF, MGPG, ACEPCP, VEGF-binding, VEGF or VEGF-binding proteins which are secreted by determined by immunolocalization and/or by determined by immunolocalization as determined by immunolocalization, or by immunology-PCR, (C-PCR) and/or by localization, and (T-PCR, and (T-binding to a receptor protein receptor binding proteins which are expressed by a receptor, and (or by a receptor which are involved in a receptor,FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2, uPAR, and/or galectin-1, for example, into the medium in which the cells are grown; (f) expression of micrornas: the level of miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92 and/or miR-296 is higher than that of a corresponding number of bone marrow-derived mesenchymal stem cells; (g) expression of micrornas: the level of miR-20a, miR-20b, miR-221, miR-222, miR-15b, and/or miR-16 is lower than that of a comparable number of bone marrow-derived mesenchymal stem cells; (h) and (3) expressing miRNA: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, miR-296, miR-221, miR-222, miR-15b, and/or miR-16; and/or (i) to 21% O₂The following expression of CD202b, IL-8 or VEGF when cultured in less than about 5% O₂Express increased levels of CD202b, IL-8, or VEGF. Further provided are cell populations comprising AMDACs, for example AMDACs, having one or more of the above-described properties.

In another embodiment, any of the above cell populations comprising amnion derived adherent cells secrete angiogenic factors. In particular embodiments, the population of cells secretes Vascular Endothelial Growth Factor (VEGF), Epithelial Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), basic fibroblast growth factor (bFGF), and/or interleukin-8 (IL-8). In other specific embodiments, the population of cells comprising amnion-derived angiogenic cells secrete one or more angiogenic factors to induce migration of human endothelial cells in an in vitro wound healing assay. In other specific embodiments, the population of amnion-derived adherent cells induces maturation, differentiation, or proliferation of human endothelial cells, endothelial progenitor cells, muscle cells, or myoblasts.

In another embodiment, any of the above cell populations comprising amnion derived adherent cells are in the presence of extracellular matrix proteins such as collagen types I and IV and/or one or more angiogenic factors such as VEGF, EGF, PDGF or bFGF, for example, placental collagen or MATRIGEL^TMWhen cultured on the substrate of (2), the acetylated Low Density Lipoprotein (LDL) is absorbed.

In another embodiment, the invention provides a population of cells comprising amnion derived adherent cells, wherein said cells are attached to a tissue culture plastic, and wherein said cells are OCT-4 as determined by RT-PCR^–And VEGFR2/KDR as determined by immunolocalization⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺Or VE-cadherin^–. In specific embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are amnion-derived cells that are OCT-4 as determined by RT-PCR^–And measured by immunolocalization as VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺Or VE-cadherin^–. In another specific embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in the population are amnion-derived cells that are OCT-4 as determined by RT-PCR^–And measured by immunolocalization as VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺And VE-cadherin^–. In another specific embodiment, said RT-PCR assay is OCT-4^–And VEGFR2/KDR as determined by immunolocalization⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺Or VE-cadherin^–Does not express CD34 after 4-21 days of exposure to 1-100ng/mL VEGF as determined by immunolocalization. In another specific embodiment, said cell is also VE-cadherin^–。

The cell populations provided herein, comprising amnion derived adherent cells, are capable of forming a bud or tube-like structure, similar to a blood vessel or a vascular tube. In one embodiment, the population of cells comprising amnion derived adherent cells forms a bud or tube-like structure when cultured in the presence of an angiogenic moiety (motif) such as VEGF, EGF, PDGF or bFGF. In a more specific embodiment, the assay is performed by RT-PCROCT-4^–And VEGFR2/KDR as determined by immunolocalization⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺Or VE-cadherin^–The amniotic membrane derived cells of (a), which form a bud or tube-like structure when the cell population is cultured in the presence of Vascular Endothelial Growth Factor (VEGF).

The amnion-derived adherent cells described herein exhibit the above-described properties, e.g., a combination of cell surface markers and/or gene expression profiles, and/or angiogenic ability and function, in primary culture or during propagation in a medium suitable for stem cell culture, including, for example, media containing 1-100% DMEM-LG (Gibco), 1-100% MCDB-201(Sigma), 1-10% Fetal Calf Serum (FCS) (Hyclone Laboratories), 0.1-5 × insulin-transferrin-selenium supplement (ITS, Sigma), 0.1-5 × linoleic acid-bovine serum albumin (LA-BSA, Sigma), 10^-5-10^-15M dexamethasone (Sigma), 10^-2-10^-10M ascorbic acid 2-phosphate (Sigma), 1-50ng/mL Epidermal Growth Factor (EGF) (R)&DSYSTEMS), 1-50ng/mL platelet-derived growth factor (PDGF-BB) (R)&D SYSTEMS), and a medium of 100U penicillin/1000U streptomycin in one embodiment, the medium contains 60% DMEM-LG (Gibco), 40% MCDB-201(Sigma), 2% Fetal Calf Serum (FCS) (Hyclone Laboratories), 1 × insulin-transferrin-selenium supplement (ITS), 1 × linoleic acid bovine serum albumin (LA-BSA), 10U penicillin/1000U streptomycin^-9M dexamethasone (Sigma), 10^-4M ascorbic acid 2-phosphate (Sigma), Epidermal Growth Factor (EGF)10ng/ml (R)&D SYSTEMS), platelet derived growth factor (PDGF-BB)10ng/ml (R)&D SYSTEMS), and 100U penicillin/1000U streptomycin. Other suitable media are described in detail below.

An isolated amnion-derived adherent cell population provided herein can, for example, comprise about, at least about, or no more than about 1 × 10 in one container⁵、5×10⁵、1×10⁶、5×10⁶、1×10⁷、5×10⁷、1×10⁸、5×10⁸、1×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹Or more amnion derived adherent cells. In various embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the isolated population of cells provided herein are amnion derived adherent cells. That is, an isolated population of amnion-derived adherent cells can include, for example, up to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% non-stem cells.

Amnion-derived adherent cells provided herein can be cultured on a substrate. In various embodiments, the substrate can be any surface upon which culture and/or selection of amnion derived adherent cells can be performed. Typically, the substrate is a plastic, for example, a tissue culture dish or multi-well plate plastic. Tissue culture plastics may be treated, coated or blotted with biomolecules or synthetic mimic reagents, e.g., CELLTART^TM、MESENCULT^TMACF-substrate, ornithine, or polylysine, or an extracellular matrix protein, e.g., collagen, laminin, fibronectin, vitronectin, and the like.

Amnion-derived cells, e.g., amnion-derived adherent cells and populations of such cells, provided herein, can be isolated from one or more placentas. For example, an isolated amnion-derived cell population provided herein can be a placental cell population, including those derived from or contained within disrupted amnion tissue, e.g., tissue digest (i.e., cells collected by enzymatic digestion of the amnion), wherein the cell population is enriched for amnion-derived cells, and wherein the tissue is derived from a single placenta or from two or more placentas. The isolated amnion-derived cells can be cultured and expanded to produce the population of cells. A placental cell population containing amnion-derived adherent cells can also be cultured and expanded to produce an amnion-derived adherent cell population.

In certain embodiments, AMDACs displaying any of the above-described markers and/or gene expression characteristics have been passaged at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, or more. In certain other embodiments, AMDACs displaying any of the above-described markers and/or gene expression characteristics have been passaged in culture at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or at least 50 times, or more.

5.2 amnion derived adherent cell populations containing other cell types

An isolated population of cells comprising amnion derived adherent cells described herein can comprise a second cell type, e.g., placental cells that are not amnion derived adherent cells, or, e.g., cells that are not placental cells. For example, an isolated population of amnion-derived adherent cells can comprise, e.g., can be combined with a population of a second cell type, wherein the second cell type is, e.g., an embryonic stem cell; blood cells (e.g., placental blood cells, umbilical cord blood cells, peripheral blood cells, nucleated cells from placental blood, umbilical cord blood, or peripheral blood, etc.); stem cells isolated from blood (e.g., stem cells isolated from placental blood, umbilical cord blood, or peripheral blood); placental stem cells (e.g., placental stem cells described in U.S. patent No. 7,468,276 and U.S. patent application publication No. 2007/0275362, the disclosures of which are incorporated by reference in their entirety); nucleated cells from placental perfusate, e.g., total nucleated cells from placental perfusate; cord blood stem cells; a population of blood-derived nucleated cells; bone marrow-derived stromal cells; bone marrow-derived mesenchymal stem cells; bone marrow-derived hematopoietic stem cells; coarse bone marrow; adult (somatic) stem cells; a population of stem cells contained in a tissue; cultured cells, e.g., cultured stem cells; a fully differentiated cell population (e.g., chondrocytes, fibroblasts, amniotic cells, guerbet cells, myocytes, cardiomyocytes, etc.); peripheral cells, and the like. In a specific embodiment, the population of cells comprising amnion-derived adherent cells comprises placental stem cells or stem cells from umbilical cord blood. In certain embodiments, the second cell type is blood or blood cells, wherein red blood cells have been removed from the cell population.

In a specific embodiment, the second cell type is a hematopoietic stem cell. The hematopoietic stem cells may be present, for example, in raw placenta, umbilical cord blood, or peripheral blood; total nucleated cells from placental blood, umbilical cord blood, or peripheral blood; CD34 isolated from placental, umbilical cord or peripheral blood⁺A population of cells; in unprocessed bone marrow; total nucleated cells from bone marrow; CD34 isolated from bone marrow⁺Cell populations, etc.

In another embodiment, the isolated amnion derived adherent cell population is combined with a plurality of adult or progenitor cells from the vascular system. In various embodiments, the cell is an endothelial cell, an endothelial progenitor cell, a muscle cell, a cardiomyocyte, a peripheral cell, a hemangioblast, a myoblast, or a cardiac myoblast.

In another embodiment, the second cell type is a non-embryonic cell type that has been treated in culture to express pluripotency and functional markers associated with embryonic stem cells.

In particular embodiments of the isolated population of amnion-derived adherent cells described above, one or both of the amnion-derived adherent cells and the second type of cells are autologous or heterologous to the intended recipient of the cells.

Further provided herein is a composition comprising amnion derived adherent cells and a plurality of stem cells other than amnion derived adherent cells. In a specific embodiment, the composition comprises a stem cell from the placenta, i.e., placental stem cells, e.g., as described in U.S. Pat. nos. 7,045,148, 7,255,879, and 7,311,905; and placental stem cells of U.S. patent application publication No. 2007/0275362, the disclosures of any of which are incorporated herein by reference in their entirety. In a specific embodiment, the placental stem cells are CD200⁺And HLA-G⁺；CD73⁺、CD105⁺And CD200⁺；CD200⁺And OCT-4⁺；CD73⁺、CD105⁺And HLA-G⁺；CD73⁺And CD105⁺And, when said population is cultured under conditions that allow the formation of embryoid-like bodies, facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell; or OCT-4⁺And, when said population is cultured under conditions that allow the formation of embryoid-like bodies, facilitates the formation of one or more embryoid-like bodies in a population of placental cells comprising said stem cell; or any combination thereof. In a more specific embodiment, the CD200⁺、HLA-G⁺The stem cell is CD34^–、CD38^–、CD45^–、CD73⁺And CD105⁺. In another more specific embodiment, said CD73⁺、CD105⁺And CD200⁺The stem cell is CD34^–、CD38^–、CD45^–And HLA-G⁺. In another more specific embodiment, the CD200⁺、OCT-4⁺The stem cell is CD34^–、CD38^–、CD45^–、CD73⁺、CD105⁺And HLA-G⁺. In another more specific embodiment, said CD73⁺、CD105⁺And HLA-G⁺The stem cell is CD34^–、CD45^–、OCT-4⁺And CD200⁺. In another more specific embodiment, said CD73⁺And CD105⁺The stem cell is OCT-4⁺、CD34^–、CD38^–And CD45^–. In another more specific embodiment, the OCT-4⁺The stem cell is CD73⁺、CD105⁺、CD200⁺、CD34^–、CD38^–And CD45^–. In another more specific embodiment, the placental stem cells are maternal-derived (i.e., have a maternal genotype). In another more specific embodiment, the placental stem cells are paternally derived (i.e., have a paternal genotype).

In another specific embodiment, the composition comprises amnion derived adherent cells and embryonic stem cells. In another specific embodiment, the composition comprises amnion-derived adherent cells and mesenchymal cells or stem cells, e.g., bone marrow-derived mesenchymal cells or stem cells. In another specific embodiment, the composition comprises bone marrow-derived hematopoietic stem cells. In another specific embodiment, the composition comprises amnion derived adherent cells and hematopoietic progenitor cells, e.g., hematopoietic progenitor cells from bone marrow, fetal blood, umbilical cord blood, placental blood, and/or peripheral blood. In another specific embodiment, the composition comprises amnion derived adherent cells and somatic stem cells. In a more specific embodiment, the somatic stem cell is a neural stem cell, a hepatic stem cell, a pancreatic stem cell, an endothelial stem cell, a cardiac stem cell, or a muscle stem cell.

In other specific embodiments, the second cell type comprises about, at least, or no more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells in the population. In other specific embodiments, the AMDACs in the composition comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the cells in the composition. In other specific embodiments, amnion derived adherent cells comprise about, at least, or no more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% of the cells in the population.

The cells in the isolated population of amnion-derived adherent cells can be combined with a plurality of cells of another type, e.g., a population of stem cells in a ratio to the total number of nucleated cells in each population of about 100,000,000:1, 50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 2:1, 1:2, 1:5, 1:10, 1:100, 1:200, 1: 500:1, 1,000:1, 10:1,000, 1,000: 000, 1,000:1, 20:1,000: 1, 5:1,000: 1, 2:1,000, 1,000:1, 2:1,000, 1,000:1, 100, 200, 1:1, 100: 000, 1, 100, 200, 1:1, 500:1, 1,000, 1, or about 000:1,000, 1,000:1, 1. The cells in the isolated amnion derived adherent cell population can also be combined with multiple cells of multiple types.

5.3 growth culture

For any mammalian cell, the growth of the amnion derived adherent cells described herein depends on the particular medium selected for growth. Under the most preferred conditions, amnion derived adherent cells typically multiply in number after about 24 hours. During culture, the amnion-derived adherent cells described herein adhere to a culture substrate, such as the surface of a tissue culture vessel (e.g., tissue culture dish plastic, fibronectin-coated plastic, etc.) and form a monolayer. Typically, cells are stably established in culture within 2-7 days after amniotic membrane digestion. It proliferates at a rate of about 0.4-1.2 population doublings per day and can undergo at least 30-50 population doublings. The cells exhibit a mesenchymal/fibroblast-like phenotype in the half-full (confluent) and expanded states and a cuboidal/cobblestone-like appearance in the full (confluent) state, and culture proliferation is strongly contact inhibited. The population of amnion-derived angiogenic cells can form embryoid bodies during culture expansion.

5.4 method of obtaining amnion-derived angiogenic cells

Amnion derived adherent cells and cell populations comprising amnion derived adherent cells can be prepared, for example, by isolating from other cells or cell populations, for example, by digesting the amnion tissue by a particular method and optionally testing the resulting cells or cell populations for the presence or absence of a marker or combination of markers, the identity of amnion derived adherent cells, or by obtaining amnion cells and selecting amnion derived adherent cells based on the marker.

The amnion-derived adherent cells provided herein and the isolated cell population containing amnion-derived adherent cells can be prepared, for example, by digesting amnion tissue and further selecting adherent cells. In one embodiment, for example, an isolated amnion derived adherent cell or isolated population of cells comprising amnion derived adherent cells can be prepared by: (1) digesting the amniotic tissue with a first enzyme to dissociate cells from the amniotic epidermal layer on the amniotic mesenchymal cells; (2) then digesting the amniotic mesenchymal layer with a second enzyme to form a single cell suspension; (3) culturing cells in the single cell suspension on a tissue culture surface, such as tissue culture plastic; and (4) selecting cells that attach to the surface after medium replacement, thereby preparing an isolated population of cells comprising amnion-derived adherent cells. In a specific embodiment, the first enzyme is trypsin. In a more specific embodiment, the trypsin is used at a concentration of 0.25% trypsin (w/v) for 5-20 ml, e.g., 10 ml of solution per gram of amniotic membrane tissue to be digested. In another more specific embodiment, the tryptic digestion is performed at 37 ℃ for about 15 minutes and repeated up to 3 times. In another specific embodiment, the second enzyme is collagenase. In a more specific embodiment, the collagenase is used at a concentration of 50 to 500U/L for 5mL per gram of amniotic membrane tissue to be digested. In another more specific embodiment, the digestion with collagenase is carried out at 37 ℃ for about 45 to 60 minutes. In another specific embodiment, the single cell suspension formed after collagenase digestion is filtered between step (2) and step (3) using, for example, a 75 μ M-150 μ M filter. In another specific embodiment, the first enzyme is trypsin and the second enzyme is collagenase.

In another embodiment, the isolated population of cells comprising amnion-derived adherent cells can be obtained by selecting cells from the amnion, e.g., by digesting amniotic tissue as described elsewhere herein, which exhibit one or more amnion-derived adherent cell characteristics. In one embodiment, for example, the cell population is prepared by a method comprising selecting amniotic cells comprising: (a) OCT-4 is negative by RT-PCR, and (b)Positive for one or more of VEGFR2/KDR, CD9, CD54, CD105, CD200 as determined by immunolocalization; and isolating the cells from other cells to form a cell population. In a specific embodiment, the amniotic cells are also VE-cadherin^–. In a specific embodiment, the cell population is prepared by selecting placental cells that are: (a) OCT-4 negative by RT-PCR and VE-cadherin negative by immunolocalization, and (b) positive for one or more of VEGFR2/KDR, CD9, CD54, CD105, CD200 by immunolocalization; and isolating the cells from other cells to form a cell population. In certain embodiments, selection is performed by immunolocalization prior to selection by RT-PCR. In another specific embodiment, said selecting comprises selecting cells that do not express the cell marker CD34 after 4-21 days of culture in the presence of 1-100ng/mL VEGF.

In another embodiment, for example, the cell population is prepared by a method comprising selecting a cell population that is attached to tissue culture plastic and has been determined to be OCT-4 by RT-PCR^–And VEGFR1/Flt-1 as determined by immunolocalization⁺And VEGFR2/KDR⁺And isolating a cell population formed from said cells from other cells. In a specific embodiment, the cell population is prepared by a method comprising selecting for OCT-4 as determined by RT-PCR^–And VEGFR1/Flt-1 as determined by immunolocalization⁺、VEGFR2/KDR⁺And HLA-G^–The amniotic cells of (1). In another specific embodiment, the cell population is prepared by a method wherein the selection is further CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And/or CXCR4^–(chemokine (C-X-C motif) receptor 4) and isolating said cells from cells that do not exhibit one or more of these properties. In another specific embodiment, the cellThe population is determined by selecting whether it is VE-cadherin or not by immunolocalization^–From the amniotic cells of (a) and from VE-cadherin⁺Isolating the cell of (a) and preparing. In another specific embodiment, the population of cells is determined by selecting whether it is CD105 or an immunolocalized assay⁺And CD200⁺From CD105, and^–or CD200^–Isolating the cell of (a). In another specific embodiment, the cells do not express CD34 after 4-21 days of exposure to 1-100ng/mL VEGF as determined by immunolocalization.

In cell selection, it is not necessary to detect amnion-derived adherent cell characteristics throughout the cell population. Instead, one or more portions (e.g., about 0.5% -2%) of the cells of the population can be tested for these properties and the results used to calculate the remaining cells in the population.

Selected cells can be identified as amnion derived adherent cells as provided herein by: in the presence of VEGF (e.g., about 50ng/mL) in a matrix such as MATRIGEL^TMThe cell sample (e.g., about 10)⁴To about 10⁵Cells) for 4-14 days, e.g., 7 days, and visually observing cell budding and/or the appearance of a cell network.

Amnion derived adherent cells can be selected by the above markers using any method known in the art of cell selection. For example, adherent cells can be screened using an antibody or antibodies against one or more cell surface markers, e.g., in flow cytometry or FACS immunolocalization. Selection can be performed by using antibodies bound to magnetic beads. Antibodies specific for a label are known in the art and are commercially available, for example, anti-CD 9(Abcam), CD54(Abcam), CD105 (Abcam; Biodesign International, Saco, ME, etc.), CD200(Abcam) cytokeratin (SigmaAldrich) antibodies. Antibodies against other markers are also commercially available, e.g., CD34, CD38, and CD45 are available, e.g., from StemCell Technologies or BioDesign International. Primers for OCT-4 sequences suitable for RT-PCR are available, for example, from Millipore or Invitrogen, or can be readily derived from the human sequence of GenBank accession No. DQ 486513.

Methods for obtaining placenta and amniotic membrane tissue and detailed methods for treating the tissue for obtaining amnion-derived adherent cells are provided below.

5.4.1 cell Collection compositions

Generally, the cells can be obtained from the amniotic membrane of a mammalian placenta, e.g., a human placenta, using a physiologically acceptable solution, e.g., a cell harvesting composition. Preferably, the cell collection composition prevents or inhibits apoptosis, prevents or inhibits cell death, lysis, decomposition, and the like. Cell collection compositions are described in detail in related U.S. patent application publication No. 2007/0190042, entitled "Improved Medium for Collecting planar Stem Cells and Preserving organisms," the disclosure of which is incorporated herein by reference in its entirety.

The cell collection composition can include any physiologically acceptable solution suitable for collecting and/or culturing amnion-derived adherent cells, for example, a salt solution (e.g., phosphate buffered saline, Kreb solution, modified Kreb solution, Eagle solution, 0.9% NaCl, etc.), a culture medium (e.g., DMEM, h.dmem, etc.), and the like, with or without the addition of a buffering component, e.g., 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES).

