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WO2009089035A1 - Procédés et systèmes pour multiplier des cellules ac133+ et induire leur différentiation - Google Patents

Procédés et systèmes pour multiplier des cellules ac133+ et induire leur différentiation Download PDF

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WO2009089035A1
WO2009089035A1 PCT/US2009/000113 US2009000113W WO2009089035A1 WO 2009089035 A1 WO2009089035 A1 WO 2009089035A1 US 2009000113 W US2009000113 W US 2009000113W WO 2009089035 A1 WO2009089035 A1 WO 2009089035A1
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cells
cdl
blood
certain embodiments
subject
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Ramasamy Sakthivel
Donald J. Brown
Hai-Quan Mao
Luc Douay
Vincent Pompili
Kevin Mcintosh
Hiranmoy Das
Yukang Zhao
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Universite Pierre et Marie Curie
Johns Hopkins University
Ohio State University
Arteriocyte Inc
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Universite Pierre et Marie Curie
Johns Hopkins University
Ohio State University
Arteriocyte Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/14—Erythropoietin [EPO]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • Atherosclerotic cardiovascular disease is a leading cause of morbidity and mortality in the industrialized western hemisphere.
  • Coronary artery disease the pathologic process of arterial luminal narrowing by atherosclerotic plaque resulting in obstruction of blood flow to the heart, accounts for about half of the deaths.
  • catheter-based revascularization or surgery-based treatment approaches have been successful in restoring blood flow to ischemic myocardium in the majority of cases, the treatments are inadequate for a significant number of patients who remain incompletely revascularized.
  • the ramifications of treatment limitations may be significant in patients who have large areas of ischemic, but viable myocardium jeopardized by the impaired perfusion supplied by vessels that are poor targets for conventional revascularization techniques.
  • Atherosclerosis of the extremities is a leading cause of occlusive arterial disease of the extremities in patients over age 40.
  • Peripheral vascular occlusive disease and its complications, including ulcers and even necrosis of the affected limb, is also common.
  • percutaneous transluminal angioplasty and aorto-bifemoral bypass procedures are associated with acceptable morbidity and mortality risk and are usually initially successful, these interventions have not been shown to be effective long-term.
  • vascular endothelial growth factor VEGF
  • bFGF basic fibroblast growth factor
  • an alternative therapy that of supplying an exogenous source of endothelial precursor cells (EPCs), may be optimal for cellular therapeutics to enhance vasculogenesis and collateralization around blocked/narrowed vessels to relieve ischemia.
  • EPCs endothelial precursor cells
  • cellular hemoglobin-based blood substitutes are not suitable due to their short circulatory life span of less than 48 hours, compared to 42-120 days for human red blood cells.
  • hematopoietic stem cells generate progenitor cells that undergo terminal differentiation, resulting in mature circulating blood cells. 22.
  • Prior culturing techniques for expanding stem cells use a variety of approaches including co- culture with bone marrow stromal cells which rarely show more than 3-5 fold increase in cell number.
  • Clinical grade media-only based culturing systems are inefficient due to the requirement for multiple media changes, and have demonstrated difficulty maintaining the starting populations in undifferentiated states.
  • co-culture methods have not been shown to effectively produce clinical grade stem cells.23-27
  • the failure of erythroid development which normally culminates in mature enucleated reticulocytes and red blood cells in vitro or ex vivo is articulated to the need for cell-cell interactions.
  • Giarratana et al 12 describe a promising technology that permits for the first time both the extensive expansion of CD34+ stem cells and their total conversion ex vivo into mature enucleated red blood cells.
  • the work of Giarratana et al. is a significant milestone in red blood cell engineering, its practical implications are limited due to the constraints in obtaining enough erythrocyte progenitor cells using the co-culture techniques.
  • the complex engineering needed to implement the three-step protocol would make each unit of blood produced prohibitively expensive.
  • developing a practical method for producing red blood cells on a scale that is robust and economical would be ideal.
  • the invention provides a method for expanding CD 133+ cells, comprising providing CDl 33+ cells and culruring said CDl 33+ cells on polymeric nanofibers.
  • said expansion comprises increasing the number of CD133+ cells by at least 300-fold.
  • the CD133+ cells are CD133+CD34- cells, CD133+CD34+ cells, or combinations thereof.
  • the CDl 33+ cells are CD133+CD34+KDR-CXCR4- cells.
  • at least 10% of the total cells are CDl 33+.
  • said expansion occurs in about 10 days.
  • said polymeric nanofibers are random nanof ⁇ ber meshes or films.
  • said polymeric nanofibers are aligned nanof ⁇ ber meshes or films.
  • said polymeric nanofibers are polyethersulfone (PES) meshes or films.
  • said polymeric nanofibers are surface-conjugated with functional groups.
  • said functional groups are selected from the group consisting of: hydroxyl, carboxyl, and amino groups.