The cell collection composition can comprise one or more components that protect the cells (e.g., amnion derived adherent cells), i.e., prevent cell death, or delay cell death, reduce the number of cells in a dead cell population, from the time of collection to culture, etc. The component can be, for example, an apoptosis inhibitor (e.g., a caspase inhibitor or a JNK inhibitor); vasodilators (e.g., magnesium sulfate, antihypertensive agents, Atrial Natriuretic Peptide (ANP), adrenocorticotropic hormone releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, indomethacin or magnesium sulfate, phosphodiesterase inhibitors, etc.); necrosis inhibitors (e.g., 2- (1H-indol-3-yl) -3-pentylamine-maleimide, pyrrolidine dithiocarbamate or clonazepam); a TNF-alpha inhibitor; and/or an oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide, perfluorodecane bromide, etc.).

The cell collection composition can include one or more tissue degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, an rnase, or a dnase, among others. Such enzymes include, but are not limited to, collagenase (e.g., collagenase I, II, III, or IV, collagenase from Clostridium histolyticum, etc.); dispase, thermolysin, elastase, trypsin, LIBERASE^TMHyaluronidase, and the like. The use of the cell collection composition containing tissue digesting enzymes is described in detail below.

The cell collection composition can comprise a bactericidally or bacteriostatic effective amount of an antibiotic. In some non-limiting embodiments, the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime, or cefadroxil), clarithromycin, erythromycin, a penicillin (e.g., penicillin V), or a quinolone (e.g., ofloxacin, ciprofloxacin, or norfloxacin), tetracycline, streptomycin, and the like. In a particular embodiment, the antibiotic activity is against Gram (+) and/or Gram (-) bacteria, such as pseudomonas aeruginosa (pseudomonas aeruginosa), Staphylococcus aureus (Staphylococcus aureus), and the like.

The cell collection composition may further comprise one or more of the following compounds: adenosine (about 1mM to about 50 mM); d-glucose (about 20mM to about 100 mM); magnesium ions (about 1mM to about 50 mM); macromolecules having a molecular weight greater than 20,000 daltons, in one embodiment in an amount sufficient to maintain endothelial integrity and cell viability (e.g., synthetic or naturally occurring colloids, polysaccharides such as dextran or polyethylene glycol in an amount of about 25g/l to about 100g/l, or about 40g/l to about 60 g/l); antioxidants (e.g., butylhydroxyanisole, butylhydroxytoluene, glutathione, vitamin C, or vitamin E, in an amount of about 25M to about 100M); a reducing agent (e.g., N-acetylcysteine, in an amount of about 0.1mM to about 5 mM); agents that prevent calcium entry into cells (e.g., verapamil, in an amount of about 2M to about 25M); nitroglycerin (e.g., about 0.05g/L to about 0.2 g/L); an anticoagulant in an amount sufficient to help prevent clotting of blood residues (e.g., heparin or hirudin in a concentration of about 1000 units/l to about 100,000 units/l); or an amiloride-containing compound (e.g., amiloride, ethylisopropylamiloride, hexametholonidine, dimethylammonimidine or isobutylamiloride, in an amount of about 1.0M to about 5M).

Amnion derived adherent cells described herein can also be collected in a simple physiologically acceptable buffer, e.g., phosphate buffered saline, 0.9% NaCl solution, cell culture medium, etc., during and after digestion, e.g., as described below.

5.4.2 Collection and treatment of placenta

Typically, human placenta is collected within a short time after its birth, either by expulsion from the body or after, for example, caesarean section. In a preferred embodiment, the placenta is recovered after informed consent is given to the patient and a complete medical record of the patient is obtained and associated with the placenta. Preferably, the medical history is continuously tracked after delivery. The medical history can be used to adjust subsequent uses of the placenta or cells obtained therefrom. For example, human placental cells, e.g., amnion-derived adherent cells, can be used for personalized administration of infants, or close relatives related to the placenta, or parents, siblings, or other relatives of the infant, according to medical history.

Cord blood and placental blood are removed prior to recovery of amnion-derived adherent cells. In certain embodiments, cord blood is recovered from the placenta after delivery. The placenta can be used in conventional recovery process of umbilical cord blood. The placenta is typically bled by gravity using a needle or cannula (see, e.g., Anderson, U.S. patent No. 5,372,581; Hessel et al, U.S. patent No. 5,415,665). The needle or cannula is typically placed in the umbilical vein and the placenta may be gently squeezed to help evacuate the umbilical cord blood from the placenta. This Cord Blood recovery can be carried out commercially, for example, in LifeBank USA, Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. Preferably, the placenta is emptied by gravity without further manipulation, thereby minimizing tissue damage in cord blood recovery.

Typically, the placenta is transferred from the delivery room or delivery room to another location, e.g., a laboratory, for recovery of cord blood and collection of cells, e.g., by perfusion or tissue dissociation. The placenta is preferably transferred to a sterile, insulated transfer apparatus (maintaining the placenta temperature at 20-28℃.), for example, by placing the placenta with the proximally clamped cord blood in a sterile zippered plastic bag, which is then placed in an insulated container. In another embodiment, the placenta is transferred using an umbilical cord blood collection kit substantially as described in U.S. patent No. 7,147,626. Preferably, the placenta is transferred to the laboratory 4-24 hours after delivery. In certain embodiments, it is preferred to clamp the proximal umbilical cord 4-5cm from) insertion of the cord into the placenta prior to cord blood recovery. In other embodiments, the proximal umbilical cord is clamped after cord blood recovery and prior to further processing of the placenta.

The placenta can be stored under sterile conditions and at a temperature, for example, of 4-25 ℃ (celsius), e.g., room temperature, prior to cell collection. The placenta may be stored, for example, for 0-24 hours, up to 48 hours, or more than 48 hours, before the perfused placenta removes any residual cord blood. In one embodiment, the placenta is harvested from about 0 hours to about 2 hours after draining. The placenta can be stored in the anticoagulant solution at a temperature of, for example, 4-25 ℃ (celsius). Suitable anticoagulant solutions are well known in the art. For example, sodium citrate, heparin or warfarin sodium solution may be used. In a preferred embodiment, the anticoagulant solution comprises a heparin solution (e.g., 1% w/w in a 1:1000 solution). Preferably, the bled placenta is stored for no more than 36 hours prior to collection of the cells.

5.4.3 physical disruption and enzymatic digestion of amniotic Membrane tissue

In one embodiment, the amniotic membrane is isolated from the remaining placenta, e.g., by blunt dissection, e.g., using a finger. The amniotic membrane may be cut into, for example, small pieces or tissue fragments prior to enzymatic digestion and adherent cell recovery. Amnion-derived adherent cells can be obtained from an intact amniotic membrane, or from a small portion of the amniotic membrane, for example, a portion of the amniotic membrane having an area of about 1, 2, 3,4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 square millimeters.

Amnion-derived adherent cells can generally be collected from the placental amnion or portion thereof, which can occur at any time within about the first 3 days of expulsion, but preferably are from about 0 hours to 48 hours after expulsion, or from about 8 hours to about 18 hours after expulsion.

In one embodiment, the amnion-derived adherent cells are isolated from the amniotic membrane tissue by enzymatic digestion using one or more tissue digesting enzymes. The amniotic membrane or a portion thereof may be digested, for example, using one or more enzymes that are solubilized or mixed into the cell collection composition as described above.

In certain embodiments, the cell collection composition comprises one or more tissue-destructive enzymes. The enzymatic digestion preferably uses a combination of enzymes, e.g., a combination of matrix metalloprotease and neutral protease, e.g., a combination of dispase and collagenase, e.g., sequentially. When more than one protease is used, the proteases may be used simultaneously or sequentially to digest amniotic membrane tissue. In one embodiment, for example, the amniotic membrane tissue is digested 3 times with trypsin and then once with collagenase.

In one embodiment, the amniotic membrane tissue is enzymatically digested with one or more of a matrix metalloproteinase, a neutral protease, and a mucolytic enzyme. In a specific embodiment, the amniotic membrane tissue is digested with a combination of collagenase, dispase, and hyaluronidase. In another specific embodiment, the amniotic membrane tissue is treated with LIBERASE^TM(Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidaseOther enzymes that may be used to destroy amniotic membrane tissue include papain, deoxyribonuclease, serine proteases such as trypsin, chymotrypsin, or elastase serine proteases can be inhibited in serum by α 2 microglobulin, so that the media used for digestion may be serum-free in certain embodiments.

Typical concentrations of tissue digesting enzymes include, for example, 50-200U/mL collagenase I and collagenase IV, 1-10U/mL dispase, and 10-100U/mL elastase. The proteases may be used in combination, i.e., two or more proteases are used in the same digestion reaction, or may be used sequentially to isolate amnion-derived adherent cells. For example, in one embodiment, the amniotic membrane tissue or portion thereof is first digested with a suitable amount of trypsin at a concentration of about 0.25%, 37 ℃, e.g., 15 minutes, and then digested with about 1 to about 2mg/ml of collagenase I, e.g., 45 minutes.

In one embodiment, amnion derived adherent cells can be obtained as follows. The amniotic membrane is cut into pieces of 0.1 "x 0.1" to about 5 "x 5", e.g., 2 "x 2" size. The epithelial monolayer was removed from the embryonic side of the amniotic membrane by three trypsinization as follows. The amniotic membrane fragments are placed in a container containing a warm (e.g., about 20 ℃ to about 37 ℃) trypsin-EDTA solution (0.25%). The volume of trypsin can be from about 5mL per gram of amniotic membrane to about 50mL per gram of amniotic membrane. The vessel is shaken for about 5 minutes to about 30 minutes, for example, 15 minutes, and the temperature is held constant. The amniotic fragments are then separated from the trypsin solution, wherein the amniotic fragments may be removed by any suitable method, for example manually, or by filtration. The trypsin digestion step may be repeated at least more than once.

After the end of the tryptic digest, the amniotic membrane pieces were returned to a container containing a warm trypsin neutralizing solution such as Phosphate Buffered Saline (PBS)/10% FBS, PBS/5% FBS, or PBS/3% FBS. The container is shaken for about 5 seconds to about 30 minutes, for example, 5 minutes. The amniotic membrane fragments were then separated from the trypsin neutralizing solution as described above and placed in a container with warmed pH 7.2 PBS. The container is shaken for about 5 seconds to about 30 minutes to separate the amniotic membrane fragments from PBS as described above.

The amniotic membrane fragments are then placed in a container containing a warm (e.g., about 20 ℃ to about 37 ℃) digestion solution. The volume of the digestion solution can be from about 5mL per gram of amniotic membrane to about 50mL per gram of amniotic membrane. The digestion solution contains digestive enzymes in a suitable medium such as DMEM. Typical digestion solutions include collagenase type I (about 50U/mL to about 500U/mL); collagenase type I (about 50U/mL to about 500U/mL) plus dispersing enzyme (about 5U/mL to about 100U/mL); and collagenase type I (about 50U/mL to about 500U/mL), dispase (about 2U/mL to about 50U/mL), and hyaluronidase (about 3U/mL to about 10U/mL). The container is shaken at 37 ℃ until the amniotic membrane digestion is substantially complete (about 10 minutes to about 90 minutes). Warmed PBS/5% FBS was then added to the container in a ratio of about 1mL per gram of amniotic tissue to about 50mL per gram of amniotic tissue. The vessel is shaken for about 2 minutes to about 5 minutes. The cell suspension was then filtered through a 40 μm-100 μm filter to remove any undigested tissue. The cells are suspended in warm PBS (about 1mL to about 500mL) and then centrifuged at 200 Xg to about 400 Xg for about 5 minutes to about 30 minutes, e.g., 300 Xg at 20 ℃ for about 15 minutes. After centrifugation, the supernatant is removed and the cells are resuspended in a suitable medium. The cell suspension may be filtered (40 μm-70 μm filter) to remove any remaining undigested tissue, resulting in a single cell suspension.

In this embodiment, the cells in suspension will be collected and cultured as described elsewhere herein to produce isolated amnion-derived adherent cells and cell populations thereof. In this embodiment, the remaining undigested amniotic membrane may be discarded. Cells released from amniotic membrane tissue may be collected by, for example, centrifugation and cultured in standard cell culture media.

In any of the digestion procedures herein, the cell suspension obtained from the digestion may be filtered, for example, through a filter containing pores from about 50 μm to about 150 μm, e.g., from about 75 μm to about 125 μm. In a more specific embodiment, the cell suspension may be filtered through two or more filters, for example, a 125 μm filter and a 75 μm filter.

Along with any of the methods described herein, AMDACs can be isolated from released cells during digestion by selecting cells capable of expressing one or more characteristics of AMDACs, as described above in section 5.1.

AMDACs can also be isolated using, for example, a particular two-step separation method, which includes trypsin digestion followed by collagenase digestion. Thus, in another aspect, the invention provides a method of isolating amnion-derived adherent cells, comprising trypsinizing the amnion, or a portion thereof, to release epithelial cells from the amnion; removing the amniotic membrane or a portion thereof from the epithelial cells; further digesting the amniotic membrane or a portion thereof with collagenase to release amnion-derived adherent cells from the amniotic membrane or portion thereof; and isolating the amnion-derived adherent cells from the amnion. In a specific embodiment, the digestion of the amniotic membrane or portion thereof is repeated at least once. In another specific embodiment, the collagenase digestion of the amniotic membrane or a portion thereof is repeated at least once. In another specific embodiment, the trypsin is about 0.1% to 1.0% (final concentration). In a more specific embodiment, the trypsin is about 0.25% (final concentration). In another specific embodiment, the collagenase is about 50U/mL to about 1000U/mL (final concentration). In a more specific embodiment, the collagenase is about 125U/mL (final concentration). In another specific embodiment, the method of isolating further comprises culturing the amnion derived adherent cells in a cell culture medium and isolating the non-adherent cells in the culture from the amnion derived adherent cells to produce a population enriched for amnion derived adherent cells. In more specific embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the cells in the enriched population of amnion derived adherent cells are the amnion derived adherent cells.

In a more specific embodiment of the above method, the amnion derived adherent cells are OCT-4 negative as determined by RT-PCR and HLA-G negative as determined by flow cytometry⁺、CD90⁺、CD105⁺And CD117^–One or more of (a).

5.4.4 isolation, Classification and characterization of amnion derived adherent cells

The cell pellet may be resuspended in a fresh cell collection composition as described above, or in a medium suitable for maintaining the cells, for example, Dulbecco's Modified Eagle Medium (DMEM); iscove's Modified Dulbecco's Medium (IMDM), such as IMDM serum-free medium containing 2U/mL heparin and 2mM EDTA (GibcoBRL, NY); buffer (e.g., PBS, HBSS) mixed with FBS (e.g., 2% v/v); and the like.

Amnion derived adherent cells cultured on, for example, a tissue culture plastic surface, whether or not they have additional extracellular matrix coating, such as fibronectin, can be passaged or isolated by differential adherence. For example, a cell suspension of amniotic membrane tissue digested with collagenase as described in section 5.4.3 above can be obtained and cultured in medium on tissue culture plastic, for example, for 3-7 days. During the culture process, a plurality of cells in the suspension are attached to the culture surface, and after continuous culture, amnion derived adherent cells are formed. Non-adherent cells that do not form amnion derived adherent cells are removed when the medium is changed.

Monitoring of the number and type of cells collected from the amniotic membrane may be performed, for example, by detecting changes in cell morphology and surface markers using standard cell detection techniques such as immunolocalization, e.g., flow cytometry, cell sorting, immunocytochemistry (e.g., tissue-specific or cell marker-specific antibodies)Staining) Fluorescence Activated Cell Sorting (FACS), Magnetic Activated Cell Sorting (MACS), examining cell morphology by using optical or confocal microscopy, and/or detecting changes in gene expression by using techniques well known in the art, such as PCR and gene expression profiling. These techniques can also be used to identify cells that are positive for one or more specific markers. For example, using one or more CD34 antibodies, one can use the techniques described above to determine whether a cell contains a detectable amount of CD 34; if so, then the cell is CD34⁺。

Amnion-derived cells, e.g., cells isolated by Ficoll separation, differential wallcovering, or a combination of both, can be sorted using a Fluorescence Activated Cell Sorter (FACS). Fluorescence Activated Cell Sorting (FACS) is a well-known method for separating particles, involving the separation of cells based on the fluorescent properties of the particles (see, e.g., Kamarch, 1987, methods enzymol, 151: 150-. Laser excitation of the fluorescent portion of each particle results in a small charge that allows electromagnetic separation of positive and negative particles from the mixture. In one embodiment, the cell surface marker specific antibody or ligand is labeled with a different fluorescent label. Cells are processed through a cell sorter, allowing for separation of cells based on their ability to bind the antibody used. FACS sorted particles can be stored directly into individual wells of 96-well or 384-well plates for ease of isolation and cloning.

In one sorting protocol, cells from the placenta, e.g., amnion derived adherent cells, may be sorted based on the expression of the markers CD49f, VEGFR2/KDR, and/or Flt-1/VEGFR 1. Preferred cells are identified as OCT-4^–For example, the expression of OCT-4 in a cell sample is detected by RT-PCR for identification, wherein the cell is OCT-4 if the cell in the sample fails to exhibit detectable mRNA synthesis of OCT-4 after 30 cycles^–. For example, VEGFR2/KDR from amniotic membrane⁺And VEGFR1/Flt-1⁺The cells may be derived from cells having VEGFR2/KDR^–And VEGFR1/Flt-1⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺And/or VE-cadherin^–Sorting is performed in the cells of one or more of (a). In a specific embodiment, the amniotic membrane is derived from CD49f⁺、VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺And/or VE-cadherin^–Tissue culture of plastic-adherent cells, or VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺And VE-cadherin^–Cells, sorted and selected from cells that do not express one or more of these markers. In another specific embodiment, will additionally be CD31⁺、CD34⁺、CD45⁺、CD133^–And/or Tie-2⁺CD49f (see above)⁺、VEGFR2/KDR⁺、VEGFR1/Flt-1⁺The cells of one or more or all of (a) are sorted from cells that have not exhibited one or more or any of these properties. In another specific embodiment, will additionally be CD9⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺、TEM-7⁺、CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And/or CXCR4^–One or more or all of VEGFR2/KDR of⁺、VEGFR1/Flt-1⁺Cells, from which sorting has never been performed, exhibit one or more or any of these properties.

The selection of amnion derived adherent cells can be performed on a cell suspension resulting from digestion or on isolated cells collected from the digest, for example, by centrifugation or using a flow cytometer. Selection by expression markers can be done alone or, for example, in conjunction with a cell selection procedure based on the adherent properties in cell culture. For example, adherent screening can be performed before or after sorting based on marker expression.

For antibody-mediated placental cell detection and sorting, any antibody specific for a particular marker can be used, which is specific for the markerIn combination with any fluorophore or other tag suitable for cell detection and sorting (e.g., fluorescence activated cell sorting). Antibodies/fluorophores conjugated to specific labels include, but are not limited to, Fluorescein Isothiocyanate (FITC) -conjugated anti-CD 105 monoclonal antibodies (available from R)&D SYSTEMSICN, Minneapolis, Minnesota), Phycoerythrin (PE) conjugated anti-CD 200 monoclonal antibodies (BD Biosciences Pharmingen), VEGFR 2/KDR-biotin (CD309, Abcam), and the like, antibodies to any of the labels disclosed herein can be labeled with any standard label for antibodies that facilitates detection of the antibodies, including, for example, horseradish peroxidase, alkaline phosphatase, β -galactosidase, acetylcholinesterase streptavidin/biotin, avidin/biotin, umbelliferone, Fluorescein Isothiocyanate (FITC), rhodamine, dichlorotriazamine fluorescein, dansyl chloride, or Phycoerythrin (PE), luminol, luciferase, fluorescein, and aequorin, and examples of suitable radioactive materials, including examples of suitable radioactive materials¹²⁵I、¹³¹I、³⁵S or³H。

Amnion derived adherent cells can be labeled with a single labeled antibody and detected and sorted based on the single label; or multiple antibodies labeled with multiple different labels simultaneously and sorted based on the multiple labels.

In another embodiment, magnetic beads can be used to isolate cells, e.g., amnion derived adherent cells described herein are isolated from other amnion cells. Cells can be sorted using Magnetic Activated Cell Sorting (MACS) technology, a method for separating particles based on their ability to bind magnetic beads (0.5-100 μm diameter). A variety of useful modifications can be made to the magnetic microspheres, including the covalent addition of antibodies that specifically recognize particular cell surface molecules or haptens. The magnetic beads are then mixed with the cells for binding. The cells are then passed through a magnetic field to isolate cells having specific cell surface markers. In one embodiment, these cells may then be separated and the magnetic beads coupled with antibodies against other cell surface markers may be remixed. The cells were again passed through a magnetic field to separate the cells bound to the two antibodies. The cells may then be diluted in a different tray, such as a microtiter tray, for clonal isolation.

The viability, proliferation potential and longevity of amnion derived adherent cells can be assessed using standard techniques known in the art, such as trypan blue exclusion experiments, fluorescein diacetate uptake assays, propidium iodide uptake assays (assessing viability); and thymine uptake assays or MTT cell proliferation assays (to assess proliferation). Cell life can be determined by methods well known in the art, for example by measuring the maximum population doubling in extension culture.

Amnion derived adherent cells can also be isolated from placental cells using other techniques known in the art, e.g., selective growth of desired cells (positive selection), selective destruction of undesired cells (negative selection); isolation based on agglutination of differentiated cells in a mixed population of, for example, soybean lectins; freezing and thawing; filtering; conventional and zonal centrifugation; centrifugal elutriation (countercurrent centrifugation); separating by unit gravity; counter-current distribution; electrophoresis; and so on.

5.5 culture of amnion-derived adherent cells

5.5.1 culture Medium

Isolated amnion-derived adherent cells, or populations of such cells, can be used to initiate cell culture or to inoculate cell cultures. The cells are typically transferred to a sterile tissue culture vessel that is either not coated or coated with an extracellular matrix or biomolecules, such as laminin, collagen (e.g., native or denatured), gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane proteins (e.g., MATRIGEL)^TM(BD Discovery Labware，Bedford，Mass.))。

AMDACs can be, for example, cultured in a medium suitable for stem cell culture. The culture medium may, for example, contain EGM-2 medium (Lonza), DMEM + 10% FBS, or 60% DMEM-LG (Gibco), 40% MCDB-201(Sigma),2% Fetal Calf Serum (FCS) (Hyclone Laboratories), 1 × insulin-transferrin-selenium supplement (ITS), 1 × linoleic acid-bovine serum albumin (LA-BSA), 10^-9M dexamethasone (Sigma), 10^-4M ascorbic acid 2-phosphate (Sigma), Epidermal Growth Factor (EGF)10ng/ml (R)&D SYSTEMS), platelet derived growth factor (PDGF-BB)10ng/ml (R)&D SYSTEMS), and 100U penicillin/1000U streptomycin medium (referred to herein as "standard medium").

Amnion derived adherent cells can be cultured in any medium recognized in the art as useful for cell culture of, for example, adherent placental stem cells, and under any conditions. Preferably, the medium contains serum. In various embodiments, the culture medium for the culture or subculture of AMDACs comprises(Invitrogen)、MSCM-sf(ScienCell、Carlsbad、CA)、ACF medium (StemCell Technologies, Vancouver, Canada), standard medium lacking EGF, standard medium lacking PDGF, DMEM + 10% FBS, EGM-2(Lonza), EGM-2MV (Lonza), 2%, 10% and 20% ES medium, ES-SSR medium or α -MEM-20% FBS^TMCulture medium (Hyclone), KNOCKOUT^TMDMEM (Invitrogen), L-15 medium of Leibovitz, MCDB, DMEM/F12, RPMI 1640, modified DMEM (Gibco), DMEM/MCDB201(Sigma), CELL-GRO FREE, and the like. In various embodiments, for example, DMEM-LG (Dulbecco's modified essential Medium, Low glucose)/MCDB 201 (Chicken fibroblast minimal Medium) contains ITS (insulin-transferrin-selenium supplement), LA + BSA (linoleic acid-bovine serum Albumin), glucose, L-ascorbic acidPDGF, EGF, IGF-1, and penicillin/streptomycin, DMEM-HG (high glucose) contains about 2 to about 20% fetal bovine serum, e.g., about 10% fetal bovine serum (FBS; e.g., defined fetal bovine serum, Hyclone, Logan Utah), DMEM-HG contains about 2 to about 20% FBS, e.g., about 15% FBS, IMDM (Iscove's modified Dulbecco's medium) contains about 2 to about 20% FBS, e.g., about 10% FBS, about 2 to about 20% horse serum, e.g., about 10% horse serum, and hydrocortisone, M199 contains about 2 to about 20%, e.g., about 10% FBS, EGF and heparin, α -MEM (minimal essential medium) contains about 2 to about 20%, e.g., about 10% FBS, GLUTAMAX^TMAnd gentamicin; DMEM containing 10% FBS, GLUTAMAX^TMAnd gentamicin, DMEM-LG containing from about 2 to about 20%, e.g., about 15% (v/v) fetal bovine serum (e.g., defined fetal bovine serum, Hyclone, Logan Utah), antibiotics/antifungals (e.g., penicillin about 100 units/microliter, streptomycin 100 micrograms/microliter, and/or amphotericin B0.25 micrograms/microliter (Invitrogen, Carlsbad, Calif.)), and 0.001% (v/v) β -mercaptoethanol (Sigma, St.Louis Mo.); KNOCKOUT supplemented with 2-20% FBS, non-essential amino acids (Invitrogen), β -mercaptoethanol^TMDMEM minimal medium supplemented with KNOCKOUT^TMThe serum substitute of (1), KNOCKOUT containing α -MEM containing 2-20% FBS^TMEBM2 supplemented with EGF, VEGF, bFGF, R3-IGF-1, hydrocortisone, heparin, ascorbic acid, FBS, gentamicin in minimal medium^TMMinimal media, and the like.

The medium can be supplemented with one or more components, including, for example, serum (e.g., FCS or FBS, e.g., about 2-20% (v/v); horse serum (ES); Human Serum (HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one or more growth factors, e.g., Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), and Erythropoietin (EPO); amino acids, including L-valine; and one or more antibiotics and/or antifungal agents to control microbial contamination, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, which may be used alone or in combination.

Amnion derived adherent cells (AMDAC) can be cultured under standard tissue culture conditions, e.g., in tissue culture dishes or multiwell plates⁴Cells per mL, and one or more drops of culture medium are placed in the lid of a tissue culture vessel, e.g., in a 100mL petri dish. These droplets may be, for example, a single droplet, or multiple droplets from, for example, a multichannel pipette. The lid is carefully inverted and placed on top of the bottom of the dish, containing a volume of liquid, e.g., sterile PBS, sufficient to maintain a moist environment in the dish, and the cells are then cultured. AMDAC can also be cultured in standard or large volume or high yield culture systems, such as T-shake flasks, CorningCell factors (Nunc), 1-, 2-, 4-, 10-or 40-track Cell stack, and the like.

In one embodiment, the amnion derived adherent cells are cultured in the presence of a compound capable of maintaining the undifferentiated phenotype of the cells. In a specific embodiment, the compound is a substituted 3, 4-dihydropyridinol [4,5-d ] pyrimidine. In a more specific embodiment, the compound is a compound having the following chemical structure:

the compound can be contacted with an amnion-derived adherent cell or population of cells at a concentration of, for example, about 1 μ M to about 10 μ M.

5.5.2 amplification and proliferation of amnion-derived adherent cells

Once the amnion derived adherent cells or the population of cells are isolated (e.g., amnion derived adherent cells or the population of cells isolated from at least 50% of the amnion cells, with which the cell or population of cells is typically associated in vivo), the cells can be propagated and expanded in vitro. For example, adherent cells or amnion-derived adherent cell populations can be cultured in a tissue culture vessel, e.g., a petri dish, a shake flask, a multi-well plate, etc., for a time sufficient for the cells to proliferate to a confluency of 40-70% (confluency), i.e., until the cells and their progeny occupy 40-70% of the culture surface area of the tissue culture vessel.

Amnion derived adherent cells can be seeded in a culture vessel at a density that allows for cell growth. For example, it may be at low density (e.g., about 400 to about 6,000 cells/cm)²) To high densities (e.g., about 20,000 or more cells/cm)²) The cells were inoculated. In a preferred embodiment, the cells are cultured in CO₂From about 0% to about 5% air. In some preferred embodiments, the cells are cultured in about 0.1% to about 25% O₂Preferably from about 5% to about 20% O₂In the air of (2). The cells are preferably cultured at about 25 ℃ to about 40 ℃, preferably about 37 ℃.

The cells are preferably cultured in an incubator. The culture medium may be static or agitated during the culturing process, for example, by using a bioreactor during the culturing process. Amnion derived adherent cells are preferably grown under low oxidative pressure (e.g., with the addition of glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, etc.).

Although the amnion-derived angiogenic cells may grow to full, the cells preferably do not grow to full. For example, once a confluency of 40% to 70% is achieved, the cells can be passaged. For example, cells may be enzymatically treated using techniques well known in the art, e.g., trypsinization, and isolated from tissue culture surfaces. After the cells are aspirated and counted, about 20,000-100,000 cells, preferably about 50,000 cells, or about 400 to about 6,000 cells/cm can be used²Passage to freshA new culture container of culture medium. Typically, the new medium is of the same type as the medium from which the cells are removed. Amnion derived adherent cells may be passaged at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, or more. AMDACs may be doubled in culture by at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or at least 50 fold or more.

5.6 preservation of amnion-derived adherent cells

Amnion-derived adherent cells can be stored, i.e., placed under conditions that allow long-term storage, or that inhibit cell death, such as apoptosis or necrosis, prior to, for example, a harvesting process or use of a method such as described herein to produce a composition described herein.

Amnion derived adherent cells can be preserved using, for example, compositions comprising an apoptosis inhibitor, a necrosis inhibitor, and/or an oxygen-carrying perfluorocarbon, as described in U.S. application publication No. 2007/0190042, the disclosure of which is incorporated herein by reference in its entirety. In one embodiment, the method of preserving the cell or population of cells comprises contacting the cell or population of cells with a cell collection composition comprising an apoptosis inhibitor and an oxygen-carrying perfluorocarbon, wherein the apoptosis inhibitor is used in an amount and for a time sufficient to reduce or prevent apoptosis in the population of cells compared to a population of cells not contacted with the apoptosis inhibitor. In a specific embodiment, the apoptosis inhibitor is a caspase inhibitor. In another specific embodiment, the apoptosis inhibitor is a JNK inhibitor. In a more specific embodiment, the JNK inhibitor does not modulate differentiation or proliferation of amnion derived adherent cells. In another embodiment, the cell collection composition comprises the apoptosis inhibitor and the oxygen-carrying perfluorocarbon is in a different phase. In another embodiment, the cell collection composition contains the apoptosis inhibitor and the oxygen-carrying perfluorocarbon in an emulsion. In another embodiment, the cell collection composition further comprises an emulsifier, e.g., lecithin. In another embodiment, the apoptosis inhibitor and the perfluorocarbon are from about 0 ℃ to about 25 ℃ when contacted with a cell. In another more specific embodiment, the apoptosis inhibitor and the perfluorocarbon are about 2 ℃ to 10 ℃, or about 2 ℃ to about 5 ℃ at the time of contacting the cell. In another more specific embodiment, said contacting is performed during delivery of said population of cells. In another more specific embodiment, said contacting is performed during freeze-thawing of said cell population.

Amnion-derived adherent cell populations can be preserved, for example, by a method comprising contacting the cell population with an apoptosis inhibitor and an organ preservation compound, wherein the apoptosis inhibitor is used in an amount and for a time sufficient to reduce or prevent apoptosis in the cell population as compared to a cell population not contacted with the apoptosis inhibitor. In a specific embodiment, the organ preservation compound is a UW solution (described in U.S. Pat. No. 4,798,824; also known as ViaPasan; see also Southard et al, Transplantation 49(2):251-257(1990)) or a solution described in Stern et al, U.S. Pat. No. 5,552,267. In another embodiment, the organ preservation compound is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof. In another embodiment, the cell collection composition further comprises an oxygen-carrying perfluorocarbon, either in two phases or in an emulsion.

In another embodiment of the method, the amnion derived adherent cells are contacted with a cell collection composition comprising an apoptosis inhibitor and an oxygen-carrying perfluorocarbon, an organ preservation compound, or a combination thereof during perfusion. In another embodiment, the amnion derived adherent cells are contacted with the cell collection composition during tissue disruption, e.g., during enzymatic digestion of the amnion tissue. In another embodiment, the amnion derived adherent cells are contacted with the cell collection composition after collection by, for example, enzymatic digestion of tissue disruption of the amnion tissue.

Generally, it is preferred to minimize or eliminate cell stress due to hypoxia and mechanical stress during collection, enrichment and isolation of amnion-derived adherent cells. Thus, in another embodiment of the method, the amnion derived adherent cells, or a population of cells comprising amnion derived adherent cells, are exposed to hypoxic conditions, e.g., a lower than normal atmospheric oxygen concentration, for less than 6 hours during said storing, at the time of collection, enrichment and isolation; lower than normal blood oxygen concentration, etc. In a more specific embodiment, said cell or said population of cells is exposed to said hypoxic conditions for less than 2 hours during said preservation. In another more specific embodiment, said cell or said cell population is exposed to said hypoxic conditions for less than 1 hour, or less than 30 minutes, or is not exposed to hypoxic conditions during collection, enrichment or isolation. In another specific embodiment, the cell population is not exposed to shear forces during collection, enrichment, or isolation.

Suitable cryopreservation media include, but are not limited to, media including, for example, growth media, or cell freezing media, e.g., commercially available cell freezing media, e.g., cell freezing media according to SigmaAldrich catalog number C2695, C2639(1 × cell freezing serum-free media, without DMSO), or C6039(1 × cell freezing glycerol media, with minimal essential media, glycerol, calf serum, and bovine serum), Lonza PROFREEZE^TM2 × Medium, methylcellulose, dextran, human serum albumin, fetal bovine serum, or Plasmalyte cryopreservation Medium preferably comprises DMSO (dimethyl sulfoxide) or glycerol at a concentration of, for example, about 1% to about 20%, for example, about 5% -10% (v/v), optionally fetal bovine serum or human serumAdherent cells are preferably frozen at a rate of about 1 ℃/min during cryopreservation. Preferred cryopreservation temperatures are from about-80 ℃ to about-180 ℃, preferably from about-125 ℃ to about-140 ℃. Cryopreserved cells can be transferred to the gas phase of liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoule reaches about-80 ℃, it is transferred into a liquid nitrogen storage area. Cryopreservation may also use a controlled rate refrigerator. Cryopreserved cells are preferably thawed at a temperature of about 25 ℃ to about 40 ℃, preferably to a temperature of about 37 ℃.

5.7 preparation of amnion-derived adherent cell bank

Amnion derived adherent cells can be cultured using a variety of different means to produce a series of batches (lot), e.g., a series of individually administrable doses (dose) of the cells. A series of batches of angiogenic amniotic cells obtained from a plurality of placentas may be placed in a single cell bank for, e.g., long term storage. Generally, amnion derived adherent cells are derived from a primary culture of cells to form a seed culture, which is expanded under controlled conditions to form a population of cells from approximately a significant number of doublings. The cell batch is preferably derived from tissue of a single placenta, but may also be derived from tissue of multiple placentas.

In one non-limiting embodiment, batches or doses of amnion derived adherent cells are obtained as follows. Amniotic membrane tissue is first disrupted, for example, by digestion using a series of trypsin and collagenase digestion procedures as described in section 5.4.3 above. Cells from the collagenase digested tissue are cultured for a period of, for example, about 1-3 weeks, preferably about 2 weeks. After removal of non-adherent cells, the high density colonies formed, for example, by trypsinization, are collected. These cells were collected and resuspended in an appropriate volume of medium and defined as passage 0 cells.

The 0 generation cells can then be used to inoculate the expanded culture. The expansion culture may be an isolated cell culture device arranged arbitrarily, e.g. NUNC^TMCell Factory of (1). 0 generation of cultured cells can be subdivided intoTo what extent the expanded culture is inoculated, e.g. 1 × 10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴Or 10 × 10⁴Preferably, about 1 × 10 is used³To about 3 × 10⁴0 passage cells of (a) were inoculated into each of the expanded cultures. The number of expanded cultures may depend on the number of 0 generation cells, and may be more or less depending on the particular placenta from which the adherent cells are derived.

The expanded culture may then be grown until the cell density in the culture reaches a particular value, for example, about 1 × 10⁵Cells/cm². At this point the cells can be harvested and cryopreserved, or passaged to a new expanded culture as described above. Cells may be passaged, for example, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times prior to use. Preferably, the cumulative number of population doublings is continuously recorded in expanded culture. The 0 generation cultured cells can expand 2, 3,4, 5,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 fold, or up to 60 fold. Preferably, however, the population doubling number is about 15 to about 30 fold before dividing the cell population into individual doses. The cells may be cultured continuously during the expansion process, or may be frozen at one or more time points during the expansion process.

Cells for individual doses may be frozen, e.g., cryopreserved for later use. An individual dose may include, for example, about 1 million to about 5 million cells per mL, and may include about 10⁶To about 10¹⁰Total amount of cells.

In one embodiment, a cell bank containing amnion derived adherent cells can be prepared by a method comprising: from placenta of human postpartumAugmenting adherent cells from cultured amnion for a first plurality of population doublings; cryopreserving the cells to form a primary cell bank; optionally expanding a plurality of cells from the primary cell bank for a second plurality of population doublings; cryopreserving the expanded cells to form a working cell bank; optionally expanding a plurality of expanded amnion derived adherent cells from the working cell bank for a third plurality of population doublings; and cryopreserving the resulting expanded cells in individual doses, wherein the individual doses together comprise a cell bank. The cell bank may contain a dose or batch of amnion derived adherent cells alone, or may contain a combined batch of amnion derived adherent cells and a batch or dose of another cell, e.g., another stem cell or progenitor cell. Preferably, each individual dose contains only amnion derived adherent cells. In another specific embodiment, all of said cells in said primary culture are from the same placenta. In another specific embodiment, the individual dose contains about 10⁴To about 10⁵A cell. In another specific embodiment, the individual dose contains about 10⁵To about 10⁶A cell. In another specific embodiment, the individual dose contains about 10⁶To about 10⁷A cell. In another specific embodiment, the individual dose contains about 10⁷To about 10⁸A cell. In another specific embodiment, the individual dose contains about 10⁸To about 10⁹A cell. In another specific embodiment, the individual dose contains about 10⁹To about 10¹⁰A cell.

In certain embodiments, amnion derived adherent cells can be thawed from a working cell bank and cultured for a plurality of population doublings. When the desired number of cells is produced, or when the desired number of population doublings occur, adherent cells can be collected, for example, by centrifugation, and resuspended in a solution containing, for example, dextran, e.g., 5% dextran. In certain embodiments, the glucan is glucan-40. In certain embodiments, the cells are harvested and resuspended in a solution containing dextran and a cryopreservative, e.g., a 5% dextran (e.g., dextran-40) solution containing 10% HAS and 2% -20% DMSO, e.g., 5% DMSO, and cryopreserved. Cryopreserved amnion-derived adherent cells can be thawed, e.g., immediately prior to use.

In a preferred embodiment, at least one pathogen is detected on a donor (e.g., mother) of the placenta. In certain embodiments, if the mother tests positive for a pathogen, the entire batch from the placenta is discarded. The assay can be performed at any time during batch production of amnion derived adherent cells, including before and after 0 passage cell culture, or during expansion culture. Pathogens to be detected may include, but are not limited to, hepatitis a, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human immunodeficiency virus (types I and II), cytomegalovirus, herpes virus, and the like.

5.8 uses of amnion derived adherent cells

The invention provides compositions comprising amnion derived adherent cells. Examples of such compositions include pharmaceutical compositions (see section 5.8.1, below); stroma and scaffold (see section 5.8.2 below), and amnion-derived adherent cell conditioned medium (see section 5.8.3 below).

5.8.1 composition comprising amnion derived adherent cells

In certain embodiments, the amnion-derived adherent cells are comprised by or are a component of a pharmaceutical composition. The cells, e.g., amnion derived adherent cells, can be prepared in a manner that facilitates administration to an individual and placed in a container suitable for medical use. The container can be, for example, a syringe, sterile plastic bag, flask, jar, or other container in which the population of amnion-derived angiogenic cells can be readily dispensed. For example, the container may be a blood bag or other plastic, medically acceptable bag suitable for intravenous administration of a liquid to a subject. In certain embodiments, the container allows cryopreservation of the cells. The cells in the compositions, e.g., pharmaceutical compositions, provided herein can include amnion-derived adherent cells derived from a single donor or multiple donors. The cells may be completely HLA-matched, or partially or completely HLA-mismatched to the target recipient.

In a more particular embodiment, the bag is adapted to permit or facilitate intravenous administration of the adherent cells, e.g., by intravenous infusion, bolus injection, etc., the bag may include a plurality of chambers or compartments in communication with one another to permit mixing of the cells with one or more other solutions, e.g., a drug, prior to or during administration, prior to or during cryopreservation, the solution containing the adherent cells from the amniotic membrane comprises one or more compounds that facilitate cryopreservation of the cells⁶The cell, 5 × 10⁶The cell, 1 × 10⁷The stem cell, 5 × 10⁷The cell, 1 × 10⁸The cell, 5 × 10⁸The cell, 1 × 10⁹The cell, 5 × 10⁹The cell, or 1 × 10¹⁰The cell is described. In other embodiments with respect to any of the foregoing cryopreservation populationsIn one embodiment, the cells have been passaged about, at least, or no more than 5 times, no more than 10 times, no more than 15 times, or no more than 20 times in another specific embodiment for any of the foregoing cryopreserved cells, the cells have been expanded in the container⁵、5×10⁵、1×10⁶、5×10⁶、1×10⁷、5×10⁷、1×10⁸、5×10⁸、1×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹Or more amnion derived adherent cells.

In certain embodiments, the pharmaceutical compositions provided herein comprise an amnion-derived adherent cell population that includes 50% viable cells or more (i.e., at least 50% of the cells in the population are functional or viable). Preferably, at least 60% of the cells in the population are viable cells. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the population of pharmaceutical compositions are viable cells.

5.8.2 matrices containing amnion derived adherent cells

Further provided herein are compositions comprising a matrix, a hydrogel, a scaffold, and the like. The composition may be used to replace or assist the cells in a liquid suspension.

The matrix may be, for example, permanent or degradable decellularized tissue, e.g., decellularized amniotic membrane, or a synthetic matrix. The matrix may be a three-dimensional scaffold. In a more specific embodiment, the matrix comprises collagen, gelatin, laminin, fibronectin, pectin, ornithine, or vitronectin. In another more specific embodiment, the substrate is an amniotic membrane or an amniotic membrane derived biomaterial. In another more specific embodiment, the matrix comprises an extracellular membrane protein. In another more specific embodiment, the matrix comprises a synthetic compound. In another more specific embodiment, the matrix comprises a biologically active compound. In another more specific embodiment, the biologically active compound is a growth factor, cytokine, antibody, or organic molecule of less than 5,000 daltons.

Amnion-derived adherent cells described herein can be seeded onto a natural substrate, for example, placental biological material such as amniotic membrane material. The amniotic membrane material may be, for example, an amniotic membrane obtained by directly cutting a mammalian placenta; the fixed or heat-treated amniotic membrane, substantially dry (i.e.,<20％H₂o) an amniotic membrane, a chorion, a substantially dry amniotic membrane, a chorion, and the like. Preferred placental biomaterials that can be seeded with the amnion-derived adherent cells provided herein are described in Hariri, U.S. application publication No. 2004/0048796, the disclosure of which is incorporated herein by reference in its entirety.