  • said polymeric nanofibers are surface-conjugated with fibronectin.
  • said CDl 33+ cells are mammalian cells. In certain embodiments, said CDl 33+ cells are human cells.
  • the CD 133+ cells are isolated from umbilical cord blood, bone marrow or peripheral blood or combinations thereof. In certain embodiments, the CD 133+ cells are enriched from umbilical cord blood, bone marrow or peripheral blood or combinations thereof. In certain embodiments, the composition comprises CD133+ enriched at least 2-fold over bone marrow mononuclear cells. In certain embodiments, the expanded CDl 33+ cells are recultured for further expansion.
  • the invention provides a method further comprising directing differentiation of the CDl 33+ cells.
  • said CDl 33+ cells differentiate into smooth muscle cells.
  • said CD 133+ cells differentiate into endothelial cells.
  • said CDl 33+ cells differentiate into red blood cells.
  • the red blood cells are O Rh negative cells.
  • said CDl 33+ cells differentiate into platelets.
  • the total number of cells is increased by about 4500-fold. In certain embodiments, differentiation occurs in about 6-28 days.
  • directing differentiation comprises sequentially exposing cells to media comprising: a) Stem Cell Growth Factor 1 , Erythropoietin and Interleukin 3 and b) EPO. In certain embodiments, directing differentiation comprises sequentially exposing cells to media comprising: a) Stem Cell Growth Factor 1, Erythropoietin and Interleukin 3, b) SCF and EPO and c) EPO.
  • the CDl 33+ cells or differentiated cells express a recombinant transgene. In certain embodiments, the CDl 33+ cells or differentiated cells express a recombinant proangiogenic growth factor. In certain embodiments, the proangiogenic growth factor is VEGF 164, PDGF-BB, or both. In certain embodiments, VEGF 164 and PDGF-BB are expressed in a bicistronic co-delivery vector.
  • the invention provides a method further comprising administering a composition comprising said CDl 33+ cells to a subject in need thereof.
  • said subject in need thereof is suffering from a condition selected from the group consisting of: ischemia, diabetes, a wound in need of healing, a need for tissue or organ replacement, a need for dialysis, need for blood transfusion, a blood condition, blood disease and blood loss.
  • the CDl 33+ cells are autologous to said subject.
  • the CDl 33+ cells are allogeneic to said subject.
  • the CDl 33+ cells are administered by infusion into an artery.
  • the composition comprises a matrix in which the CDl 33+ cells are embedded.
  • the matrix comprises polyethylene glycol (PEG), collagen, fibrin, fibronectin, gelatin, poly-lysine, laminin, heparan sulfate proteoglycan, entactin, elastin, nidogen, hyaluronin, or a combination thereof.
  • the fibrin matrix is polymerized from a solution that contains from about 50 mg/ml to about 400 mg/ml fibrinogen and from about 250 units/mL to about 2000 units/mL thrombin.
  • the CD 133+ cells are held in a solution comprising buffered saline for 6-36 hours prior to administering to the subject.
  • the composition further comprises soluble human fibronectin, hyaluronan, type I collagen, fibrin, gelatin, poly-lysine, laminin, heparan sulfate proteoglycan, entactin, elastin, nidogen, or a combination thereof.
  • the method further comprises administering to the subject a cytokine, chemokine or growth factor.
  • the growth factor is PDGF or VEGF.
  • the method further comprises pre- incubating said CD 133+ cells with a cytokine, chemokine, or growth factor before administering said CDl 33+ cells to the subject.
  • the growth factor is PDFG or VEGF.
  • the method further comprising directing differentiation in any of the ways described above of said CD133+ cells before administering said CDl 33+ cells to the subject.
  • the method further comprises administering to the subject an anticoagulant.
  • at least 10% of cells in the composition are CD133+ cells.
  • the CD133+ cells are isolated from umbilical cord blood, bone marrow or peripheral blood or combinations thereof.
  • the CDl 33+ cells are enriched from umbilical cord blood, bone marrow or peripheral blood or combinations thereof.
  • the composition comprises CDl 33+ enriched at least 2-fold over bone marrow mononuclear cells.
  • the CDl 33+ cells are recultured for further expansion prior to administering to the subject.
  • administering the composition to the subject consists of a single administration of the composition to the subject.
  • administering the composition to the subject consists of multiple administrations of the composition to the subject.
  • administering the composition reduces, delays or eliminates the need for surgical or pharmaceutical intervention.
  • administering the composition increases by at least 25% the likelihood that the subject will survive over a one- year period following treatment.
  • the invention provides a bioreactor system comprising polymeric nanofibers and CDl 33+ cells.
  • cells are contacted with one or more of: SCF, IL-3, EPO, and TPO.
  • said cells are expanded.
  • said system directs differentiation after expansion.