In another specific embodiment, the matrix is a composition comprising an extracellular matrix. In a more specific embodiment, the composition is MATRIGEL^TM(BD Biosciences)。

The isolated amnion-derived adherent cells described herein can be suspended, for example, in a hydrogel solution suitable for injection. Hydrogels are, for example, organic polymers (natural or synthetic) that are cross-linked by covalent, ionic, or hydrogen bonds to form a three-dimensional open lattice structure, surrounding water molecules to form a gel. Hydrogels suitable for the composition include self-assembling peptides, such as RAD 16. In one embodiment, the cell-containing hydrogel solution may be allowed to harden, for example, in a mold to form a matrix with dispersed cells for transplantation. The amnion-derived adherent cells in the matrix can also be cultured, for example, prior to transplantation, so that the cells undergo mitotic expansion. Hydrogel-forming materials include polysaccharides such as alginic acid and salts thereof, polypeptides, polymers containing phosphorus and nitrogen chains, and polyacrylates, which are ionically crosslinked; or block copolymers, such as polyethylene oxide-polypropylene alcohol block copolymers, which are crosslinked by temperature or pH, respectively. In some embodiments, the hydrogel or matrix is biodegradable.

In certain embodiments, the cell-containing compositions provided herein comprise an in situ polymerizable gel (see, e.g., U.S. patent application publication No. 2002/0022676; Anseth et al, J.control Release,78(1-3):199-209 (2002); Wang et al, Biomaterials, 24(22):3969-80 (2003); in some embodiments, the polymer is at least partially soluble in an aqueous solution such as water, buffered saline solution, or aqueous alcoholic solution having charged side chain groups, or monovalent ionic salts thereof, examples of polymers having acidic side chain groups that are reactive with cations are poly (phosphazenes), poly (acrylic acid), poly (methacrylic acid), copolymers of acrylic and methacrylic acids, poly (vinyl acetate), and sulfonated polymers such as sulfonated polystyrene. copolymers having acidic side chain groups can also be used, formed by the reaction of acrylic or methacrylic acid with a vinyl ether monomer or polymer. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

In a particular embodiment, the substrate is a felt, which can be made of multifilament yarns made of bioabsorbable materials such as PGA, PLA, PCL copolymers or blends or hyaluronic acid. The yarns are felted using standard textile processing techniques including crimping, cutting, carding and knitting. In another preferred embodiment, the cells of the invention are seeded onto a foam scaffold which may be a composite structure. In addition, the three-dimensional frame may be molded into a useful shape, such as a particular bodily structure that requires repair, replacement, or expansion. Other examples of scaffolds that may be used include non-woven mats, porous foams, or self-assembling polypeptides. Non-woven mats may be made using fibers of absorbable copolymers containing synthetic glycolic and lactic acid copolymers (e.g., PGA/PLA) (VICRYL, Ethicon, inc., Somerville, n.j.). Foams composed of poly (-caprolactone)/poly (glycolic acid) (PCL/PGA) copolymers prepared by processes such as freeze-drying or lyophilization (see, e.g., U.S. patent No. 6,355,699) can also be used as scaffolds.

Amnion derived adherent cells described herein can be seeded onto a three-dimensional framework or scaffold and implanted into the body. The frame may be implanted with any one or more growth factors, cells, drugs or other components that, for example, stimulate tissue formation, such as bone formation or vascularization.

In another embodiment, the placental amnion-derived adherent cells provided herein can be seeded on a foam scaffold, which can be a composite structure. The foam scaffold may be molded into a useful shape, such as a portion of a particular bodily structure that requires repair, replacement, or expansion. In some embodiments, the frame is treated with, for example, 0.1M acetic acid and then incubated in polylysine, PBS, and/or collagen prior to cell seeding to enhance cell adsorption. The outer surface of the matrix may be modified to enhance cell adsorption or growth and tissue differentiation, for example by coating the matrix with plasma, or by adding one or more proteins (e.g., collagen, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.), cell matrix and/or other materials such as, but not limited to, gelatin, alginic acid, agar, agarose, and vegetable gums, among others.

In some embodiments, the matrix comprises, or is treated with, a material that renders it non-embolic. These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of such materials and treatments include, but are not limited to, natural materials such as basement membrane proteins, e.g., laminin and type IV collagen; synthetic materials such as EPTFE; and split polyurethaneurea silicones, e.g. PURSPAN^TM(The Polymer Technology Group, Inc., Berkeley, Calif.). The base may also include antithrombotic agents such as heparin; the scaffold can also be treated to change surface charge (e.g., plasma coating) prior to seeding with adherent cells provided herein.

The framework can be treated to enhance cell attachment prior to seeding with amnion derived adherent cells provided herein. For example, prior to seeding the cells of the invention, a nylon matrix may be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon. Polystyrene can be similarly treated with sulfuric acid.

Additionally, the outer surface of the three-dimensional framework may be modified to enhance cell adsorption or growth and tissue differentiation, for example, by coating the framework with plasma, or by adding one or more proteins (e.g., collagen, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate), cell matrix, and/or other materials, such as, but not limited to, gelatin, alginic acid, agar, agarose, or vegetable gums.

In some embodiments, the matrix comprises, or is treated with, a material that renders it non-embolic, such as natural materials, e.g., basement membrane proteins, e.g., laminin and type IV collagen; and synthetic materials such as EPTFE or cut polyurethaneurea silicones such as PURSPAN (The Polymer Technology Group, inc., Berkeley, Calif.). The material may be further treated to render the scaffold non-embolic, for example, by treatment with heparin, and by treatment to alter the surface charge of the material, for example, plasma coating.

The therapeutic cell composition comprising amnion derived adherent cells can also be provided in the form of a matrix-cell complex. The matrix may comprise a biocompatible scaffold, lattice, self-assembled structure, or the like, whether bioabsorbable or not, or a liquid, gel, or solid. Such matrices are known in the art for use in therapeutic cell processing, surgical recovery, tissue engineering and wound healing. In certain embodiments, the cells are attached to a substrate. In other embodiments, the cells are embedded or contained within a stromal space. Most preferred are matrix-cell complexes in which the cells grow in close association with the matrix and, when used therapeutically, stimulate and support the ingrowth of recipient cells, or stimulate or support angiogenesis. The matrix-cell composition may be introduced into the subject by any method known in the art, including but not limited to implantation, injection, surgical attachment, transplantation of other tissues, injection, and the like. In some embodiments, the matrix is formed in vivo or in situ. For example, in situ polymerizable gels may be used in conjunction with the present invention. Examples of such gels are known in the art.

In some embodiments, the cells provided herein are seeded onto the three-dimensional matrix, such as a scaffold, and implanted in vivo, wherein the seeded cells proliferate on or within the framework or help to cultivate replacement tissue in vivo, with or without the synergistic effect of other cells. Growth of amnion-derived adherent cells or co-cultures thereof on a three-dimensional framework preferably results in the formation of a three-dimensional tissue or base thereof, which can be utilized in vivo, for example, for repair of damaged or diseased tissue. For example, three-dimensional stents may be used to form tubular structures, such as for repairing blood vessels; or aspects of the circulatory system or coronary structures. According to one aspect of the invention, amnion derived adherent cells or co-cultures thereof are seeded onto a three-dimensional framework or matrix, such as a scaffold, foam or hydrogel. The frame may be provided in different shapes, for example substantially flat, substantially cylindrical or tubular, or may be entirely free-form, as it may be necessary or desirable to have a structure that is modifiable under consideration. In some embodiments, amnion derived adherent cells grow in a three-dimensional structure, while in other embodiments, the cells are only marginally viable or even dead, but stimulate or promote new tissue ingrowth or vascularization within the recipient.

The cells of the invention can grow freely in culture, be removed from culture and seeded onto a three-dimensional framework. Seeding the three-dimensional framework with a concentration of cells, e.g., about 10⁶To 5 × 10⁷The cultivation of the three-dimensional support preferably takes place in a relatively short time per microliter of cells. In addition, it may be preferred in some applications to use a greater or lesser number of cells, depending on the desired effect.

In one particular embodiment, the stroma may be cut into strips (e.g., rectangular in shape) having a width approximately equal to the inner diameter of the tubular organ into which it will ultimately be inserted. Amnion derived adherent cells can be seeded onto a scaffold and cultured by flowing or suspending in liquid medium. When the appropriate stage of confluence is reached, the stent can be rolled into a tube by joining the long sides together. The two edges can then be stitched together using a fibre of suitable material, of suitable diameter, to close the seam. To prevent cells from blocking the lumen, one open end of the tubular frame may be secured to the nozzle. The liquid medium may be squeezed through the nozzle from a source chamber connected to the culture chamber to form a fluid inside the tubular frame. The other open end may be fixed to a drain hole connected to the collection chamber, wherein the culture medium may be recirculated through the source chamber. When the culture is completed, the tube can be separated from the nozzle and the liquid outlet hole. See, for example, international application No. WO 94/25584.

In general, any of the following methods may be used in accordance with the present invention to combine two three-dimensional frames into a tube. Two or more planar frames may be placed on top of one another and stitched. The resulting two-layer sheet may then be rolled and joined and sealed as described above. In certain embodiments, a tubular scaffold can be seeded with amnion derived adherent cells as an inner layer and cultured. The second stent may be grown as a flat strip having a width slightly larger than the outer diameter of the tubular frame. After proper growth is achieved, the flat frame is wrapped around the outside of the tubular stent, and then the slits at both ends of the flat frame are closed and the flat frame is sealed to the inner tube. In another embodiment, two or more tubular networks of slightly different diameters may be grown separately. A frame having a smaller diameter may be inserted into the larger interior and sealed. For each such method, more layers can be added to the double-walled tube by reusing the method therein. Scaffolds may be assembled at any stage of growth of amnion-derived adherent cells, and culturing of the assembled scaffold may continue as needed.

In conjunction with the above procedures, the cells and therapeutic compositions provided herein can be used with devices capable of transplantation. For example, amnion derived adherent cells can be administered with, for example, a stent, a prosthetic valve, a ventricular assist device, an electrolytic removable coil, and the like. Since the device may constitute the primary treatment means for an individual in need of such treatment, cells and the like may be used as a secondary or secondary therapeutic agent to assist, stimulate or promote proper healing of the implanted device region. The cells and therapeutic compositions of the present invention may also be used to pre-treat certain implantable devices to minimize problems when used in vivo. The pretreated devices, including coated devices, may be better received by the patient in which they are implanted, reducing the risk of local or systemic infection, or the risk of restenosis or further occlusion of, for example, blood vessels.

5.8.3 amnion derived adherent cell conditioned medium

Further provided herein is an amnion-derived adherent cell conditioned medium, i.e., a medium comprising one or more biomolecules secreted or excreted by adherent cells. In various embodiments, the conditioned medium comprises cells grown in the medium for at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or at least 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 population doublings, or more. In other embodiments, the conditioned medium comprises medium in which amnion-derived adherent cells have been grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100% confluence. The conditioned medium can be used to support the culture of a population of cells, e.g., stem cells, e.g., placental stem cells, embryonic germ cells, adult stem cells, and the like. In another embodiment, the conditioned medium comprises amnion derived adherent cells and non-amnion derived adherent cells co-cultured in the medium.

Conditioned media can include adherent cells provided herein. Thus, the invention provides cell cultures comprising amnion derived adherent cells. In a specific embodiment, the conditioned medium comprises a plurality, e.g., amnion derived, adherent cell populations.

5.9 modified amnion derived adherent cells

5.9.1 genetically modified amnion derived adherent cells

In another aspect, the amnion-derived adherent cells described herein can be genetically modified, e.g., to produce a nucleic acid or polypeptide of interest, or to produce differentiated cells, e.g., osteoblasts, cardiomyocytes, pericytes, or angiogenic cells, that produce a nucleic acid or polypeptide of interest. For example, amnion derived adherent cells can be modified to produce angiogenic factors, such as pro-angiogenic molecules, soluble factors, and receptors or pro-migratory molecules such as chemokines, e.g., stromal cell derived factor 1(SDF-1) or chemokine receptors. Genetic modifications can be made using, for example, viral-based vectors, including, but not limited to, non-integrative replication vectors, e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors; an integrative viral vector, e.g., a retroviral vector or an adeno-associated viral vector; or a replication-deficient viral vector. Other methods of introducing DNA into cells include the use of liposomes, electroporation, gene guns, direct DNA injection, and the like.

Adherent cells provided herein can, for example, be transformed or transfected DNA that is controlled or operably linked to one or more suitable expression regulatory elements, e.g., promoter or enhancer sequences, transcription terminators, polyadenylation sites, internal nucleic acid entry sites. Preferably, the DNA comprises a selectable marker. After introduction of the exogenous DNA, the engineered adherent cells can be grown, for example, in enrichment medium and subsequently transferred to selection medium. In one embodiment, the DNA used to engineer amnion derived adherent cells comprises a nucleic acid sequence encoding a polypeptide of interest, e.g., a cytokine, growth factor, differentiation agent, or therapeutic polypeptide.

DNA used to engineer adherent cells can include any promoter known in the art to drive expression of a nucleotide sequence in a mammalian cell, e.g., a human cell. For example, promoters include, but are not limited to, the CMV promoter/enhancer, the SV40 promoter, the papilloma virus promoter, the Epstein-Barr virus promoter, the elastin gene promoter, and the like. In a particular embodiment, the promoter is regulatable such that the nucleotide sequence is expressed only when required. Promoters may be inducible (e.g., associated with metallothionein and heat shock proteins) or constitutive.

In another specific embodiment, the promoter is or exhibits tissue specificity. Examples of such promoters include, but are not limited to, the myosin light chain-2 gene control region (Shani, 1985, Nature 314:283) (skeletal muscle).

Amnion-derived adherent cells disclosed herein can be engineered or otherwise selected to "knock out" or "suppress" the expression of one or more genes in the cell. Expression of the cell's own genes can be suppressed by, for example, completely inactivating the genes by homologous recombination. In one embodiment, for example, the production of conventional mRNA from a target gene and resulting inactivation of that gene is prevented by disrupting an exon encoding a region of interest of the protein or an exon 5' to that region, e.g., by a positive selection marker such as neo. Genes can also be inactivated by creating gaps in a portion of the gene or deleting the entire gene. By using a construct with homologous regions of two genes of interest located apart from each other on the genome, the sequence between the two regions can be deleted (Mombaerts et al, 1991, Proc. Nat. Acad. Sci. U.S.A.88: 3084). Antisense, morpholine (morpholinos), dnase, small interfering RNA, short hairpin-like RNA, and ribozyme molecules that inhibit expression of a target gene can also be used to reduce target gene activity in adherent cells. For example, antisense RNA molecules that inhibit expression of major histocompatibility gene complexes (HLA) have been found to be the most versatile for immune responses. Triple helix molecules can be used to reduce the level of activity of a target gene. See, e.g., L.G.Davis et al (ed.), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2 nd edition, Appleton & Lange, Norwalk, Conn., which is incorporated herein by reference.

In a particular embodiment, amnion-derived adherent cells disclosed herein can be genetically modified with a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of interest, wherein expression of the polypeptide of interest is controllable by an exogenous factor, e.g., a polypeptide, a small organic molecule, etc. The polypeptide of interest may be a therapeutic polypeptide. In a more specific embodiment, the polypeptide of interest is IL-12 or an interleukin-1 receptor antagonist (IL-1 Ra). In another more specific embodiment, the polypeptide of interest is an interleukin-1 receptor antagonist fused to dihydrofolate reductase (DHFR) and the exogenous agent is an antifolate, e.g., methotrexate. The constructs can be used to engineer amnion derived adherent cells, which express IL-1Ra, or a fusion of IL-1Ra and DHFR, by exposure to methotrexate. The constructs are useful, for example, in the treatment of rheumatoid arthritis. In this embodiment, the IL-1Ra and DHFR fusions are up-regulated upon exposure to an antifolate such as methotrexate. Thus, in another specific embodiment, a nucleic acid for genetically engineering amnion-derived adherent cells can comprise a nucleotide sequence encoding a first polypeptide and a second polypeptide, wherein the first and second polypeptides are expressed as a fusion protein that is translationally upregulated in the presence of exogenous factors. The polypeptide may be expressed transiently or chronically (e.g., over a period of weeks or months). The nucleic acid molecule may additionally comprise a nucleotide sequence encoding a polypeptide that allows positive screening of the engineered cell or allows visualization of the engineered cell. In another more specific embodiment, the nucleotide sequence encodes a polypeptide that fluoresces under appropriate visualization conditions, such as luciferase (Luc). In a more specific embodiment, the nucleic acid molecule can include IL-1Ra-DHFR-IRES-Luc, wherein IL-1Ra is an interleukin-1 receptor antagonist, IRES is an internal ribosome entry site, and DHFR is dihydrofolate reductase.

5.9.2 immortalized amnion derived adherent cell line

Mammalian amnion-derived adherent cells can be conditionally immortalized by transfection with any suitable vector containing a growth-promoting gene, i.e., a gene encoding a protein that promotes growth of the transfected cells under appropriate conditions, such that production and/or activity of the growth-promoting protein can be modulated by external factors. In a preferred embodiment, the growth promoting gene is an oncogene, such as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma virus large T antigen, E1a adenovirus, or human papilloma virus E7 protein. In another embodiment, amnion derived adherent cells can be immortalized using cre-lox recombination as shown in the example for the human pancreatic β -cell line in Narushima, M.et al, (Nature Biotechnology, 2005, 23(10: 1274-.

External regulation of the growth-promoting proteins may be performed by placing the growth-promoting genes under the control of an externally regulatable promoter, e.g., a promoter whose activity may be controlled by, for example, changing the temperature of the transfected cells or the composition of the medium in contact with the cells. In one embodiment, a tetracycline (tet) controlled gene expression system may be employed (see Gossen et al, Proc. Natl. Acad. Sci. USA 89: 5547-. In the absence of tet, the tet-controlled transactivator (tTA) in this vector strongly activates the gene from ph_CMV*-1The latter is the minimal promoter from human cytomegalovirus, fused to tet operator sequences. tTA is a fusion protein of transposon-10 derived E.coli (Escherichia coli) tet resistance operon inhibitor (tetR) and the acidic region of herpes simplex virus VP 16. Low, non-toxic tet concentrations (e.g., 0.01-1.0. mu.g/mL) are almost completely abolished by tTA.

In one embodiment, the vector further comprises a gene encoding a selectable marker, e.g., a drug resistance-producing protein. Bacterial neomycin resistance gene (neo)^R) Is a marker that can be used in the present method. Carry neo^RThe cells can be screened by methods known to those of ordinary skill in the art, such as the addition of 100-In (1).

Transfection may be performed by methods known to those of ordinary skill in the art, including, but not limited to, retroviral infection. In general, cell cultures can be transfected by incubation with conditioned media mixture of the producer cell line collected from the vector and DMEM/F12 containing N2 supplement. For example, placental cell cultures prepared as described above can be transfected in vitro, e.g., after 5 days, by incubation with one volume of conditioned medium and two volumes of DMEM/F12 containing N2 supplement for about 20 hours. Transfected cells carrying the selectable marker may then be selected as described above.

After transfection, the cultures are passaged to a surface that allows proliferation, e.g., allowing at least 30% of the cells to multiply over a 24 hour period. Preferably, the substrate is a polyornithine/laminin substrate, including tissue culture plastic coated with polyornithine (10 μ g/mL) and/or laminin (10 μ g/mL), a polylysine/laminin substrate, or a surface treated with fibronectin. The cultures are then supplemented every 3-4 days with growth medium, which may or may not be supplemented with one or more proliferation-enhancing factors. Proliferation-enhancing factors may be added to the growth medium at a state where the culture is less than 50% confluency.

Conditionally-immortalized amnion-derived adherent cell lines can be passaged using standard techniques, e.g., by trypsinization at 80-95% confluence. In some embodiments, maintenance screening (e.g., by addition of G418 in cells containing the neomycin resistance gene) is not facilitated until about passage 20. Cells can also be frozen in liquid nitrogen for long term storage.

Clonal cell lines can be isolated from adherent cell lines immortalized according to the conditions prepared as described above. Generally, the clonal cell line can be isolated using standard techniques, such as by limiting dilution or using cloning rings (cloningring), and expanded. Clonal cell lines can generally be cultured and passaged as described above.

Conditionally-immortalized human amnion-derived adherent cell lines, which may be clonal, but need not be, can generally be induced to differentiate by inhibiting growth-promoting protein production and/or activity under culture conditions conducive to differentiation. For example, if the gene encoding a growth-promoting protein is under the control of an externally regulatable promoter, conditions such as temperature or culture medium composition may be altered to inhibit transcription of the growth-promoting gene. For the tetracycline-controlled gene expression system, differentiation can be performed by adding tetracycline to inhibit transcription of growth-promoting genes. In general, 4-5 days of 1 u g/mL tetracycline is enough to initiate differentiation. To promote further differentiation, other agents may also be included in the growth medium.

5.10 methods of treatment using amnion derived adherent cells

5.10.1 circulatory diseases

Amnion derived adherent cells, populations of such cells, and cell populations comprising amnion derived adherent cells provided herein are useful for treating individuals exhibiting a variety of disease states or conditions in which angiogenesis may be ameliorated. Examples of such disease states or conditions include myocardial infarction, stroke, congestive heart failure, peripheral arterial disease, left cardiac hypoplasia syndrome, diabetic ulcers, decubitus ulcers, venous ulcers, arterial ulcers, burns, nonunion of fractures, tumor-related osteoporosis, osteoarthritis, and oromaxillofacial bone repair. Amnion derived adherent cells, and populations of such cells, may also be used to promote angiogenesis in individuals exhibiting traumatic tissue defects or preventing scar formation, or individuals with total joint replacement or veneering.

In a more specific embodiment, the amnion derived adherent cells and populations of such cells provided herein are useful for treating individuals having circulatory insufficiency, e.g., individuals having peripheral vascular disease or coronary artery disease.

In one aspect, the invention provides a method for treating a patient having heart disease or heart damage, comprising administering to a patient having heart or circulatory disease or damage a therapeutic cell composition comprising amnion-derived adherent cells as described herein, and evaluating the improvement in cardiac function in the patient. In one embodiment, the cardiac disease is cardiomyopathy. In particular embodiments, the cardiomyopathy is a cardiomyopathy that is congenital or has a known cause. In other specific embodiments, the cardiomyopathy is ischemic or non-ischemic in nature. In another embodiment, the disease of the heart or circulatory system comprises one or more of angioplasty, aneurysm, angina (angina pectoris), aortic valve stenosis, aortic inflammation, arrhythmia, arteriosclerosis, arteritis, asymmetric ventricular septal hypertrophy (ASH), atherosclerosis, atrial fibrillation and flutter, bacterial endocarditis, Barlow syndrome (mitral valve prolapse), bradycardia, Buerger's disease (thromboangiitis obliterans), cardiac hypertrophy, cardiomyopathy, myocarditis, carotid artery disease, aortic stenosis, congenital heart disease (congenital heart defect), congestive heart failure (heart failure), coronary artery disease, Eisenmenger's syndrome, embolism, endocarditis, erythromelalgia, fibrillation, myofibrodysplasia, cardiac conduction block, heart murmurmur, hypertension, hypotension, congenital infant arterial calcification, coronary heart disease, angina pectoris, arrhythmia, angina pectoris, myocardial infarction, angina pectoris, myocardial infarction, kawasaki disease (mucocutaneous lymph gland syndrome, mucocutaneous lymph gland disease, polyarteritis infantis), metabolic syndrome, microvascular angina, myocardial infarction (heart attack), myocarditis, Paroxysmal Atrial Tachycardia (PAT), periarteritis nodosa (polyarteritis nodosa ), pericarditis, peripheral vascular disease, critical limb ischemia, diabetic vasculopathy, phlebitis, pulmonary stenosis (pulmonary stenosis), Raynaud's disease, renal artery stenosis, renal vascular hypertension, rheumatic heart disease, septal defect, myocardial ischemia, syndrome X, tachycardia, takayasu's arteritis, Fallo tetranectasis, large vessel displacement, tricuspid valve occlusion, arterial stem, valvular heart disease, varicose ulcer, varicose vein, vasculitis, ventricular septal defect, Wolff-Parkinson-White syndrome, or endocardial pad defect.

In other embodiments, the heart or circulatory system disorder comprises one or more of acute rheumatic fever, acute rheumatic pericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis, chronic rheumatic heart disease, mitral valve disease, mitral stenosis, rheumatic mitral insufficiency, aortic valve disorders, other endocardial structural disorders, ischemic heart disorders (acute and subacute), angina, pulmonary circulatory disorders (acute pulmonary heart disorder, pulmonary embolism, chronic pulmonary heart disorder), postspinal scoliotic heart disease, myocarditis, endocardial fibrosis, proliferation of the elastic tissue of the endocardium, atrioventricular conduction block, abnormal cardiac rhythm, myocardial degeneration, circulatory disorders including cerebrovascular disorders, anterior cerebral artery occlusion and stenosis, cerebral artery occlusion, arterial disorders, arteriolar and capillary disorders (atherosclerosis), and the like, Aneurysms), or venous and lymphatic diseases.

In one embodiment, the treatment comprises treating a patient having cardiomyopathy with a therapeutic cell composition comprising amnion-derived adherent cells, with or without another cell type. In other preferred embodiments, the patient benefits from such treatment, for example, from the ability of the amnion derived adherent cells to support the growth of other cells, including stem or progenitor cells in the heart, from tissue in the growth or vascularization of the tissue, and from the presence of beneficial cytokines, chemokines, cytokines, and the like, but without integration or expansion of the amnion derived adherent cells into the patient. In another embodiment, the patient benefits from the cell therapy, but the cells do not survive long in the patient. In one embodiment, the number, viability or biochemical activity of the cells is gradually reduced, in other embodiments the reduction of cells may precede an activity cycle, such as growth, division or biochemical activity. In other embodiments, the aged, non-viable, or even dead cells may have a beneficial therapeutic effect.

An improvement in an individual having a circulatory disease or disorder can be assessed or manifested by a detectable improvement in one or more symptoms of the circulatory disease or disorder, wherein the individual is administered amnion derived adherent cells or a therapeutic composition provided herein.

In another embodiment, improvement in an individual having a disease or disorder of the circulatory system can be assessed or manifested by a detectable improvement in one or more indicators of cardiac function, wherein the individual is administered amnion derived adherent cells or therapeutic composition provided herein, e.g., exhibits a detectable improvement in one or more of the following compared to the individual prior to administration of amnion derived adherent cells: chest Cardiac Output (CO), Cardiac Index (CI), Pulmonary Artery Wedge Pressure (PAWP), and Cardiac Index (CI),% fractional shortening (% FS), Ejection Fraction (EF), Left Ventricular Ejection Fraction (LVEF); left Ventricular End Diastolic Diameter (LVEDD), left ventricular end systolic diameter (LVEDD), contractility (e.g., dP/dt), pressure-volume loop, cardiac function detection, atrial or ventricular function enhancement; increased pump efficiency, reduced rate of pump efficiency loss, reduced hemodynamic performance loss; and reduced complications associated with cardiomyopathy.

The improvement of an individual receiving a therapeutic composition provided herein can also be assessed by subjective indicators, e.g., the individual's self-assessment of his or her health status after administration.

In certain embodiments, successful administration of the cells is not dependent on the survival of the administered amnion derived adherent cells in the individual. Rather, the success is based on one or more indicators of improvement in cardiac or circulatory health, as described above. Thus, the cells do not need to integrate and beat with the patient's heart, or enter blood vessels.

In certain embodiments, the therapeutic methods provided herein comprise inducing therapeutic amnion-derived adherent cells to differentiate according to a mesenchymal lineage, e.g., into a cardiomyocyte-like phenotype, an angiogenic phenotype, or a angiogenic phenotype, or to form cells such as muscle cells, cardioblasts, endothelial cells, endocardial cells, epicardial cells, vascular endothelial cells, smooth muscle cells (e.g., vascular smooth muscle cells).

Administration of amnion-derived adherent cells or a therapeutic composition comprising such cells to an individual in need thereof can be performed, for example, by implantation, transplantation (e.g., of the cells themselves or as part of a stroma-cell combination), injection (e.g., directly to the site of the disease or condition, e.g., directly to the site of cardiac ischemia in an individual having myocardial infarction), fusion, catheter delivery, or any other method known in the art for providing cell therapy.

In one embodiment, the therapeutic cellular composition is provided to an individual in need thereof, for example, by injection at one or more locations of the individual. In a specific embodiment, the therapeutic cellular composition is provided by intracardiac injection, for example, to an ischemic cardiac region. In other specific embodiments, the cells are injected onto the surface of the heart, adjacent regions, or even more distant regions. In a preferred embodiment, the cells may be anchored (home) to the diseased or injured area.

Individuals with a disease or condition of the coronary or vascular system may be administered amnion derived adherent cells at any time that the cells are conducive to treatment. In certain embodiments, for example, a cell or therapeutic composition of the invention is administered within 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or within 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days of myocardial infarction. For myocardial infarction, for example, administration within 1-3 or 1-7 days of the approach time is preferred over administration later, for example, 3 or 7 days after myocardial infarction. In other embodiments, the cells or therapeutic compositions of the invention are administered within 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or within 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the initial diagnosis of the disease or condition.

Kits for treating myocardial infarction are also provided herein. The therapeutic cellular composition is provided in a kit, which can be prepared in a pharmaceutically acceptable form, for example, by admixing a pharmaceutically acceptable carrier; and an applicator, and instructions for use. Ideally, the kit is useful, for example, in a physician's office, or by first aid providers for administration to patients diagnosed with myocardial infarction or similar cardiac event.

In particular embodiments of the methods of treatment provided herein, the amnion derived adherent cells are administered with stem cells (i.e., stem cells that are not amnion derived adherent cells), myoblasts, myocytes, cardiac myoblasts, cardioblasts, or myoblasts, myocytes, cardiac myoblasts, and/or progenitor cells of cardioblasts.

In a specific embodiment, the treatment methods provided herein comprise administering amnion derived adherent cells, e.g., a therapeutic composition comprising the cells, to a patient having a cardiac or circulatory disease; and evaluating the improvement in cardiac function in the patient, wherein the therapeutic cell composition is administered as a matrix-cell complex. In certain embodiments, the matrix is a scaffold, preferably bioabsorbable, comprising at least cells.

To this end, the invention provides amnion derived adherent cell populations incubated in the presence of one or more factors that stimulate differentiation of stem or progenitor cells into the angiogenic, vasculogenic, angiogenic or angiogenic pathways. Such factors are known in the art; conditions suitable for differentiation can be determined by routine experimentation. Such factors include, but are not limited to, factors such as growth factors, chemokines, cytokines, cell products, demethylating agents, and other stimulators now known or later determined to be capable of stimulating pathway or lineage differentiation such as stem cell cardiotrophicity, vasculogenesis, or angiogenicity.

Amnion derived adherent cells can be differentiated into pathways or lineages such as cardiogenic, angiogenic or angiogenetic by culturing the cells in the presence of a factor comprising at least one of a demethylating agent, BMP, FGF, Wnt factor protein, Hedgehog, and/or anti-Wnt factor.

The inclusion of a demethylating agent allows cells to differentiate towards the mesenchymal system of the cardiogenic pathway. Differentiation may be measured, for example, by expressing at least one of cardiac myosin, skeletal myosin, or GATA 4; inducing acquisition of heart rhythm, either spontaneously or otherwise; or the ability to be at least partially integrated into the myocardium of a patient without inducing arrhythmia. Demethylating agents that may be used to initiate such differentiation include, but are not limited to, 5-azacytidine, 5-aza-2' -deoxycytidine, dimethylsulfoxide, chelerythrine chloride, a retinoid or salt thereof, 2-amino-4- (ethylthio) butyric acid, procainamide, and procaine.

In certain embodiments herein, cells identified above that are induced using one or more factors can become cardiogenic, angiogenic or angiogenic cells or progenitor cells. Preferably at least some of the cells may be at least partially integrated into the cardiovascular system of the subject, including but not limited to myocardium, blood vessels and other structures of the heart, cardiovascular or peripheral blood vessels, and the like. In certain other embodiments, the differentiated amnion derived adherent cells differentiate to obtain two or more labeled cells capable of recognizing cardiogenic cells or their progenitors and are capable of integrating partially or fully into the recipient's heart or vasculature. In particular embodiments, the cells administered to the subject do not increase arrhythmia, cardiac defect, vascular defect, or other abnormality of the subject's circulatory system or health. In certain embodiments, the amnion-derived adherent cells are capable of promoting differentiation of stem cells naturally present in the myocardium, blood vessels, blood, etc., of a patient, to differentiate themselves into, for example, cardioblast cells, or at least to differentiate into cardiogenic, angiogenic, or angiogenetic lines.

Amnion derived adherent cells and populations of such cells can be provided to an individual as a treatment or prophylaxis, e.g., an individual having a heart or circulatory disease, disorder or condition, or infection. The disease, disorder or condition may include congestive heart failure, e.g., ischemic injury, e.g., from myocardial infarction or trauma (acute or chronic), caused by atherosclerosis, cardiomyopathy, or cardiac injury.

In certain embodiments, a therapeutically effective amount of amnion derived adherent cells is administered to an individual, e.g., in a cell population comprising amnion derived adherent cells. In a specific embodiment, the population comprises about 50% amnion derived adherent cells. In another specific embodiment, the population is a substantially uniform population of amnion derived adherent cells. In other embodiments, the population comprises at least about 5%, 10%, 20%, 25%, 30%, 33%, 40%, 60%, 66%, 70%, 75%, 80%, or 90% amnion derived adherent cells.

Amnion-derived adherent cells can be administered to an individual in the form of a therapeutic composition comprising the cells and another therapeutic agent, such as insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), IL-8, an antithrombotic agent (e.g., heparin derivatives, urokinase, and PPack (dextrophenylalanine proline arginine chloromethyl ketone), an antithrombin compound, a platelet receptor antagonist, an antithrombin antibody, an antiplatelet receptor antibody, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors, and/or platelet inhibitors), an anti-apoptotic agent (e.g., EPO derivatives and analogs and salts thereof, EPO, and analogs and salts thereof, and combinations thereof, TPO, IGF-I, IGF-II, Hepatocyte Growth Factor (HGF) or caspase inhibitors), anti-inflammatory agents (e.g., P38MAP kinase inhibitors, statins, IL-6 and IL-1 inhibitors, pemirolast, tranilast, dactinospora, sirolimus, non-steroidal anti-inflammatory compounds such as acetylsalicylic acid, ibuprofen, teporaline, tolmetin, or suprofen), immunosuppressive or immunomodulatory agents (e.g., calcineurin inhibitors such as cyclosporine, tarolimus, mTOR inhibitors such as sirolimus or everolimus; antiproliferative agents such as azathioprine and mycophenolate mofetil; corticosteroids, such as prednisolone or hydrocortisone; antibodies, such as monoclonal anti-IL-2R α receptor antibodies, basiliximab, Daclizuma, polyclonal anti-T-cell antibodies such as anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG), and monoclonal anti-T cell antibody OKT3, or adherent placental cells described in U.S. patent No. 7,468,276 and U.S. patent application publication No. 2007/0275362, the disclosures of which are incorporated by reference in their entirety), and/or antioxidants (e.g., probucol; vitamins A, C and E, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine, or derivatives, analogs or salts of the foregoing antioxidants). In certain embodiments, a therapeutic composition comprising amnion-derived adherent cells further comprises one or more additional cell types, e.g., adult cells (e.g., fibroblasts or endoderm cells) or stem or progenitor cells. The therapeutic agent and/or one or more additional cells may be administered separately to an individual in need thereof, or a combination of two or more of the compounds or agents.

In certain embodiments, the subject to be treated is a mammal. In a specific embodiment, the subject to be treated is a human. In particular embodiments, the subject is a livestock animal or livestock. In other specific embodiments, the subject to be treated is a horse, sheep, cow, or steer bull, pig, dog, or cat.

5.10.2 apoplexy and other ischemic diseases

In certain embodiments, the present invention provides a method of treating a subject suffering from blood flow disruption (disruption), e.g., in or around the brain or in the peripheral vasculature, comprising administering to the subject a therapeutically effective amount of an AMDAC. In certain particular embodiments, the ischemia is Peripheral Arterial Disease (PAD), e.g., Critical Limb Ischemia (CLI). In certain other embodiments, the ischemia is Central Nervous System (CNS) ischemia. In certain other embodiments, the ischemia is a peripheral arterial disease, an ischemic vascular disease, an ischemic heart disease, an ischemic brain disease, or an ischemic kidney disease.

In a specific embodiment, the disruption of blood flow is stroke. In a more specific embodiment, the stroke is an ischemic stroke. In another more specific embodiment, the stroke is a hemorrhagic stroke, e.g., intracranial cerebral hemorrhage or spontaneous subarachnoid hemorrhage. In another specific embodiment, the rupture is a hematoma. In more specific embodiments, the hematoma is a dural hematoma, a subdural hematoma, or a subarachnoid hematoma. In another specific embodiment, the hematoma is caused by external forces on the skull, such as head injury. In another specific embodiment, the disruption is a Transient Ischemic Attack (TIA), e.g., a regenerative TIA. In another specific embodiment, the disruption is vasospasm, e.g., vasospasm following hemorrhagic stroke.

In another specific embodiment of the method, the therapeutically effective amount is an amount of AMDACs that abrogates, detectably ameliorates, or reduces the severity of or slows the progression of one or more symptoms or neurological deficits that cause the individual to exhibit a disruption in or around the brain or CNS blood flow, such as hypoxic injury or hypoxic injury. In another specific embodiment, the therapeutically effective amount of the isolated AMDACs is administered prophylactically to the individual, e.g., to reduce or eliminate nerve damage caused by a secondary or secondary disruption of blood flow in or around the brain or CNS after the disruption of blood flow.

In another specific embodiment, the symptom of disruption of blood flow in or around the brain, e.g., stroke, hypoxic injury or hypoxic injury, is one or more of the following: hemiplegia (hemiparalysis); hemiparesis (hemiparalysis); facial muscle weakness; numbness; a reduction in sensation; olfactory, gustatory, auditory or visual changes; loss of smell, taste, hearing, or vision; drooping eyelids (ptosis); detectable extraocular muscle weakness; a decrease in pharyngeal reflex; a decrease in swallowing ability; a decrease in pupillary light response; a reduction in facial sensation; the balance is reduced; nystagmus; a change in breathing rate; a change in heart rate; sternocleidomastoid muscle weakness, difficulty or inability to rotate the head; the tongue is weak; aphasia (inability to speak or understand language); apraxia (altered voluntary exercise); a visual field defect; memory loss; half neglect or half-side spatial neglect (impairment of visual field spatial lack of attention on the other side); collapse of thinking; confusion of thinking; lewd gestures (hypersexual gestures); sick disappointment (adherence refusal to admit defect); difficulty in walking; a change in motor coordination; vertigo; the balance is unstable; loss of consciousness; headache; and/or vomiting.

In another specific embodiment, the above method of treatment comprises administering to the individual a second therapeutic agent. In a more specific embodiment, the second therapeutic agent is a neuroprotective agent. In a more specific embodiment, the second therapeutic agent is NXY-059 (a disulfonyl derivative of phenylbutylnitrone: disodium 4- ((t-butylimine) -methyl) benzene-1, 3-disulfonic acid N-oxide, or disodium 4- ((oxy-t-butyl-aminylidene) -methyl) benzene-1, 3-disulfonic acid; also known as disufentin). In another more specific embodiment, the second therapeutic agent is a thrombolytic agent. In a more specific embodiment, the thrombolytic agent is tissue plasminogen activator (tPA). In embodiments where the disruption of blood flow in or around the brain is hemorrhagic, the second therapeutic agent may be an antihypertensive, e.g., a beta blocker or diuretic, a combination of diuretic and potassium sparing diuretic, a combination of a, beta blocker and diuretic, a combination of Angiotensin Converting Enzyme (ACE) inhibitor and diuretic, an angiotensin-II antagonist and diuretic, and/or a calcium channel blocker and an ACE inhibitor. In another more specific embodiment, the second therapeutic agent is a calcium channel blocker, a glutamate antagonist, a gamma aminobutyric acid (GABA) agonist, an antioxidant, or a free radical scavenger.

In another specific method of treatment embodiment, the isolated AMDACs are administered to the individual within 21-30 days, e.g., within 21 days, of the individual developing one or more symptoms of blood flow disruption in or around the brain, e.g., within 21-30 days, e.g., within 21 days, of the individual developing symptoms of stroke, hypoxic injury, or hypoxic injury. In another specific method of treatment embodiment, said isolated AMDACs are administered to said individual within 14 days of one or more symptoms of blood flow disruption developed in or around the brain of said individual. In another specific method of treatment embodiment, said isolated AMDACs are administered to said individual within 7 days of one or more symptoms of blood flow disruption developed in or around the brain of said individual. In another specific method of treatment embodiment, said isolated AMDACs are administered to said individual within 48 hours of one or more symptoms of blood flow disruption developing in or around the brain of said individual. In another specific embodiment, the isolated AMDACs are administered to the individual within 24 hours of one or more symptoms of blood flow disruption developing in or around the brain of the individual. In another specific embodiment, the isolated AMDACs are administered to the individual within 12 hours of one or more symptoms of blood flow disruption developing in or around the brain of the individual. In another specific embodiment, the isolated AMDACs are administered to the individual within 3 hours of one or more symptoms of blood flow disruption developing in or around the brain of the individual.

In a specific embodiment, the disruption of blood flow is Critical Limb Ischemia (CLI). In another more specific embodiment, the CLI is a severe occlusion of a lower limb artery that significantly reduces blood flow. In another more specific embodiment, the CLI is characterized by ischemic rest pain, severe pain in the legs and feet when a person is immobilized, uncured ulcers in the legs or feet, pain or numbness in the feet, shiny, smooth, dry skin in the legs or feet, thickened toenails, absence or absence of pulse in the legs or feet, open ulcers, non-healing skin infections or ulcers, dry gangrene (dry, black skin) in the legs or feet. In another specific embodiment, the CLI may cause damage to the fingers or the entire limb. In another specific embodiment of the method, the therapeutically effective amount is an amount of AMDACs that eliminates, detectably ameliorates, reduces the severity of or slows progression of one or more symptoms of limb dysfunction or non-oxygenation (hypoxia/anoxia) caused by blood flow disruption, e.g., hypoxic injury or hypoxic injury, in or around the brain or CNS of the individual. In another specific embodiment, the therapeutically effective amount of the isolated AMDACs is administered to the individual prophylactically, e.g., to reduce or eliminate tissue damage caused by secondary or secondary disruption of blood flow in or around a limb following the disruption of blood flow.

5.10.3 dosage and route of administration

Any medically acceptable route associated with the disease or condition to be treated may be employed when administering the AMDACs to an individual in need thereof. In another specific embodiment of the above method of treatment, the AMDAC is administered by bolus injection. In another specific embodiment, the isolated AMDACs are administered by intravenous infusion. In a specific embodiment, the intravenous infusion is an intravenous infusion for about 1 to about 8 hours. In another specific embodiment, the isolated AMDACs are administered intracranially. In another specific embodiment, the isolated AMDACs are administered by intramuscular injection. In another specific embodiment, the isolated AMDACs are administered intraperitoneally. In another specific embodiment, the isolated AMDAC is administered intra-arterially. In a more specific embodiment, the isolated AMDACs are administered within an ischemic region. In another more specific embodiment, the isolated AMDACs are administered around an ischemic area. In another particular method of treatment embodiment, the isolated AMDACs are administered intramuscularly, intradermally, or subcutaneously.