  • the invention provides a method of expanding CDl 33+ cells, comprising: (a) providing a population of cells including CD 133+ cells, (b) purifying
  • CDl 33+ cells from the population, (c) providing a bioreactor system containing polymeric nanofibers, and (d) culturing said CD 133+ cells in said bioreactor.
  • said CD 133+ cells are expanded about 300-fold.
  • said method further comprises directing differentiation of the CDl 33+ cells.
  • said CD 133+ cells differentiate into erythrocytes.
  • said CD 133+ cells differentiate into platelets.
  • said CD 133+ cells differentiate into smooth muscle cells.
  • said CDl 33+ cells differentiate into endothelial cells.
  • the total number of cells is increased by at least 4500-fold.
  • said differentiation occurs in about 6-28 days.
  • the method further comprises administering a composition comprising said CDl 33+ cells to a subject in need thereof.
  • said subject in need thereof is suffering from a condition selected from the group consisting of: ischemia, diabetes, a wound in need of healing, a need for tissue or organ replacement, a need for dialysis, need for blood transfusion, a blood condition, blood disease and blood loss.
  • cellular waste production is optimized during culturing.
  • cellular waste is selected from the group consisting of ammonia and lactic acid.
  • CO2 levels, O2 levels and temperature are optimized during culturing.
  • CO2 levels, O2 levels and temperature are re-optimized during different phases of culturing.
  • FIG. 1 Isolation of cord blood derived stem cells and ex-vivo expansion on nanofiber scaffold.
  • CDl 33 positive cells were isolated from human umbilical cord blood using autoMACS machine and reagents as described in Materials and Methods. Flowcytometric analysis was performed for the evaluation of purity. Right panel indicates the purity is more than 90% and left panel is the isotype control. This is representative of at least six separate cord samples evaluated.
  • Figure 2 depicts the appearance of ACl 33+ cells plated on polymeric nanofibers by light microscopy.
  • FIG. 3 Charactererization of nanofiber scaffold expanded cells. Flowcytometric analysis was performed for the characterization of nanofiber scaffold expanded cells at day 10 using various conjugated antibodies as stated in one or two color staining. This is a representative of the three independent analyses of the expanded cells.
  • Figure 4. Expanded cells were transfected with VIP vector (see Materials and Methods for details) and after cyto-spin cells were stained for PDGF expression using immunofluorescence technique. More than 60% cells were positive for PDGF staining. One million cells were either transfected with VIP vector or empty vector and cell culture supernatants were collected. ELISA was performed with the culture supernatants after 24h and 48h of transfection for PDGF secretion. After 48h of transfection almost 4-fold induction of PDGF secretion was observed compare to the basal empty vector transfected cells.
  • FIG. 5 In vitro functional evaluation of the expanded cells.
  • A. The 10-day nanofiber expanded cells were re-cultured on a chamber slide using regular complete DMEM plus 10% FBS and PSG for 7 days. DiI-AcLDL was added to the culture and incubated for 4h at 37 C. cells were washed, fixed and mounted with DAPI. Nucleus stained with DAPI (blue) and uptaken DiI-AcLDL (red in color).
  • B Detection of stem cell migration. Five hundred thousand GFP vector or empty vector transfected nanofiber expanded stem cells were injected into each of the hind limb ischemic mice via intra- myocardial delivery. After 36h of injection mice were sacrificed and organs were harvested. Immunohistochemical detection was performed with the fixed and paraffin embedded tissue sections using anti-GFP Ab. Appropriate controls were also evaluated.
  • FIGs 6, 7, and 8. In vitro differentiation of expanded stem cells to smooth muscle cells. Expanded cells were also cultured with SMBM complete media for another 14 days. Indeed, early smooth muscle differentiated marker such as F-Phalloidin, smooth muscle myosin heavy chain (SM-MHC) and smooth muscle ⁇ -actin staining indicated that they are positive for all markers. Respective isotype controls were evaluated for representative studies.
  • Figure 9 In vitro differentiation of expanded stem cells to endothelial lineage.
  • the 10-day nanofiber expanded cells were re-cultured for another 14 days on chamber slides with EGM2 media.
  • Immunofluorescence studies were performed using endothelial specific markers to the differentiated cells.
  • Early endothelial markers, such as CD31 , vWF and ICAM-I and VCAM-I were studied, isotype-matched IgG were also used for control staining. Indeed, these cells were positive for the early endothelial markers. Isotype control was evaluated for the representative experiment.
  • FIGs 10 and 1 Genetic manipulation of expanded stem cells. Expanded cells were transfected pmaxGFP vector using Nucleofactor technology and human CD34 cell nucleofactor kit (Amaxa Inc. program U-008) following manufacturer's protocol. At 24-h post transfection cells were visualized under fluorescent microscope for green fluorescence protein (GFP) expression and under normal light for cellular morphology. Almost all cells were expressing GFP indicates an efficient transfection. We subsequently cultured these cells with regular complete DMEM media for another 14 days to evaluate cellular differentiation and viability. These cells look elongated in shape and endothelial like appearance (Figure 1 1).