In another specific embodiment of the above method of treatment, the AMDAC is administered to the individual once. In addition toIn a specific embodiment, the isolated AMDAC is administered to the individual two or more times in another specific embodiment, the administering comprises administering about 1 × 10⁴To 1 × 10⁵An isolated AMDAC, e.g., AMDAC per kilogram of the subject in another specific embodiment, the administering comprises administering about 1 × 10⁵To 1 × 10⁶In another specific embodiment, said administering comprises administering about 1 × 10⁶To 1 × 10⁷In another specific embodiment, said administering comprises administering about 1 × 10⁷To 1 × 10⁸In other specific embodiments, said administering comprises administering about 1 × 10⁶To about 2 × 10⁶Isolated placental cells per kilogram of said individual, about 2 × 10⁶To about 3 × 10⁶About 3 × 10 per kilogram of said individual isolated placental cells⁶To about 4 × 10⁶Isolated placental cells per kilogram of said individual, about 4 × 10⁶To about 5 × 10⁶Isolated placental cells per kilogram of said individual, about 5 × 10⁶To about 6 × 10⁶Isolated placental cells per kilogram of said individual about 6 × 10⁶To about 7 × 10⁶Isolated placental cells per kilogram of said individual, about 7 × 10⁶To about 8 × 10⁶Isolated placental cells per kilogram of said individual, about 8 × 10⁶To about 9 × 10⁶Isolated placental cells per kilogram of said individual, or about 9 × 10⁶To about 1 × 10⁷In another specific embodiment, said administering comprises administering to said individual about 1 × 10⁷To about 2 × 10⁷In another specific embodiment, said administering comprises administering to said individual about 1.3 × 10⁷To about 1.5 × 10⁷In another specific embodiment, said administering comprises administering to said individual up to about 3 × 10⁷Isolated placental cells per kilogram of said individual. In a specific embodimentWherein said administering comprises administering to said individual about 5 × 10⁶To about 2 × 10⁷In another specific embodiment, said administering comprises administering about 150 × 10 in about 20 milliliters of solution⁶Isolated placental cells to said individual.

In a specific embodiment, the administering comprises administering about 5 × 10⁶To about 2 × 10⁷Administering to said individual isolated placental cells, wherein said cells are contained in a solution comprising 10% dextran, e.g., dextran-40, 5% human serum albumin, and optionally an immunosuppressive agent in another specific embodiment, said administering comprises administering about 5 × 10 intravenously ⁷To 3 × 10⁹In a more specific embodiment, said administering comprises administering about 9 × 10 intravenously⁸Isolated placental cells or about 1.8 × 10⁹In another specific embodiment, said administering comprises intracranial administration of about 5 × 10⁷To 1 × 10⁸In a more specific embodiment, said administering comprises intracranial administration of about 9 × 10⁷Isolated placental cells.

5.11 differentiation of amnion-derived adherent cells

In a more specific embodiment, the endothelial cells, myocytes, or pericytes are characterized by expression of one or more of CD9, CD31, CD54, CD102, NG2 (neuroglia/glia antigen 2), or α smooth muscle actin, relative to OCT-4^–、VEGFR2/KDR⁺、CD9⁺、CD54⁺、CD105⁺、CD200⁺And VE-cadherin^–Expression is enhanced in amniotic cells. In other more special casesIn embodiments of (a), the endothelial cell, myocyte or pericyte is characterized by expressing one or more of CD9, CD31, CD54, CD102, NG2 (nerve/glial antigen 2) or α smooth muscle actin, relative to OCT-4^–、VEGFR2/KDR⁺And VEGFR1/Flt-1⁺Expression is enhanced in amniotic cells.

5.11.1 Induction of angiogenesis

Angiogenesis by amnion-derived adherent cells provided herein can be performed as follows. For example atCulturing amnion-derived adherent cells to passage 3 in endothelial cell culture medium of-2 (Lonza), or in a medium containing 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma); 2% fetal bovine serum (HycleLabs.); 1 × insulin-transferrin-selenium supplement (ITS); 1 × linoleic acid-bovine serum albumin (LA-BSA); 5 × 10^{- 9}M dexamethasone (Sigma); 10^-4M ascorbic acid 2-phosphate (Sigma); epidermal growth factor 10ng/mL (R)&DSYSTEMS); and platelet derived growth factor (PDGF-BB)10ng/mL (R)&D SYSTEMS). Then placing the cells in MATRIGEL^TMOr the matrix comprises collagen-1, e.g., in a 96-well plate, having a density of, e.g., about 1.5 × 10⁴Cells per well in the same medium or in DMEM with FBS (0-5% v/v) containing, for example, about 10-50ng per microliter of vascular endothelial growth factor (VEGF.) the medium can be changed about twice a week angiogenesis is confirmed by visual observation of cell sprouting tube-like structures and lumen formation, which can be observed under a microscope at, for example, 50 × -100 × magnification.

5.11.2 Induction of differentiation into cardiomyocytes

Myogenic (cardiogenic) differentiation of amnion-derived adherent cells provided herein can be achieved, for example, by subjecting the cells to culture conditions that induce differentiation into cardiomyocytes. Preferred cardiomyocyte culture media include DMEM/20% CBS supplemented with 1 μ M retinoid; basic fibroblast growth factor, 10 ng/mL; and transforming growth factor beta-1, 2 ng/mL; and epidermal growth factor, 100 ng/mL. KnockOut serum replacement (Invitrogen, Carlsbad, California) may be used in place of CBS. Alternatively, amnion derived adherent cells are cultured in DMEM/20% CBS supplemented with 1-100 for 24 hours, e.g., 50ng/mL of cardiotrophin-1. In another embodiment, amnion derived adherent cells can be cultured for 10-14 days, wherein the culture is in protein free medium for 5-7 days, and then stimulated with a human myocardium extract, for example, by homogenizing human myocardium in 1% HEPES buffer supplemented with 1% cord blood serum.

Differentiation can be confirmed by, for example, RT/PCR to show gene expression or by observable cell beating (waiting). Adherent cells can be considered to have differentiated into cardiomyocytes when the cells exhibit one or more of these properties.

6. Examples of the embodiments

6.1 example 1: isolation and expansion of adherent cells from amniotic membrane

This example shows the isolation and expansion of amnion derived adherent cells.

6.1.1 isolation

Amnion-derived adherent cells were isolated from amnion according to the following method. The amniotic membrane/chorion is excised from the placenta and the amniotic membrane is manually separated from the chorion. The amniotic membrane was washed with sterile PBS to remove remaining blood, blood clots, and other materials. Blood, clots, or other material not removed was washed with sterile gauze removal and the amniotic membrane was washed again with PBS. Excess PBS was removed from the membrane and the amniotic membrane was cut into 2 "by 2" pieces with a scalpel. To release the epithelial cells, the processing vessel was constructed by connecting a sterile jacketed glass processing vessel to a 37 ℃ circulating water bath using tubing and connectors, and mounted onto a stir plate. Trypsin (0.25%, 300mL) was heated to 37 ℃ in the process vessel; the amniotic membrane fragments are added and the amniotic membrane/trypsin suspension is stirred, for example, at 37 ℃ for 15 minutes at 100RPM to 150 RPM. The sterile container is placed in a sterile field adjacent to the processing container and a sterile 75-125 μm screener (Millipore, Billerica, MA) is inserted therein to assemble the sterile screening system. After agitating the amniotic pieces for 15 minutes, the contents of the processing vessel are transferred to a sizer and the amniotic pieces are transferred back to the processing vessel using, for example, sterile forceps; the trypsin solution containing the epithelial cells was discarded. The amniotic membrane pieces were again stirred with 300mL of trypsin solution (0.25%) as described above. The filter was washed with approximately 100 and 150mL PBS and the PBS solution was discarded. After agitating the amniotic pieces for 15 minutes, the contents of the processing vessel were transferred to a screener. Then transferring the amniotic membrane fragments back to a processing container; the trypsin solution containing the epithelial cells was discarded. The amniotic membrane pieces were again stirred with 300mL of trypsin solution (0.25%) as described above. About 100 and 150mL of PBS were washed with the screener and the PBS solution was discarded. After agitating the amniotic pieces for 15 minutes, the contents of the processing vessel were transferred to a screener. The amniotic membrane fragments were then transferred back to the processing vessel and the trypsin solution containing the epithelial cells was discarded. The amniotic membrane pieces were neutralized with trypsin by stirring in PBS/5% FBS (amniotic membrane in a 1:1 ratio by volume: PBS/5% FBS solution) at 37 ℃ for about 2-5 minutes. A fresh sterile screening system was assembled. After neutralization of trypsin, the contents of the processing vessel are transferred to a new screener and the amniotic membrane fragments are transferred back to the processing vessel. Sterile PBS (400mL) was added to the process vessel at room temperature, and the process vessel contents were stirred for about 2-5 minutes. The screener was washed with approximately 100 and 150mL of PBS. After stirring, the contents of the processing vessel are transferred to a screener; the treated flask was washed with PBS and the PBS solution was discarded. The processing vessel was filled with 300mL of pre-heated DMEM and the amniotic membrane fragments were transferred to DMEM solution.

In order to release the amnion-derived adherent cells, the amnion treated as described above is further treated with collagenase as follows. A sterile collagenase stock (500U/mL) was prepared by dissolving the appropriate amount of collagenase powder (varying with the activity of the collagenase lot supplied by the supplier) in DMEM. The solution was filtered through a 0.22 μm filter and dispensed into separate sterile containers. Adding CaCl₂The solution (0.5mL, 600mM) was added to each 100mL dose and each dose was frozen. Collagenase (100mL) is added to the amniotic membrane fragments in a processing vessel and the processing vessel is agitated for 30-50 minutes, or until the amniotic membrane has been digested by visual observation. After completion of the amniotic membrane digestion, 100mL of pre-heated sterile PBS/5% FBS was added to the processing vessel and the processing vessel was stirred for an additional 2-3 minutes. After stirring, the contents of the flask were transferred to a sterile 60 μm sieve and the liquid was collected by vacuum filtration. The processing container was washed with 400mL of PBS and the PBS solution was sterile filtered. The filtered cell suspension was then centrifuged at 20 ℃ for 300x g 15 minutes and the cell pellet was resuspended in pre-warmed PBS/2% FBS (ca. 10mL total).

6.1.2 cultivation

Freshly isolated angiogenic amniotic cells were added to a medium containing 60% DMEM-LG (Gibco), 40% MCBD-201(Sigma), 2% FBS (Hyclone labs), 1 × insulin-transferrin-selenium supplement (ITS), 10ng/mL linoleic acid-bovine serum albumin (LA-BSA), 1 n-dexamethasone (Sigma), 100. mu.M ascorbic acid 2-phosphate (Sigma), 10ng/mL epidermal growth factor (R)&System D); and 10ng/mL platelet-derived growth factor (PDGF-BB) (R&D System) and at 10,000 cells per cm²The inoculation density of (2) was inoculated in T-flash. Then 5% CO at 37 deg.C₂And>incubate the culture device at 90% humidity. Cell attachment, growth and morphology were monitored daily. Non-adherent cells and debris were removed in time during replacement culture. The medium was changed twice a week. Adherent cells with a typical fibroblast/spindle morphology appear a few days after the initial seeding. When the confluency reached 40% -70% (4-11 days after initial inoculation), cells were harvested by trypsinization (0.25% trypsin-EDTA) for 5 minutes at room temperature (37 ℃). After neutralization with PBS-5% FBS, the cells were centrifuged at 200 and 400g for 5-15 minutes at room temperature and then resuspended in growth medium. At this time, it can be considered that the AMDAC line has been successfully cultivated in the early generation. In some cases, primary amnion derived adherent cells are cryopreserved or expanded.

6.1.3 culture method

Amnion-derived adherent cells were cultured in the above growth medium at a density of 2000-4000 per cm²The culture apparatus is inoculated into a suitable tissue culture process. Then 5% CO at 37 deg.C₂And>incubate the culture device at 90% humidity. During culture, AMDACs will adhere and proliferate. The growth, morphology and confluency of the cells were monitored daily. If the culture extends to 5 days or more, the medium is changed twice a week to replenish fresh nutrients. When the confluency reached 40% -70% (3-7 days after inoculation), cells were harvested by trypsinization (0.05% -0.25% trypsin-EDTA) for 5 minutes at room temperature (37 ℃). After neutralization with PBS-5% FBS, the cells were centrifuged at 200 and 400g for 5-15 minutes at room temperature and then resuspended in growth medium.

AMDAC isolated and cultured according to the present method is typically inoculated at 1 × 10⁶33530 +/-15090 colony forming units (fibroblasts) were produced in the cells (CFU-F).

6.2 example 2: phenotypic characterization of amnion derived adherent cells

6.2.1 Gene and protein expression profiling

This example describes the phenotypic properties of amnion derived adherent cells, including cell surface markers, mRNA and protein expression.

Sample preparation amnion derived adherent cells were obtained as described in example 1 passage 6 cells were grown to approximately 70% confluence in the growth medium described in example 1 above, trypsinized and washed with PBS NTERA-2 cells (American type culture Collection, ATCC accession number CRL-1973) were grown in DMEM containing 4.5g/L glucose, 2mM glutamine and 10% FBS, nucleated cell counting was performed to obtain a minimum of 2 × 10⁶-1x 10⁷A cell. Cells were then lysed using a Qiagen RNeasy kit (Qiagen, Valencia, Calif.) and lysates obtained using QIAshreder. RNA was then isolated using Qiagen RNeasy kit. The quantity and quality of RNA were determined using a Nanodrop ND1000 spectrophotometer, 25ng/. mu.L RNA/reaction. The cDNA reaction was performed using a High CapacitycDNA Archive kit from Applied Biosystems (Foster City, Calif.). Using Applied BiosystemsReal-time PCR reactions were performed using Universal PCR Master mixers. Reactions were performed in 40 cycles in a standard format in an Applied Biosystems7300 real-time PCR system.

Sample analysis and results: using real-time PCR methods and specificGene expression probe and/orHuman angiogenic arrays (Applied Biosystems) characterize the expression of stem cell-associated angiogenic and cardiogenic markers of cells. The results are shown either as the relative expression of the target gene compared to the relevant cellular controls, or as the relative expression (Ct) of the target gene compared to the ubiquitous housekeeping gene (e.g., GAPDH, 18S, or GUSB).

Amnion derived adherent cells express different stem cell-associated angiogenic and cardiogenic genes and exhibit relatively absent OCT-4 expression compared to NTERA-2 cells. Table 1 summarizes the expression of selected angiogenic, cardiogenic and stem cell genes.

Table 1: and determining the gene expression profile of the amnion derived adherent cells by RT-PCR.

The column "mRNA" indicates in each case whether the mRNA of the particular marker is determined to be present.

In a separate experiment, AMDAC was also found to express the following genes: aryl hydrocarbon receptor nuclear transporter 2(ARNT2), Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Glial Derived Neurotrophic Factor (GDNF), neurotrophin 3(NT-3), NT-5, hypoxia-induced factor 1 α (HIF1A), hypoxia-induced protein 2(HIG2), heme oxygenase (decyclization) 1(HMOX1), extracellular superoxide dismutase [ Cu-Zn ] (SOD3), Catalase (CAT), transforming growth factor β 1(TGFB1), transforming growth factor β 1 receptor (TGFB1R), and hepatocyte growth factor receptor (HGFR/c-met).

6.2.2 evaluation of angiogenic Capacity of amnion-derived adherent cells Using flow cytometry

Cell identity is defined using flow cytometry as a method to quantify amnion derived adherent cell phenotypic markers cell identity is defined.cell samples are from frozen storage, pre-thaw and during reagent preparation, cell vials are maintained on dry ice.then samples are quickly thawed using a 37 ℃ water bath.a pre-frozen cell count (count) is used to calculate the number-based cell dilutions after initial thaw.cryovial is briefly thawed in a 37 ℃ water bath for about 30 seconds with gentle agitation.about 100. mu.L of a cold (2-8 ℃) thawed solution (with 2.5% albumin and 5% Gentran 40 PBS added) is added to the cryovial and mixed immediately after thawing.after gentle mixing, the entire contents of the cryovial are transferred to a 15mL conical tube containing an equal volume of the cold (2-8 ℃) thawed solution, the residual volume is measured with a pipette (estimated) at room temperature before centrifugation of 400g of myocytes in the conical tube at room temperature for 5 minutes.the residual volume and cell pellet are resuspended in PBS containing 1% concentration 250 × x10 PBS to obtain a cell concentration of 250 x10³Cells/100. mu.L buffer, for example, 1 × 10⁶Cells were resuspended in 400 μ L of 1% FBS. The cell suspension was placed in pre-labeled 5mL FACS tubes (Becton Dickinson (BD), Frankli)n Lakes, NJ) for each first antibody class, 100 μ Ι _ of cell suspension was aliquoted into one isotype control tube prior to phenotypic analysis, the concentration of all antibodies was optimized to achieve good signal/noise ratio and adequate detection of CD antigen across the 4-log dynamic range⁵Target final antibody μ g of cells) ═ added antibody # μ L, mastermix for preparation of isotype and sample antibodies by addition of appropriate amounts of antibody to each tube, staining of cells at room temperature for 15-20 minutes, after staining, unbound antibody in each sample was removed by centrifugation (400g × 5 minutes), then washed with 2mL 1% FBS PBS (room temperature) before resuspension in 150 μ L of room temperature 1% FBS PBS, then sample analysis was performed in Becton Dickinson FACSCalibur, facscantotoi or facscantotoi flow cytometer according to manufacturer's instructions, after summary of real-time device compensation parameters, multiparameter flow cytometer data sets (side scatter), Front Scatter (FSC) and integrated fluorescence spectra (FL)) were obtained using FACSDiva software to determine compensation parameters according to manufacturer's instructions.

Table 2: cell surface markers expressed in amnion derived adherent cells as determined by flow cytometry.

The column "immunolocalization flow cytometry" indicates the determination of the presence or absence of a particular marker by immunolocalization, in particular flow cytometry.

In another experiment, AMDAC cells were labeled with anti-human CD49f (phycoerythrin-conjugated clone GoH 3; BDPharmingen commercial number 555736) and analyzed by flow cytometry. About 96% of AMDAC labeled anti-CD 49f (i.e., is CD49 f)⁺)。

In additional experiments, AMDAC was also found to express CD49a, CD106, CD119, CD130, c-met (hepatocyte growth factor receptor; HGFR), CXC chemokine receptor 1(CXCR1), PDGFRA, and PDGFRB by immunolocalization. AMDAC has also been found by immunolocalization to lack the expression of CD49E, CD62E, fibroblast growth factor receptor 3(FGFR3), tumor necrosis factor receptor superfamily member 12A (TNFRSF12A), insulin-like growth factor 1 receptor (IGF-1R), CXCR2, CXCR3, CXCR4, CXCR6, chemokine receptor 1(CCR1), CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, epidermal growth factor receptor (EGF-R), insulin receptor (CD220), interleukin receptor 4(IL 4-R; CD124), IL6-R (CD126), TNF-R1a and 1b (CD120a, b) and erbB2/Her 2.

6.2.3 evaluation of angiogenic Capacity of amnion derived adherent cells Using Immunohistochemistry (IHC)/immunofluorescence chemistry (IFC)

Amnion-derived adherent cells of passage 6 were grown to approximately 70% confluence on 4-well chambered slides and fixed with 4% formalin solution for 30 minutes each, after fixation, the slides were washed twice with PBS, incubated for 20 minutes in a humidity chamber at room temperature with 10% normal serum from the same host as the second antibody, 2 × casein and 0.3% Triton X100 in PBS, excess serum was blotted and the slides were incubated in a humidity chamber with the first antibody (goat polyclonal IgG (Santa Cruz; Santa Cruz, CA)), the incubation temperature and time were dependent on the optimal conditions selected for the antibody used.The slides were then washed three times with PBS for 5 minutes each and incubated with a fluorescence-conjugated anti-immunoglobulin secondary antibody directed against the primary antibody (rabbit anti-goat antibody (Santa Cruz)) host for 20-30 minutes at room temperature in a humidity chamber. Slides were subsequently washed three times with PBS for 5 minutes each, using DAPI(Vector Labs) coverslips were mounted and the solution was added to counterstain nuclei. Cell staining can be observed using a Nikon fluorescence microscope. All images were taken with equal exposure times, normalized against the background of the corresponding category (goat igg (santa cruz)). Table 3 summarizes the results of the expression of angiogenic proteins by amnion derived adherent cells.

Table 3: markers of angiogenesis in the presence or absence of amnion-derived adherent cells.

Amnion derived adherent cells express an angiogenesis marker tumor endothelial cell marker 7(TEM-7), one of the proteins shown in table 3. See fig. 2.

6.2.4 evaluation of angiogenic Capacity of amnion-derived adherent cells Using Membrane proteomics

And (3) purifying membrane protein: amnion-derived adherent cells at passage 6 were grown to approximately 70% confluence in growth medium, trypsinized, and washed with PBS. Prior to cell lysis, cells were incubated for 15 minutes with a solution containing a protease inhibitor cocktail (P8340, Sigma Aldrich, st. The cells were then lysed by adding 10mM HCl solution (thereby avoiding the use of detergent) and centrifuged at 400g for 10 min to pellet and remove nuclei. The enucleated supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000g for 150 minutes using a WX80 ultracentrifuge with a T-1270 rotor (Thermo Fisher Scientific, Asheville, NC) to produce a membrane protein precipitate.

Production, immobilization and digestion of proteoliposomes: the membrane protein pellet was washed several times with Nanoxin buffer (10mM Tris, 300mM NaCl, pH 8). The membrane protein pellet was resuspended in 1.5mL of Nanoxin buffer, followed by VIBRA-CELL^TMVC505 ultrasonic processor (Sonics)&Materials, inc., Newtown, CT) were tip sonicated on ice for 20 minutes. Proteoliposomes were sized by staining with FM1-43 dye (Invitrogen, Carlsbad, CA) and visualized with a fluorescence microscope. The protein concentration of the proteoliposome suspension was determined by BCA assay (Thermo Scientific). The proteoliposomes were then injected into the LPI using a standard pipette tip^TMFlow Cell (Nanoxins AB, Gothenburg, Sweden) and allowed to immobilize for 1 hour. After immobilization, a series of washing steps are carried out and directed towards the LPI^TMFlow Cell injection trypsin 5. mu.g/mL (Princeton Separations, Adelphi, NJ). Incubate the chip overnight at 37 ℃ and remove trypsin from the LPI^TMEluted on-chip, and then desalted using Sep-Pak cartridges (Waters Corporation, Milford, Mass.).