  • GFP green fluorescence protein
  • RBCs were compared for erythrophagocytosis.
  • FIG. 16 Blood Group Ag Expression. RhD and A antigen expression were examined on differentiated RBCs cultured to Day 0, 4, 8, 1 1 , 14 and 16.
  • Figure 17. In vivo Fate of cRBCs. Labeled 30X10 6 Day 15 differentiated RBCs were delivered via retro-orbital injection into each NOD/SCID mouse and analyzed for LDS and the label CFSE.
  • FIG. 1 Cell Expansion. The expansion of cells from various sources were examined over a number of days.
  • CB Cord Blood
  • LK Leukocyte
  • BM Bone Marrow
  • PB Peripheral Blood.
  • FIG. 19 Production of RBC from CD 34 Hemopoietic Stem Cells. A comparison of expansion of CB and PB cells using Protocols A and B was performed.
  • CB Cord Blood
  • PB Peripheral Blood.
  • FIG. 20 Cell Expansion Results. Expansion of cells was examined at Day 5 and 10.
  • FIG. 21 Process Flow Chart for NANEX RBC Production.
  • FIG. 23 MACS Sorted CDl 33 Surface Phenotype in Primary and Expanded Cells.
  • CDl 33, CD34 and CXCR4 were compared between primary and expanded cells.
  • Figure 24 Reculture of primarily nanofiber expanded CD 133+ cells for further expansion. Expansion of recultured pre-expanded CDl 33+ cells was analyzed over 27 days.
  • Figure 25 Phenotype of Expanded Cells. Various markers were analyzed in expanded CDl 33+ cells.
  • FIG 26 Transfection of VlP Vector in CD 133+ expanded cells. PDGF was detected in VIP transfected cells.
  • Figure 27 Transwell migration of stem cells. Migration was compared between fresh and expanded cells with and without SDF.
  • Figure 28 Genetic modification on stem cells induces neovascularization. Blood flow and capillary number were examined in control, CDl 33+ cells, expanded cells and VIP transfected expanded cells.
  • FIG. 29 Timeline and Growth Factor Requirement for NANEX RBC Production.
  • S Stem Cell Growth Factor 1
  • E Erythropoietin
  • IL3 Interleukin 3.
  • FIG 38 NANEX RBC Production. A comparison was made between total cell production and RBC production using Protocols A and B.
  • FIG. 39 Metabolites Concentration per Cell in Culture. Glucose, ammoniac and lactic acid levels were analyzed during proliferation.
  • Ammonia is a Limiting Factor for Proliferation. Analysis of ammonium levels and proliferation were analyzed.
  • Figure 41 Geometric Representation For Optimal Conditions Inside Bioreactor Device.
  • FIG 44 Process Flow Chart for RBC Production using a Bioreactor Device.
  • Figure 45 A Hypothetical Bioreactor Device for NANEX RBC Production.
  • HSC endothelial progenitor cell
  • BM bone marrow
  • peripheral circulation (1, 2)
  • ECM extracellular matrix
  • HSCs Besides regulating the local presentation of growth factors, they provide the adhesive interactions or anchorages for HSCs, which are of critical importance to the survival, homing and lodging behavior of HSCs and also facilitate their interactions with stromal cells (5, 6), (7).
  • Human UCB CD34+ cells cultured on aminated nanofibers could be efficiently expanded after 10 days of culture in serum-free medium (8).
  • stromal layers are very important.
  • the stromal layer comprises a mixture of fibroblasts, macrophages, adipocytes, endothelial cells and reticular cells that can provide a large portion of the HSC niche of secreted biochemical factors.
  • the stromal layer is not defined, and it is difficult to generate the stromal layer in a reproducible fashion, which therefore renders the expansion outcome less predictable.
  • the use allogeneic cell sources is less desirable due to potential immunologic complications (20), (21 ).
  • the stromal layer may produce negative regulators of hematopoiesis such as transforming growth factor TGF- ⁇ and chemokines (4), (19).
  • stromal-free suspension culture has been rapidly adopted for HSC expansion due to their chemically defined nature.
  • This method involves the use of various combinations of growth factors and cytokines to substitute for the regulatory signals provided by stromal cells (22), (23), (24).
  • these suspension cultures offer the obvious advantage of simplicity, the expansion outcomes are less impressive (25), (26).
  • suspension culture lacks the critical HSC-extracellular matrix (ECM) interaction (27). Therefore, the new biomaterials approach aims to provide adhesion support and topographical features of the BM microenvironment.
  • VEGF Vascular endothelial growth factor
  • PDGF Platelet derived growth factor
  • PhVEGF-Ai 64 increased capillary density more than phPDGF-BB, and phPDGF-BB preferentially stimulated arteriolar growth.
  • the combination increased both capillaries and arterioles but did not enhance angiogenesis any more than single plasmid treatments did.