LTQ Linear ion trap LC/MS/MS analysis Each trypsin digested sample was analyzed at 0.2mm × 150mm 3 μmMAGIC C18 column (Michrom biolesources, Inc., Auburn, CA) directly connected to an axial desolvation vacuum assisted nanocapillary level electrospray ionization (ADVANCE) source (Michrom biolesources, Inc.). A180 min gradient was used (buffer A: water, 0.1% formic acid; buffer B: acetonitrile, 0.1% formic acid). The sensitivity comparable to conventional nano-ESI can be achieved by operating the ADVANCE source at very high flow rates of 3 μ L/min. The eluted polypeptides were analyzed on a LTQ Linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, Calif.) using 10 data dependent MS/MS scans after each full scan mass spectrum. Duplicate datasets were collected for 7 analyses for each biological sample.

Bioinformatics: search 7 RAW files corresponding to 7 analysis duplicate datasets collected from each cell line as a single against IPI people databaseSearch using Sorcerer Solo^TMSEQUEST algorithm implemented by a workstation (Sage-N Research, San Jose, Calif.). Peptide mass tolerance of 1.2amu was assigned, methionine oxidation as differential correction, and urea methylation as static correction. Membrane proteomics data were classified and resolved using scaffolding software implemented by Trans-genomic Pipeline (TPP). The analysis was performed for those proteins identified as having a peptide probability of 95%, a protein probability of 95%, and being the only peptide. Membrane proteomics datasets were compared using self-developed custom Perl scripts.

As a result: amnion derived adherent cells express different angiogenic and cardiogenic markers as shown in table 4.

Table 4: amnion derived adherent cells express cardiogenic or angiogenic markers.

6.2.5 evaluation of the angiogenic potential of amnion-derived adherent cells using secretory proteome

Protein array generation 6 amnion derived adherent cells were seeded in equal numbers to in growth medium, conditioned medium was collected after 4 days, simultaneous qualitative analysis of multiple angiogenic cytokines/growth factors in cell conditioned medium was performed using RayBiotech angiogenic protein array (Norcross, GA). briefly, the protein array was incubated with 2mL 1 × blocking buffer (Ray Biotech) for 30 minutes (min) at room temperature to block the membrane, then the blocking buffer was poured out and the membrane was incubated with 1mL samples (4 days with respective cell conditioned growth medium) for 1-2 hours at room temperature, then the samples were poured out and the membrane was incubated with 2mL 1 × wash buffer i (Ray Biotech) for 3 × 5min at room temperature, then the membrane was washed with 2mL 1 × wash buffer ii Ray Biotech (2 × 5 min) at room temperature, after which 1mL of diluted biotin-Ray antibody (y Biotech) was coupled to each membrane for 1-2 hours, and added to each membrane as above, and the conditioned medium was then added to the membraneThe washing buffer was used for washing. Diluted HRP-conjugated streptavidin (2mL) was then added to each membrane and the membranes were incubated at room temperature for 2 hours. Finally, the membrane was washed again with ECL as per the instructions^TMThe detection kit (Amersham) was incubated and the results were visualized and analyzed using a Kodak Gel Logic 2200 imaging system. Various angiogenic proteins secreted by AMDAC are shown in figure 3.

ELISA: using commercially available R&Briefly, ELISA assays were performed according to manufacturer's instructions and the amount of each angiogenic growth factor in conditioned media was normalized to 1 × 10⁶A cell. Amnion derived adherent cells (n ═ 6) showed approximately 4500pg VEGF per million cells and approximately 17,200pg IL-8 per million cells.

Table 5: ELISA results for angiogenic markers

In another experiment, AMDAC was also confirmed to secrete pro-angiogenic protein factor-1, pro-angiogenic protein factor-2, PECAM-1(CD 31; platelet endothelial cell adhesion molecule), laminin and fibronectin.

6.2.6 micro RNA expression of AMDAC confirmed angiogenic Activity

This example shows that AMDACs express higher levels of certain micro-rnas (mirnas) and lower levels of certain other mirnas, which are associated with angiogenic function, respectively, than bone marrow-derived mesenchymal stem cells.

Pro-angiogenic miR-296 is known to modulate angiogenic function by modulating growth factor receptor levels. For example, miR-296 in endothelial cells contributes significantly to angiogenesis by directly targeting hepatocyte growth factor-regulated tyrosine kinase matrix (HGS) mRNA, resulting in reduced HGS levels and thus reduced HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRb. See Hurdinger et al, Cancer Cell 14:382-393 (2008). In addition, miR-15b and miR-16 have been shown to control the expression of VEGF, a pro-angiogenic factor involved in angiogenesis, and hypoxia-induced decrease of miR-15b and miR-16 results in an increase in VEGF, a pro-angiogenic cytokine. See Kuelbacher et al, Trends in pharmaceutical Sciences, 29(1):12-15 (2007).

AMDAC was prepared according to the method described in example 1 above. Using MIRVANA^TMMiRNA isolation kit (Ambion, Cat #1560) preparation of microRNA (miRNA) using AMDAC and BM-MSC cells (for comparison) 0.5 × 10⁶-1.5×10⁶Cells were disrupted in denaturing lysis buffer. The sample is then extracted with acid-phenol + chloroform to isolate RNA that is highly enriched for small RNA molecules. 100% ethanol was added so that the sample contained 25% ethanol. After the lysate/ethanol mixture is passed through a glass fiber filter, the large RNA is immobilized and the small RNA molecules are collected in the filtrate. The ethanol concentration of the filtrate was then increased to 55% and the mixture was passed through a second glass fiber filter, where the small RNA molecules were immobilized. The RNA was washed and eluted using a low ionic strength solution. The concentration and purity of the recovered small RNA were determined by measuring its absorbance at 260 and 280 nm.

AMDAC was found to be able to express the following angiogenic mirnas: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, (members of angiogenesis miRNA cluster 17-92), miR-296, miR-221, miR-222, miR-15b and miR-16. AMDAC was also found to express higher levels than bone marrow-derived mesenchymal stem cells (BM-MSCs) of the following angiogenic mirnas: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92 (a member of angiogenesis miRNA cluster 17-92), miR-296. These results are in good agreement with the observed high level of AMDAC expression of VEGFR2/KDR (see above). In contrast, AMDAC was found to express lower levels than BM-MSC of the following angiogenic mirnas: miR-20a, miR-20b, (members of angiogenesis miRNA cluster 17-92), miR-221, miR-222, miR-15b and miR-16. The decreased expression of miR-15b and miR-16 is consistent with the observed higher level of VEGF expression in AMDAC.

6.3 example 3: functional characterization of amnion-derived adherent cells

This example shows the different properties of AMDACs related to angiogenic and differentiative capabilities.

6.3.1 HUVEC tube lumen formation assessment of angiogenic potential of amnion derived adherent cells

Human Umbilical Vein Endothelial Cells (HUVECs) were sub-cultured in EGM-2 medium (Cambrex, East Rutherford, NJ) for 3 days at passage 3 or less and harvested at a confluence of approximately 70% -80%. HUVEC were washed once with minimal medium/antibiotic (DMEM/F12(Gibco)) and resuspended in the same medium at the desired concentration. Used within 1 hour of HUVEC preparation. Human Placental Collagen (HPC) was made up to a concentration of 1.5mg/mL with 10mM HCl (pH 2.25), "neutralized" with buffer to pH 7.2 and placed on ice until use. HPC and HUVEC suspensions were mixed to a final cell concentration of 4000 cells/. mu.l. The resulting HUVEC/HPC suspension was immediately pipetted into a 96-well plate, 3 μ l per well (plates were pre-filled with sterile PBS around to prevent evaporation, n ═ 5 under each condition). HUVEC droplets were incubated at 37 ℃ and 5% CO without medium addition₂75-90 minutes to allow collagen to polymerize. After the "dry" incubation was complete, wells were gently filled with 200 μ l conditioned AMDAC medium (n-5 cell line) or control medium (e.g., DMEM/F12 as negative control, EGM-2 as positive control) and incubated at 37 ℃ and 5% CO₂Incubate for 20 hours. Preparing a conditioned medium by incubating amnion-derived adherent cells of generation 6 in a growth medium for 4-6 hours; after adherence and spreading, the medium was replaced with DMEM/F12 for 24 hours. After incubation, the medium was removed from the wells without disturbing the HUVEC droplets, and the wells were washed once with PBS. The HUVEC droplets were then fixed for 10 seconds and stained using a Diff-Quik cell staining kit for 1 minute, followed by 3 washes with sterile water. The stained droplets were air dried and the wells were harvested using a Zeiss SteReo Discovery V8 microscopeAnd (4) an image. The images were then analyzed using the computer software package "ImageJ" and/or MatLab. The image is converted into 8-bit gray scale image from color, and the image is converted into black and white image by setting a critical value. The image is then analyzed using the particle analysis features, which provide pixel density data including counts (number of individual particles), total area, average size (of individual particles), and area fraction, which is equivalent to the number of endothelial lumen formations in the analysis.

Conditioned medium had an angiogenic effect on endothelial cells as shown by the formation of a lumen inducing proliferation (see figure 4).

6.3.2 HUVEC migration assay

HUVEC were tested by incubating "wounded" cells with 5 amnion-derived adherent cell lines obtained for 3 days of growth in serum-free conditioned medium (EBM 2; Cambrex). The cell-free EBM2 medium was used as a control.15 hours later, cell records (n ═ 3) migrated to acellular regions were recorded using an inverted microscope, and then images were analyzed using computer software packages "ImageJ" and/or MatLab @. the images were converted from color to 8-bit gray scale images and set up a threshold to black and white images.The images were then analyzed using particle analysis features that provide pixel density data including counts (number of individual particles), total area, (average size of individual particles) and fraction of injured cells, which is equivalent to the extent of migration of endothelial cells recorded in the assay, and the results were recorded as 1 × 10 relative to the initial migration of endothelial cell counts, and the results were recorded as × relative to the initial migration of endothelial cell counts⁶A cell.

Trophic factors secreted by amnion-derived adherent cells have angiogenic effects on endothelial cells, as shown by the induction of cell migration (figure 5).

In a separate experiment, HUVEC were grown to half-full on FN-coated 96-well platesInduced proliferation was examined by incubating the cells with serum-free conditioned medium from 5 respective amnion derived adherent cell lines (EBM-2 medium, 3 days). EBM-2 medium was used as a negative control and EGM-2 as a positive control. After 48 hours, by using Promega cell TiterEvaluation of DNA content by AZ One Solution Cell Proliferation Assay (Promega, Madison, Wis.) Cell Proliferation was scored for Cell Proliferation. error bars represent standard errors for analysis of replicate-parallel (n-3), and results were normalized to 1 × 10⁶A cell.

The trophic factors secreted by amnion-derived adherent cells resulted in an increase in DNA concentration, which is indicative of the proliferation of HUVECs. See FIG. 6, where "CM" is conditioned media.

6.3.3 evaluation of angiogenic potential of amnion-derived adherent cells Using the uptake of acetylated Low Density lipoprotein (AcLDL)

Cultured endothelial cells and microglia can be identified by their ability to take up fluorescent AcLDL. If the lysine residues of the LDL apolipoprotein are acetylated, the LDL complex no longer binds to the LDL receptor, but instead it can be taken up by endothelial cells and macrophages in a highly cell-specific manner.

Amnion derived adherent cells were grown either in growth medium without VEGF addition or in EGM2-mv (cambrex) with VEGF addition to evaluate the general angiogenic capacity of amnion derived adherent cells and the effect of VEGF on the differentiation potential of amnion derived adherent cells. Cells were cultured in the respective media on 12-well plates for 4-7 days until 70-80% confluence was reached, then incubated overnight with 10 μ g/mL acetylated ldl (invitrogen). Cells were then counterstained with calcein am (invitrogen) and their acetylated LDL uptake was evaluated using a fluorescence microscope. HUVEC as acetylated LDL uptake control cells were grown in EGM2-MV and analyzed as described above. Amnion derived adherent cells exhibit minimal uptake of acetylated LDL under normal growth conditions but induce/differentiate to an enhanced uptake capacity upon stimulation with VEGF. See fig. 7.

6.3.4 evaluation of angiogenic potential of amnion-derived adherent cells using luminal formation

Amnion derived adherent cells were grown either in growth medium without VEGF addition or in EGM2-MV with VEGF addition to evaluate the general angiogenic capacity of amnion derived adherent cells and the effect of VEGF on the differentiation potential of amnion derived adherent cells. HUVECs were grown in EGM2-MV as lumen-forming control cells. Cells were cultured in respective media for 4-7 days until 70-80% confluency was achieved. Cold (4 ℃) MATRIGEL^TMThe solution (50. mu.L; BD Biosciences) was dispersed into the wells of a 12-well plate, and the plate was incubated at 37 ℃ for 60min to allow the solution to gel. AMDAC and HUVEC cells were trypsinized, resuspended in appropriate media (with or without VEGF), and 100. mu.L of diluted cells (1-3X 10)⁴Cells) to each containing MATRIGEL^TMIn the hole of (a). In polymerized MATRIGEL^TMThe cells above, in the presence or absence of 0.5-100ng VEGF, were placed at 37 ℃ in 5% CO₂The incubator lasts for 4-24 hours. After incubation, the cells were evaluated for signs of luminal formation using standard light microscopy.

Amnion derived adherent cells exhibit minimal luminal formation in the absence of VEGF, but will induce/differentiate to form tube-like structures upon stimulation by VEGF. See fig. 8.

6.3.5 evaluation of angiogenic Capacity of amnion derived adherent cells Using hypoxia response

To assess the angiogenic function of endothelial cells and/or endothelial progenitor cells, the ability of the cells to secrete angiogenic growth factors under hypoxic and normoxic conditions can be assessed. Culturing under hypoxic conditions typically induces increased secretion of angiogenic growth factors by endothelial cells or endothelial progenitor cells, which can be detected in conditioned medium. The amnion derived adherent cells were added in the same amount as in the standard mediumSeeded and grown to approximately 70-80% confluence. The cells were then switched to serum-free medium (EBM-2) and incubated in normoxia (21% O)₂) Or hypoxia (1% O)₂) Incubate under conditions for 48 hours. The conditioned medium was collected and used with commercially available R&ELISA assays were performed according to the manufacturer's instructions and the amount of each angiogenic growth factor (VEGF and IL-8) in conditioned media was normalized to 1 × 10⁶A cell.

Amnion derived adherent cells exhibit increased secretion of angiogenic growth factors under hypoxic conditions. See fig. 9.

In a separate experiment, AMDACs were inoculated in equal amounts to their standard media and grown to approximately 70-80% confluence. The cells were then switched to serum-free medium (EBM-2) and incubated in normoxia (21% O)₂) Or hypoxia (1% O)₂) Incubate under conditions for 48 hours. Flow cytometric analysis of cells was performed for their cell marker CD202b (also known as Tie2, Tek or pro-angiogenic protein factor-1 receptor), a receptor involved in vascular development and angiogenesis. The conditioned medium was collected and used with commercially available R&The ELISA kit of D SYSTEMS analyzes the secretion of angiogenic growth factors flow cytometry analysis was performed as described above, ELISA analysis was performed according to the manufacturer's instructions, the amount of each angiogenic growth factor (VEGF and IL-8) in conditioned media was normalized to 1 × 10⁶A cell. AMDAC exhibits increased expression of CD202b under hypoxic conditions as compared to normoxic conditions. See fig. 10.

6.3.6 evaluation of angiogenic Capacity of amnion derived adherent cells Using cardiogenic differentiation

To induce differentiation of progenitor cells into cardiomyocyte lineage, a combination of hanging drop culture (HD, stopping cell proliferation and starting differentiation process) followed by treatment of growth-restricted cells with specific factors was performed at several stages. After hanging drop culture, activin A, bone morphogenetic protein 4(BMP4), and alkaline fibroblast were usedCombinations of cell growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA), and dickkopf homolog 1(DKK1) induced cells for 16 days. Briefly, amnion derived adherent cells were grown to approximately 70% confluence in standard growth media. The cells were then trypsinized and washed with the buffer described previously. Mu.l of a drop containing 700 cells were suspended in the relevant medium and placed inside the lid of a 100mm Petri dish using a multichannel pipettor. Carefully invert the lid and place it on top of the dish with 25mL of sterile PBS to prevent the drops from drying. Hanging drop cultures 5% CO at 37 ℃₂The incubator (2) was incubated for 48 h. The aggregated cells were then re-seeded to 0.1% gelatin-coated plates containing cell line-specific differentiation media for further induction.

The stimulating steps are as follows: stage 1, 4 days BMP4(0.5 ng/mL); stage 2, 5 days BMP4(10ng/mL), bFGF (5ng/mL), activin A (3 ng/mL); stage 3, 3 days VEGF (10ng/mL), DKK1(150 ng/mL); stage 4, 4 days VEGF (10ng/mL), DKK1(150ng/mL), bFGF (5g/mL) (+/-5-10nM 5-aza-cytidine). Total RNA from the treated cells was then prepared and analyzed for cardiogenic markers by qRT-PCR as previously described.

The results show that amnion derived adherent cells can be induced/differentiated to express different cardiomyocyte markers. See fig. 11.

6.3.7 HUVEC response of AMDAC-conditioned Medium

AMDAC was prepared in a medium containing 60% DMEM-LG (Gibco), 40% MCBD-201(Sigma), 2% FBS (HycleLabs), 1 × insulin-transferrin-selenium supplement (ITS), 10ng/mL linoleic acid-bovine serum albumin (LA-BSA), 1 n-dexamethasone (Sigma), 100. mu.M ascorbic acid 2-phosphate (Sigma), 10ng/mL epidermal growth factor (R)&System D); and 10ng/mL platelet-derived growth factor (PDGF-BB) (R&D system) for 48 hours, and then for another 48 hours in serum-free medium. Conditioned media from AMDAC cultures were collected and used to stimulate serum-starved HUVECs 5, 15, and 30 minutes. Then cracking HUVEC andusing BD^TMCba (cytometric Bead assay) Cell Signaling Flex kit (BD Biosciences) stains phosphoprotein, which is known to play a role in angiogenic pathway Signaling. AMDAC has been found to be a potent activator of the cell proliferation pathway of AKT-1 (which inhibits the apoptotic process), AKT-2 (which is an important signaling protein for the insulin signaling pathway), and ERK 1/2 of HUVEC. These results further demonstrate the angiogenic capacity of AMDACs.

6.4 example 4: AMDAC-induced angiogenesis

This example shows proangiogenesis of AMDACs in an in vivo assay using chicken chorioallantoic membrane (CAM).

Two separate sets of CAM analyses were performed. In the first set of CAM analyses, whole cell pellets from different AMDACs preparations were evaluated. In a second set of CAM analyses, supernatants from different AMDACs preparations were evaluated. Fibroblast growth factor (bFGF) was used as a positive control and MDA-MB-231 human breast cancer cells as a reference (negative control). The end point of the study was to determine the vascular density of all treatment and control groups.

6.4.1 CAM analysis Using cells

3 batches of AMDAC cell preparations, herein designated Lot 1, Lot 2 and Lot 3, prepared and cryopreserved according to the above method were used. AMDACs were thawed for administration and the number of cells used for CAM administration was determined.

Research and design: the study included 7 groups of 10 embryos each. The design of this study is described in table 6.

Table 6: study group, chicken chorioallantoic membrane angiogenesis assay.

CAM assay methods: fresh fertilized eggs were incubated for 3 days at 37 ℃ in a standard egg incubator. On day 3, eggs were broken under aseptic conditions and embryos placed in 20 10Culturing in a 0mm plastic plate at 37 deg.C in an embryo incubator, storing water on the bottom frame, continuously introducing air into the stored water using a small pump so that the incubator humidity is maintained constant, on day 6, placing a sterile "O" shaped silica gel ring at each CAM, and aseptically placing AMDAC at a density of 7.69 × 10⁵Cells/40. mu.L Medium/MATRIGEL^TMThe mixture (1:1) was transferred to each "O" ring. Tables 2A and 2B show the number of cells used and the amount of medium added to each cell preparation for administration. Vector control embryos 40. mu.L of vector (PBS/MATRIGEL) was added to the embryos^TM1:1), adding 40 μ L DMEM medium/MATRIGEL to the positive control^TM100ng/ml bFGF in the mixture (1:1), and media controls with only 40. mu.L DMEM media added, the embryos returned to each incubator after each administration was completed, the embryos were removed from the incubator and placed at room temperature on day 8, and the vascular density of each "O" ring was determined using an image capture system with magnification of 100 ×.

The number of vessels at the CAM treatment location is shown by detecting the density of the resulting vessels using a scoring system with positive real numbers of 0-5, or exponential vessels of 1-32. A higher score indicates higher vascular density and 0 indicates no angiogenesis. The percent inhibition for each application site was calculated using the score recorded for that site/average score obtained for the control sample in each individual experiment. The percent inhibition for each administration of a given compound was calculated by combining all results from 8-10 embryo administrations.

Table 7: the amount of medium added to each cell preparation in order to normalize the cell suspension ultimately administered

All cells used were at passage 6.

Results

The results of the vessel density score values are shown in FIG. 12. The results clearly show that the vascular density score of chicken chorioallantoic membranes treated with stem cell suspensions, or 100ng/mL bFGF, or MDAMB231 breast cancer cell suspensions, respectively, was statistically significantly higher than the CAM of vehicle control group (P <0.001, T-test). The medium used to culture the stem cells had no effect on the vascular density. Furthermore, the AMDAC preparation induced vascular density showed some variation, but these were not statistically significant. This indicates that the respective inducibility of the 5 stem cell preparations was essentially the same.

6.4.2 CAM analysis Using AMDAC cell supernatants

Supernatant samples from MDA-MB-231 cells and different stem cell preparations each described in the CAM assay of AMDAC above were used for the second CAM assay. Like the CAM assay of AMDAC, bFGF and MDA-MB-231 cells were used as positive controls.

Research and design: the study included 7 groups of 10 embryos each. The design of this study is described in table 8.

Table 8: study design-CAM analysis Using cell supernatants

The AMDAC cells used were passage 6.

CAM assay methods: the assay method is the same as the CAM assay of the AMDACs described above. The only difference was that supernatants from each stem cell preparation or from MDA-MB-231 cells were used as test material. For the dosage, each supernatant was mixed with MATRIGEL^TMMixing was performed (at a 1:1 volume ratio) and 40. mu.L of the mixture was applied to each embryo.

Results

The vascular density score (see figure 13) shows that the induction of vascularization varies from one stem cell preparation supernatant to another. Samples of AMDAC supernatant from three batches showed significant effects on vascular induction, P <0.01, P <0.001 and P <0.02 (T-test), respectively. As expected, the positive control bFGF also showed strong induction of angiogenesis as shown in CAM assay No. 1(P <0.001, T-test) above. However, MDA-MB-231 human breast cancer cell supernatant showed no significant induction of angiogenesis relative to the vector control group. As indicated above, the culture was essentially without any effect.

6.5 example 5: AMDAC exhibits neuroprotective effects

This example, using oxygen-glucose deprivation (OGD) injury (intult) analysis, shows that AMDAC has neuroprotective effects under hypoxic and low glucose conditions and reduces reactive oxygen species. These results indicate that AMDACs are effective for treating ischemic conditions such as stroke or peripheral vascular disease and can protect reperfusion injury caused by ischemic symptoms.

Human neurons (ScienCell, catalog number 1520) were cultured according to the manufacturer's recommendations. Briefly, the culture vessel was coated with poly-L-lysine (2. mu.g/mL) in sterile distilled water at 37 ℃ for 1 hour. The vessel was washed 3 times with double distilled water. Neuronal medium (ScienCell) was added to the vessel and equilibrated to 37 ℃ in an incubator. The neurons were thawed and added directly to the vessel without centrifugation. In the subsequent culture, the medium was changed on the day after the initial culture, and then every other day. Neurons can typically be analyzed for damage at day 4.

OGD medium (Dulbecco's modified Eagle Medium-without glucose) was prepared by first heating the medium in a water bath to reduce the oxygen solubility in the liquid medium to some extent. 100% nitrogen was bubbled through the medium for 30 minutes using 0.5 μm diffusing stone to remove dissolved oxygen. HEPES buffer was added to a final concentration of 1 mM. At the end of the sparging, the medium was added directly to the neurons. A small sample of the medium was taken and oxygen levels were confirmed using a sink-type oxygen sensor. The oxygen level is typically reduced to 0.9% to about 5.0% oxygen.

The anoxic tank is prepared by placing the tank in an incubator at 37 ℃ for at least 4 hours (preferably overnight) before aeration. RemovingThe medium in the culture vessel was replaced with degassed medium and the culture vessel was placed in an anoxic tank. Passing 95% N through the system in an anoxic tank at a rate of 20-25Lpm₂/5％CO₂The gas is allowed to flow for at least 5 minutes. The system was incubated in an incubator at 37 ℃ for 4 hours and after 1 hour the chamber was again degassed.

At the end of the injury procedure, OGD medium is aspirated and warmed medium is added to the neurons. After 24-28 hours, AMDACs and neurons were seeded in 6-well plates in equal numbers of 100,000 cells per well, suspended in neuronal medium, which was added to neurons and co-cultured for 6 days.

Micrographs were taken of random areas of the 6-well plate under each condition. Cells with typical neuronal morphology were identified and neurite length was recorded. The average length of neurites was positively correlated with neuronal health, which was longer in cocultures of neurons and AMDACs, indicating that AMDACs protected cells from damage.

Active oxygen analysis

AMDAC was determined to express superoxide dismutase, catalase, and heme oxygenase genes under hypoxic conditions. The ability of AMDACs to take up reactive oxygen species and protect cells from such conditions was determined in an assay using hydrogen peroxide as the active oxygen generator.

Description of the analysis: target cells (astrocytes, science cell Research Laboratories) were seeded into 96-well black-well plates using 6000/cm²poly-L-lysine was pre-coated. Astrocytes were plated overnight at 37 ℃ and 5% carbon dioxide in growth medium. The following day, the medium was removed and the cells were incubated with the cell-penetrating dye DCFH-DA (dichlorofluorescein diester), which is a fluorescent probe. Excess dye was removed by washing with Dulbecco's phosphate buffer solution or Hank's buffered saline solution. Cells were then damaged by addition of 1000 μ M hydrogen peroxide for 30-60 minutes. The hydrogen peroxide-containing medium was then removed and replaced with serum-free, glucose-free growth medium. AMDAC (cells of Lot 1 or Lot 2)) Or BM-MSC to 6000/cm²And the cells were cultured for another 24 hours. The cells were then read using a standard fluorescence plate reader at 480Ex and 530 Em. The active oxygen content of the medium is directly proportional to the level of DCFH-DA in the cytosol. The active oxygen content is determined by comparison with a predetermined standard curve of DCF. N-24 in all experiments.

For analysis, 1 × DCFH-DA was prepared prior to use, in which 20 × DCFH-DA stock solution was diluted to 1 × in cell culture medium without fetal bovine serum and stirred well₂0₂) Dilutions were prepared in DMEM or DPBS as needed. A standard curve was prepared as follows: 100. mu.L of standard DCF was transferred to a 96-well plate suitable for fluorescence detection by diluting 1mM standard DCF with cell culture medium, performing serial dilutions at 1:10 in the concentration range of 0. mu.M to 10. mu.M, and adding 100. mu.L of cell lysis buffer. Fluorescence was read at 480Ex and 530 Em.

As a result: both batches of AMDACs used significantly reduced the concentration of active oxygen in the astrocyte co-culture. See fig. 14A and 14B. In contrast, BM-MSC did not significantly reduce the concentration of reactive oxygen species in astrocyte co-cultures.

6.6 methods of treatment Using amnion derived adherent cells

6.6.1 treatment of myocardial infarction

An adult male in the middle age 50 shows sustained chest pain over 20 minutes and radiates to the left arm, shortness of breath, nausea, palpitation, sweating, etc. from the electrocardiogram results and the rise and fall of the creatine kinase concentration in the blood, it was differentially diagnosed as (transmural) myocardial infarction of the anterior wall of the heart, after the individual was stabilized with nitroglycerin and thrombolysin, the individual was given a local anesthetic heart injection of 1 × 10 in 0.9% saline directly⁸To 5 × 10⁸To the lesion area. The individual was monitored on an emergency basis for the next 72 hours. The individual is further monitored by electrocardiographic and/or dye visualization techniques within the next 3 months after treatment to assess the extent of revascularization in the infarcted area. If the electrocardiogram effect is better than the effect of the application of AMDACThe treatment effect was confirmed before, significantly closer to normal, or if angiogenesis was visibly evident in the infarcted area.

6.6.2 treatment of cardiomyopathy

The individual exhibited shortness of breath, swelling of the legs and ankles, and irregular heartbeats, excluding other causes, and was diagnosed with cardiomyopathy according to electrocardiographic confirmation, ultrasonography confirmed that the individual had congestive cardiomyopathy, and the individual was administered 1 × 10 in 0.9% saline directly using local anesthetic cardiac injection⁸To 5 × 10⁸AMDAC to cardiac pulse. The subjects were monitored for changes in their sonograms over the next 3 months for evidence of more normal blood flow, improved sensation of shortness of breath and reduced swelling of the legs and ankles. If any of these individual signs during the period of care show improvement, the effect of the treatment is confirmed.

6.6.3 treating peripheral vascular disease

The subject exhibited cold tingling in the feet and redness when suspended, as well as pain, weakness and fatigue in the legs, after diabetes was excluded, peripheral artery disease was diagnosed, the subject was intravenously administered 1 × 10mL in 0.9% saline 450⁹To 5 × 10⁹And two weeks of monitoring during the next 3 months. If any of the above symptoms show improvement during the period of care, the therapeutic effect is confirmed.

6.6.4 treating peripheral vascular disease

The subject exhibited cold tingling in the feet and redness when suspended, as well as pain, weakness and fatigue in the legs, after diabetes was excluded, peripheral artery disease was diagnosed, the subject was intravenously administered 1 × 10mL in 0.9% saline⁸To 5 × 10⁸And/or intravenous or intra-arterial administration of a significant amount locally between toes and bi-weekly monitoring over the next 3 months. If any of the above symptoms show improvement during the period of care, the therapeutic effect is confirmed.

6.6.5 combination for treating peripheral vascular disease

The subject exhibited cold tingling in the feet and redness when suspended, as well as pain, weakness and fatigue in the legs, after diabetes was excluded, peripheral artery disease was diagnosed, the subject was intravenously administered 1 × 10mL in 0.9% saline 450⁹To 5 × 10⁹And two weeks of monitoring during the next 3 months. Cilostazol is also prescribed to the individual at 100mg twice daily. If any of the above symptoms show improvement during the period of care, the therapeutic effect is confirmed.

6.6.6 combination therapy for peripheral vascular disease

The individual exhibited cold tingling in the feet and redness when suspended, and pain, weakness and fatigue in the right leg, after diabetes was excluded, peripheral artery disease was diagnosed, the individual was angioplasty and stenting was surgically placed in the femoral artery, and the individual was then intravenously administered 1 × 10mL in 0.9% saline 450⁹To 5 × 10⁹And two weeks of monitoring during the next 3 months. If any of the above symptoms show improvement during the period of care, the therapeutic effect is confirmed.

6.6.7 use of AMDAC for treating apoplexy

A52 year old male presented with left side physical hemiplegia and partial aphasia, diagnosed with ischemic stroke, after determination of ischemic area using magnetic resonance imaging, the individual was prepared for surgery, and after opening of the injured side skull, 1-2mL of 5 × 10 in 0.9% saline solution⁷To 1 × 10⁸AMDACs are administered to ischemic areas. The individuals were monitored for the next 7-14 days to see if there was any sign of improvement in stroke, particularly in any symptoms such as hemiplegia or aphasia. If any of the above symptoms show improvement during the period of care, the therapeutic effect is confirmed.

6.6.8 use of AMDAC for treating apoplexy

A52 year old male presented with left side physical hemiplegia and partial aphasia. Diagnosing ischemic stroke. After the ischemic region is determined using magnetic resonance imaging, the individual is prepared for surgery, at the injured lateral craniumBone opening after opening, 1 × 10 in 450mL of 0.9% saline solution was administered intravenously⁹To 5 × 10⁹An AMDAC. The individuals were monitored for the next 7-14 days to see if there was any sign of improvement in stroke, particularly in any symptoms such as hemiplegia or aphasia. If any of the above symptoms show improvement during the period of care, the therapeutic effect is confirmed.

And (3) equivalence:

the present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are also intended to fall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entirety.

Claims (38)

1. An isolated amnion derived adherent cell, wherein said cell is attached to a tissue culture plastic, and wherein said cell is OCT-4 as determined by RT-PCR^–(octamer binding protein 4).

2. The isolated cell of claim 1, wherein the cell is HLA-G as determined by RT-PCR^–。

3. The isolated cell of claim 1, wherein said cellTranscytosis flow cytometry also CD49f⁺。

4. The isolated cell of claim 3, wherein the cell is OCT-4^–、HLA-G^–And CD49f⁺。

5. The isolated cell of claim 1, wherein the cell is CD90 as determined by flow cytometry⁺、CD105⁺Or CD117^–。

6. The isolated cell of claim 5, wherein said cell is CD90⁺、CD105⁺And CD117^–。

7. The isolated cell of claim 6, wherein said cell is OCT-4 as determined by RT-PCR^–And HLA-G^–And is CD49f as determined by flow cytometry⁺、CD90⁺、CD105⁺And CD117^–。

8. The isolated cell of claim 1, wherein said cell is VEGFR1/Flt-1 as determined by immunolocalization⁺(vascular endothelial growth factor receptor 1) and VEGFR2/KDR⁺(vascular endothelial growth factor receptor 2).

9. The isolated cell of claim 1, wherein the cell has CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺(angiogenin receptor), TEM-7⁺(tumor endothelial cell marker 7), CD31^–、CD34^–、CD45^–、CD133^–、CD143^–(angiotensin I converting enzyme, ACE), CD146^–(melanoma cell adhesion molecule) or CXCR4^–(chemokine (C-X-C motif) receptor 4).

10. The isolated cell of claim 1, wherein the cell is CD9 as determined by immunolocalization⁺、CD10⁺、CD44⁺、CD54⁺、CD98⁺、Tie-2⁺(angiogenin receptor), TEM-7⁺(tumor endothelial cell marker 7), CD31^–、CD34^–、CD45^–、CD133^–、CD143^–、CD146^–And CXCR4^–。

11. The isolated cell of claim 1, wherein said cell is VE-cadherin as determined by immunolocalization^–。

12. The isolated cell of claim 1, wherein said cell is further CD105 as determined by immunolocalization⁺And CD200⁺And (4) positive.

13. The isolated cell of claim 1, wherein the cell does not express CD34 in an immunolocalization assay after 7 days of exposure to 50 ng/mlegf.

14. An isolated cell population comprising the cell of claim 1.

15. The isolated population of cells of claim 14, wherein at least 50% of the cells in said population are the cells of claim 1.

16. The isolated population of cells of claim 14, wherein at least 90% of the cells in said population are the cells of claim 1.

17. An isolated population of cells comprising the cell of claim 7.

18. The isolated population of cells of claim 17, wherein at least 50% of the cells in said population are the cells of claim 7.

19. The isolated population of cells of claim 11, wherein at least 90% of the cells in said population are the cells of claim 7.

20. An isolated population of cells comprising the cell of claim 4, wherein the isolated population of cells is not amniotic membrane.

21. The isolated population of cells of claim 14, wherein at least 50% of the cells in said population are the cells of claim 4.

22. The isolated population of cells of claim 14, wherein at least 90% of the cells in said population are the cells of claim 4.

23. A composition comprising the isolated amnion derived adherent cells of claim 1 or 7.

24. The isolated population of cells of claim 14, wherein said second cell type is an embryonic stem cell, a blood cell, a stem cell isolated from peripheral blood, a stem cell isolated from placental perfusate, a stem cell isolated from placental tissue, a stem cell isolated from umbilical cord blood, an umbilical cord stem cell, a bone marrow-derived mesenchymal cell, a hematopoietic stem cell, a somatic stem cell, a chondrocyte, a fibroblast, a muscle cell, an endothelial cell, an angioblast, an endothelial progenitor cell, a pericyte, a cardiomyocyte, a muscle cell, a cardioblast, a myoblast, an embryonic stem cell, or a cell processed to resemble an embryonic stem cell.

25. The isolated population of cells of claim 24, wherein said second cell type comprises at least 10% of the cells in said population.

26. The isolated population of cells of claim 24, wherein said second cell type comprises at least 25% of the cells in said population.

27. The isolated population of cells of claim 24, wherein said second cell type is a hematopoietic stem or progenitor cell.

28. The isolated population of cells of claim 27, wherein said hematopoietic stem or progenitor cells are CD34⁺A cell.

29. An isolated amnion derived adherent cell, wherein said cell is attached to a tissue culture plastic, and wherein said cell is OCT-4 as determined by RT-PCR^–And is CD49f as determined by immunolocalization⁺、HLA-G^–、CD90⁺、CD105⁺And CD117^–And wherein the cell:

(a) (ii) expresses one or more of CD9, CD10, CD44, CD54, CD98, CD200, Tie-2, TEM-7, VEGFR1/Flt-1, or VEGFR2/KDR (CD309) as determined by immunolocalization;

(b) (ii) does not express CD31, CD34, CD38, CD45, CD133, CD143, CD144, CD146, CD271, CXCR4, HLA-G, or VE-cadherin as determined by immunolocalization, or does not express SOX2 as determined by RT-PCR;

(c) the following mRNAs were expressed: ACTA, ADAMTS, AMAT, ANG, ANGPT, ANGPTL, BAI, CD200, CEACAM, CHGA, COL15A, COL18A, COL4A, CSF, CTGF, CXCL, DNMT3, ECGF, EDG, EDIL, ENPP, EPHB, FBLN, F, FGF, FIGF, FLT, FN, FST, FOXC, GRN, HGF, HEY, HSPG, IFNB, IL12, ITGA, ITGAV, ITGB, MDK, MMP, MYOZ, NRP, PDGFB, PDGFRA, PDGFRB, PECAM, PF, PGK, PROX, PTN, SEMA3, SERPINB, SERPINI, SERPINF, TIMB, TIMP, TGTGTGTGTGTF, TGTF, TNFSF, TNFSK, TFSC/or VEGF/VEGFR;

(d) expressing one or more of the following proteins: CD49d, connexin-43, HLA-ABC, β 2-microglobulin, CD349, CD318, PDL1, CD106, galectin-1, ADAM17, angiotensinogen precursor, filamin A, α -actinin 1, megalin, macrophage acetylated LDL receptors I and II, activin receptor type IIB precursor, Wnt-9 protein, glial fibrillary acidic protein, astrocytes, myosin-binding protein C or myosin heavy chain, non-muscle type A;

(e) secreting VEGF, HGF, IL-8, MCP-3, FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2, uPAR or galectin-1 into the medium of the cell growth;

(f) the following micrornas were expressed: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92 or miR-296, and the expression level is higher than that of the mesenchymal stem cells with equivalent quantity;

(g) the following micrornas were expressed: miR-20a, miR-20b, miR-221, miR-222, miR-15b or miR-16, the expression level is lower than that of the mesenchymal stem cells of equivalent bone marrow source;

(h) the following mirnas were expressed: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, miR-296, miR-221, miR-222, miR-15b or miR-16; or

(i) When less than about 5% O₂At the time of culture, with 21% O₂Is increased as compared to expression of CD202b, IL-8 or VEGF in CD202b, IL-8 or VEGF expressed therein.

30. The isolated amnion derived adherent cell of claim 29, wherein the cell is OCT-4 as determined by RT-PCR^–And CD49f as determined by immunolocalization⁺、HLA-G^–、CD90⁺、CD105⁺And CD117^–And wherein the cell:

(a) expression of CD9, CD10, CD44, CD54, CD98, CD200, Tie-2, TEM-7, VEGFR1/Flt-1 and/or VEGFR2/KDR (CD309) as determined by immunolocalization;

(b) (ii) does not express CD31, CD34, CD38, CD45, CD133, CD143, CD144, CD146, CD271, CXCR4, HLA-G, and/or VE-cadherin as determined by immunolocalization, and/or does not express SOX2 as determined by RT-PCR;

(c) the following mRNAs were expressed: ACTA, ADAMTS, AMAT, ANG, ANGPT, ANGPTL, BAI, CD200, CEACAM, CHGA, COL15A, COL18A, COL4A, CSF, CTGF, CXCL, DNMT3, ECGF, EDG, EDIL, ENPP, EPHB, FBLN, F, FGF, FIGF, FLT, FN, FST, FOXC, GRN, HGF, HEY, HSPG, IFNB, IL12, ITGA, ITGAV, ITGB, MDK, MMP, MYOZ, NRP, PDGFB, PDGFRA, PDGFRB, PECAM, PF, PGK, PROX, PTN, SEMA3, SERPINB, SERPINI, SERPINF, TIMB, TIMP, TGTGTGTGTGTF, TGTF, TNFSF, TNFSK, TFSC/or VEGF/KDF;

(d) expressing one or more of CD49d, connexin-43, HLA-ABC, β 2-microglobulin, CD349, CD318, PDL1, CD106, galectin-1, ADAM17, pro-angiotensin precursor, filamin a, α -actinin 1, megalin, macrophage acetylated LDL receptors I and II, activin receptor type IIB precursor, Wnt-9 protein, glial fibrillary acidic protein, astrocytes, myosin-binding protein C and/or myosin heavy chain, non-muscle type a;

(e) secreting one or more of VEGF, HGF, IL-8, MCP-3, FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB, TIMP-2, uPAR and galectin-1 into the medium of cell growth;

(f) the following micrornas were expressed: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92 and/or miR-296, the expression level is higher than that of the mesenchymal stem cells with equivalent quantity;

(g) the following micrornas were expressed: miR-20a, miR-20b, miR-221, miR-222, miR-15b and/or miR-16, the expression level is lower than that of the mesenchymal stem cells with equivalent quantity; and

(h) the following mirnas were expressed: miR-17-3p, miR-18a, miR-18b, miR-19b, miR-92, miR-20a, miR-20b, miR-296, miR-221, miR-222, miR-15b and/or miR-16; and/or

(i) When less than about 5% O₂At the time of culture, with 21% O₂Expresses increased levels of CD202b, IL-8 or VEGF as compared to CD202b, IL-8 or VEGF expressed therein.

31. The isolated population of cells of claim 29.

32. The isolated population of cells of claim 30.

33. A permanent or degradable decellularized or synthetic matrix or scaffold comprising the cell of any one of claims 1, 2, 4,7 or 9.

34. The substrate or scaffold of claim 33, wherein the substrate or scaffold is an amniotic membrane; dehydrating the extracellular matrix; placental collagen, or placental extracellular membrane.

35. A method of treating an individual having a disease or disorder of the circulatory system, comprising administering to the individual the population of cells of claim 1 or claim 7 in an amount and for a time sufficient to substantially ameliorate one or more symptoms of the disease or disorder.

36. A method of treating an individual having a disease or disorder of the circulatory system, comprising administering to the individual the population of cells of claim 1 or claim 7 in an amount and for a time sufficient to significantly improve one or more cardiac function indicators, wherein the cardiac function indicators are thoracic Cardiac Output (CO), Cardiac Index (CI), Pulmonary Artery Wedge Pressure (PAWP), Cardiac Index (CI),% fractional shortening (% FS), Ejection Fraction (EF), Left Ventricular Ejection Fraction (LVEF); left Ventricular End Diastolic Diameter (LVEDD), Left Ventricular End Systolic Diameter (LVESD), contractility (dP/dt), decreased atrial or ventricular function, increased pump efficiency, decreased rate of pump efficiency loss, decreased hemodynamic function loss, or decreased complications associated with cardiomyopathy.

37. A method of treating an individual having a disruption of blood flow within or around the CNS comprising administering a therapeutically effective amount of the amnion derived adherent cells of claim 1 or claim 7.

38. A method of treating an individual having a disruption of blood flow in or around a limb comprising administering a therapeutically effective amount of the amnion derived adherent cells of claim 1 or claim 7.