  • VEGF-Ai 64 and the combination of ph VEGF- Ai 64 and phPDGF-BB counteracted left ventricular dilatation after 1 week but did not counteract further deterioration (35).
  • RBCs red blood cells
  • donor RBC unit supplies in the military are limited in the battlefield environment due to the inherent liabilities of the donor system coupled with the global nature of U.S. military operations.
  • CONUS sourced which poses significant logistic and cost challenges.
  • several recent studies suggest that the storage lesions can have deleterious effects in recipients.
  • An effective development of manufacturing capabilities to grow universal donor "O- negative" red blood units would allow direct application in acute care settings of dramatic blood loss, without violating the FDA's concerns involving patient consent. This solution is both technologically attainable and desirable from a clinical and regulatory standpoint. This is be the focus of Applicants' methods to generate RBCs from hematopoietic progenitor cells using ex vivo nanofiber-based expansion.
  • UCB stem cells are more proliferative and are immunologically more naive than BM or PB stem cells.
  • Clinical studies in leukemia have shown that CB needs less HLA matching for graft survival.
  • Kelly et al found CB transplants in Leukemia demonstrated recipient conversion to donor blood type, indicating the donor unit converting to complete production of the recipient's hematopoietic system 30.
  • the capacity of these CDl 33+ and CD34+ progenitor cells to differentiate down the erythrocyte progenitor lineage reinforces these cells as appropriate starting populations for Blood Pharming.31 ,32
  • Nanex cell expansion culture can be used as automatic self selection of cells toward the erythroid lineage as the differentiating cells detach from the scaffold and become suspended, facilitating an easy removal (i.e. simple transfer of the detached cells out of the scaffold containing compartment of the bioreactor, while keeping the majority of adherent cells in a self renewing system.
  • Cytomatrix had reported the ability to differentiate CD34+ hematopoietic progenitor cells toward the erythrocyte lineage using stem cell factor (500pg/ml) or TPO (500 pg/ml) utilizing their CellFoam Biomatrix. They reported total cell (TC) culture expansion efficiency of 375-fold and erythrocyte expansion 555-fold after 4 weeks of culturing. (42nd annual meeting of the ASH, 2000)
  • an element means one element or more than one element.
  • Nanofiber Compositions The invention pertains to methods and compositions for the expansion and differentiation of stem cells.
  • the instant methods rely on the isolation of stem cells from any of a number of sources and the subsequent use of the compositions and methods of the instant invention to expand and/or differentiate these stem cells.
  • Stem cells can be isolated from any of a number of sources and techniques known to those of skill in the art.
  • U.S. Pat. No. 5,061 ,620 describes a substantially homogeneous human hematopoietic stem cell composition and the manner of obtaining such composition.
  • cells are cultured on nanofiber film.
  • cells are cultured on nanofiber mesh.
  • cells are cultured on film and mesh.
  • the nanofiber film and mesh are those described in US Patent Application Publication No. 20080153163, herein incorporated by reference in its entirety.
  • CDl 33+ cells are cultured on unmod.
  • CD 133+ cells are cultured on TCPS surface.
  • HGFs hematopoietic growth factors
  • the matrix bound growth factors will mimic the native presentation pattern of the cytokines in BM during early hematopoiesis, where the cytokines interact with HSCs in a membrane bound format.
  • HGFs are directly conjugated to the polymer fibers.
  • multiple cytokines are conjugated to the same set of fibers.
  • multiple cytokines are conjugated to different fibers.
  • the fibers are arranged into defined patterns.
  • the fibers are conjugated to fibronectin.
  • the electrospun nanofibers used in the methods and compositions of the invention can be natural or synthetic. In one embodiment, the electrospun nanofibers are comprised of natural polymers.
  • Exemplary natural polymers include cellulose acetate (CA), chitin, chitosan, collagen, cotton, dextran, elastin, fibrinogen, gelatin, heparin, hyaluronic acid (HA), poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), regenerated cellulose (RC), silk, and zein.
  • CA cellulose acetate
  • chitin chitosan
  • collagen collagen
  • cotton dextran
  • elastin fibrinogen
  • gelatin heparin
  • HA hyaluronic acid
  • PHBV poly 3-hydroxybutyrate-co-3-hydroxyvalerate
  • RC regenerated cellulose
  • silk and zein.
  • the electrospun nanofibers are made of degradable or non- degradable synthetic polymer material.
  • exemplary degradable polymers include poly(.epsilon.-caprolactone) (PCL), poly(.epsilon.-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid-co-.epsilon.-caprolactone) (PLACL), and polydioxanone (PDO).
  • PCL poly(.epsilon.-caprolactone)
  • PCLEEP poly(.epsilon.-caprolactone-co-ethyl ethylene phosphate)
  • PLA poly(lactic acid)
  • PLA poly(lactic-co-glycolic acid)
  • PLACL poly(lactic acid-co-.epsilon.-caprolactone)
  • PDO polydiox
  • non-degradable polymers include poly acrylamide (PAAm), poly acrylic acid (PAA), poly acrylonitrile (PAN), poly amide (Nylon) (PA, PA-4,6, PA-6,6), poly aniline (PANI), poly benzimidazole (PBI), poly bis(2,2,2-trifluoroethoxy) phosphazene, poly butadiene (PB), poly carbonate (PC), poly ether amide (PEA), poly ether imide (PEI), poly ether sulfone (PES), poly ethylene (PE), poly ethylene-co-vinyl acetate (PEVA), poly ethylene glycol (PEG), poly ethylene oxide (PEO), poly ethylene terephthalate (PET), poly ferrocenyldimethylsilane (PFDMS), poly 2-hydroxyethyl methacrylate (HEMA), poly 4- methyl-1 -pentene (TpX), poly methyl methacrylate (pMMA), poly p-phenylene
  • the electrospun nanofiber compostions of the invention can be made of any one of polymers identified herein.
  • the electrospun nanofiber compostions of the invention can also be made of any combination of the polymers identified herein.
  • Electrospun matrices can be formed of electrospun fibers of synthetic polymers that are biologically compatible.
  • biologically compatible includes copolymers and blends, and any other combinations of the forgoing either together or with other polymers. The use of these polymers will depend on given applications and specifications required. A more detailed discussion of these polymers and types of polymers is set forth in Brannon- Peppas, Lisa, "Polymers in Controlled Drug Delivery,” Medical Plastics and Biomaterials, November 1997, which is incorporated herein by reference.
  • the compounds to be electrospun can be present in the solution at any concentration that will allow electrospinning.
  • the compounds may be electrospun are present in the solution at concentrations between 0 and about 1.000 g/ml.
  • the compounds to be electrospun are present in the solution at total solution concentrations between 10-15 w/v % (100-150 mg/ml or 0-0.1 g/L).
  • the compounds can be dissolved in any solvent that allows delivery of the compound to the orifice, tip of a syringe, under conditions that the compound is electrospun. Solvents useful for dissolving or suspending a material or a substance will depend on the compound.
  • fibers having different physical or chemical properties may be obtained. This can be accomplished either by spinning a liquid containing a plurality of components, each of which may contribute a desired characteristic to the finished product, or by simultaneously spinning fibers of different compositions from multiple liquid sources, that are then simultaneously deposited to form a matrix.
  • the resulting matrix comprises layers of intermingled fibers of different compounds.
  • This plurality of layers of different materials can convey a desired characteristic to the resulting composite matrix with each different layer providing a different property, for example one layer may contribute to elasticity while another layer contributes to the mechanical strength of the composite matrix.
  • the electrospun nanofiber has an ultrastructure with a three-dimensional network that supports cell expansion, growth, proliferation, and/or differentiation.
  • This three dimensional network is similar to the environment where many of these stem cells naturally occur, e.g., in bone marrow.
  • the spatial distance between the fibers plays an important role in cells being able to obtain nutrients for growth as well as for allowing cell-cell interactions to occur.
  • the distance between the fibers may be about 50 nanometers, about 100 nanometers, about 150 nanometers, about 200 nanometers, about 250 nanometers, about 300 nanometers, about 350 nanometers, about 600 nanometers, about 750 nanometers, about 800 nanometers, about 850 nanometers, about 900 nanometers, about 950 nanometers, about 1000 nanometers (1 micron), 10 microns, 10 microns, 50 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, or about 500 microns.
  • the distance between the fibers may be less than 50 nanometers or greater than 500 microns and any length between the quoted ranges as well as integers.
  • the fibers can have a diameter of about 50 nanometers, about 100 nanometers, about 150 nanometers, about 200 nanometers, about 250 nanometers, about 300 nanometers, about 350 nanometers, about 600 nanometers, about 750 nanometers, about 800 nanometers, about 850 nanometers, about 900 nanometers, about 950 nanometers, about 1000 nanometers (1 micron), 50 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, or about 500 microns, or the diameter may be less than 50 nanometers or greater than 500 microns and any diameter between the quoted ranges as well as integers.
  • a preferred fiber diameter is between 100- 700 nm.
  • the pore size in an electrospun matrix can also be controlled through manipulation of the composition of the material and the parameters of electrospinning.
  • the electrospun matrix has a pore size that is small enough to be impermeable to one or more types of cells.
  • the average pore diameter is about 500 nanometers or less.
  • the average pore diameter is about 1 micron or less.
  • the average pore diameter is about 2 microns or less.
  • the average pore diameter is about 5 microns or less.
  • the average pore diameter is about 8 microns or less.
  • the matrix has a pore size between about 0.1 and about 100 .mu.m.sup.2. In another embodiment, the matrix has a pore size between about 0.1 and about 50 .mu.m.sup.2. In another embodiment, the matrix has a pore size between about 1.0 .mu.m and about 25 .mu.m. In another embodiment, the matrix has a pore size between about 1.0 .mu.m and about 5 .mu.m.
  • the mechanical properties of the matrix or core will depend on the polymer molecular weight and polymer type/mixture. It will also depend on orientation of the fibers (preferential orientation can be obtained by changing speed of a rotating or translating surface during the fiber collection process), fiber diameter and entanglement.
  • the cross- linking of the polymer will also effect its mechanical strength after the fabrication process.
  • the electrospun nanofiber core can be comprised of parallel or randomly oriented fibers.
  • a polymer is grafted onto the electrospun nanofiber core.
  • Exemplary polymers that can be grafted onto the electrospun core include, but are not limited to, polymers having functional groups which can be initiated by free radicals, e.g., free radicals formed on the surface of the electrospun core.
  • Exemplary grafted polymers include poly(acrylic acid) and derivatives and copolymers thereof, e.g., polymethacrylic acid and poly(acrylic acid-co-hydroxyethylmethacrylic acid), polyallylamine and derivatives and copolymers thereof.
  • the polymers grafted on the electrospun nanofiber core are derivatized.
  • the polymers are derivatized so that cells, e.g., stem cells, are better able to interact with the compositions of the invention.
  • the polymers are derivatized to have a positive charge.
  • the polymers are derivatized to have a negative charge.
  • Exemplary derivatives include carboxylic, hydroxyl and amino moieties.
  • the polymers are derivatized with a biological agent, e.g., a nucleic acid, peptide or polypeptide.
  • a biological agent e.g., a nucleic acid, peptide or polypeptide.
  • the peptide or polypeptide is a cell adhesion peptide or heparin.
  • the compositions of the invention comprise a spacer molecule between the electrospun nanofiber and the derivatized moiety.
  • the spacer molecule can allow for improved functionality of the compositions of the invention.
  • the spacer is a ethylene, propylene, butylenes, hexylene moiety.
  • a self-renewing progenitor cell nano fiber based culturing system that expands CD 133+ progenitor cells about 300 fold in about 10 days.
  • the disclosed methods produce about 4500 fold expansion of total cells.
  • the self-renewing progenitor cell nano fiber based culturing system yields about 4500 fold expansion in about 20 days.
  • expansion occurs in about 12, 16, 18 20, 22, 24, 26 or 28 days of culture. In certain embodiments, expansion occurs between 1 to 10 days of culturing. In certain embodiments, expansion occurs between 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 6 to 10 days, 7 to 10 days, 8 to 10 days, or 9 to 10 of culturing. In certain embodiments, expansion occurs between 10-28 days of culturing. In certain embodiments, expansion occurs between 10-28 days, 10-12 days, 10- 14 days, 10- 16 days, 10- 18 days, 10-20 days, 10-22 days, 10-24 days, or 10-26 days of culturing. In certain embodiments, expansion occurs on a scaffold. In certain embodiments, expansion occurs in culture on nanofiber mesh and/or film. In certain embodiments, expansion occurs in a bioreactor.
  • the present invention discloses methods and a processes to obtain whole blood through the ex vivo expansion and differentiation of stem cells.
  • One embodiment of the invention is to isolate stem cells from a biologic source such as peripheral blood, umbilical cord blood, bone marrow, and embryonic fluid.
  • a further embodiment of the invention is to spray a nanofiber, incorporated herein, onto a hollow fiber substrate to function as a component in cell culturing. Said stem cells are applied to said nanofiber and are allowed to expand for a period of time.
  • cell differentiation media is filtered into said hollow fiber as a delivery method to culture said expanded stern cells, differentiating said stem cells into erythrocytes.
  • cultured cells according to the methods of the application have a significant differentiation commitment towards the myeloblast / monoblast lineage.
  • cultured cells according to the methods of the application have a significant differentiation commitment towards the erythrocyte lineage. In certain embodiments, cultured cells according to the methods of the application have a significant differentiation commitment towards platelets. In certain embodiments, 80% or more of nanofiber expanded cells are differentiated to erythrocyte phenotype in about 26 days in liquid culture. In certain embodiments, 80% or more, 90% or more, or 95% or more of nanofiber expanded cells are differentiated to erythrocyte phenotype in about 10, 12, 14, 16, 18, 20, 22, 24 or 28 days in liquid culture. In certain embodiments, expanded cells successfully reconstitute hematopoiesis in at efficiency rates higher than current culturing methods. In certain embodiments, clinical products are generated from a 60cc initial harvest.
  • 1 PBSC CDl 33+ cell generates 1-2 x 10 5 cRBC. In certain embodiments, 1 PBSC CD34+ cell generates 1-2 x 10 6 cRBC. In certain embodiments, 1 expanded cell generates 0.2-1 x 10 7 cRBC. In certain embodiments, 1 expanded cell generates 3.5 x 10 7 cRBC. In certain embodiments, 1 CB CD34+ cell generates 2-10 x 10 6 cRBC. In certain embodiments, 1 leukapheresis or 1 cord blood can generate in vitro the equivalence of 4 to 10 standard units of packed RBC. In certain embodiments, total cells are expanded about 50,000 fold over about 27 days.
  • CDl 33+ cells are differentiated in defined medium for RBC production according to UPMC protocol comprising IL3, SCF and EPO.
  • IL3 continues until Day 8. In certain embodiments, IL3 continues until Day 9. In certain embodiments, IL3 continues up to about Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or 28.
  • SCF is maintained up to Dayl 1. In certain embodiments, SCF is maintained up to Day 12. In certain embodiments, SCF is maintained up to Dayl 6.
  • SCF is maintained up to about Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or 28.
  • EPO is maintained up to about Day 15. In certain embodiments, EPO is maintained up to about Day 16. In certain embodiments, EPO is maintained up to about Day 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or 28.
  • CD 133+ cells are differentiated using feeder systems according to published methods such as those described in, Koury et al. In vitro maturation of nascent reticulocytes to erythrocytes. Blood.
  • Applicants disclose a portable, automated system that produces universal donor O Rh- red blood units that may, for example, be used for advanced theater battlefield trauma care using a renewable source of progenitor cells from O Rh negative donors, eliminating RBC antigen presentation issues associated with prior studies. It is possible that other RBC antigens will be expressed during the culture period, based on the work of others, 16. This may be verified using standard blood bank hemagglutination assays or flow cytometry.
  • BM and peripheral blood derived CD133+ cells, as well as UCB derived CD133+ cells could readily differentiate to endothelial lineage and contribute to endothelium and other cell types.17, 18. It is expected that NANEX expanded CD133+/CD34+ cells will preserve this functional ability and differentiate into enucleated RBC upon stimulation. In addition, the mechanism of erythroblast enucleation has been widely demonstrated . Recent studies have clearly articulated that the cell-cell interaction (via co-culturing) can be eliminated by providing appropriate growth factors and cytokine cocktail in the culture 19,20.
  • O Rh(-) Genotype/Phenotype Universal Donor
  • An alternative approach would be to use a higher starting progenitor cell population by resetting TO to T+10 days to have enough starting cell population. It may prove beneficial to use routine experimentation to modify additional procedures in order to optimize the method for differentiating nanofiber expanded CD133+/CD34+ progenitor cell into fully mature RBCs.
  • SCF vascular endothelial growth factor
  • EPO vascular endothelial growth factor
  • IL-3 vascular endothelial growth factor
  • IGF-II insulin-like growth factor-II
  • Human UCB CDl 33+ and CD34+ cells may be cultured on novel aminated nanofibers. These cells can be efficiently expanded by 100- to 200-fold after 10 days of culture in serum-free medium, and the expanded CD34+ cells successfully reconstitute hematopoiesis in NOD/SCID mice at efficiencies higher than that of standard suspension culture on tissue culture plates.1 ,28 Applicants' evaluation of this novel animated nanofiber system has confirmed that currently >20% of expanded UCB CD133+/CD34+ cells express RBC phenotype.
  • the approach of using nanofiber based ex vivo stem cell expansion technology to generate functional RBC; and potential to make an automated system for production and keeping the self renewing stem cell population in the system is very innovative.
  • the CDl 33+ cells are placed in a device to optimize the cell culture parameters allowing cell expansion and maturation.
  • Said device can allow for optimization of oxygen levels, carbon dioxide levels, temperature, pH levels, and the level of cell waste including ammonia or other ammonia related waste products referred to as ammoniac.
  • said device allows disposable, elongated, components to be inserted within said device.
  • the inserted components are lined with a hollow fiber structure to allow for interlumen access for cell media gas exchange allowing optimization of gaseous levels within the device.
  • the nano fiber complex disclosed in 1 1/975,492 is applied to the fiber structure to create an environment to allow for the expansion and differentiation of hemopoietic and/or precursor stem cells.
  • the device creates an environment to allow for media exchange necessary to allow the stem cells to expand and differentiate in an automated system, while maintaining an environment optimal for cell expansion and differentiation as disclosed herein.
  • said device allows for continual flow of media necessary for differentiation. Said device contains a mechanism to flush and collect expanded and differentiated cells.
  • the process occurs within a bioreactor specifically designed to allow the exchange or combination of cell media, nanofiber, and hollow fiber environments.
  • Said hollow fiber functions as a scaffold for said nanofiber to provide interlumen access for cell media gas exchange etc.
  • CellExpand scaffold is used.
  • Another embodiment of the invention includes expansion and terminal differentiation of said isolated stem cells within the same bioreactor.
  • a bioreactor may be readily used with Nanex system for RBC differentiation of Nanex expanded CD133+/CD34+ hemopoietic progenitor stem cells.
  • Another embodiment of the invention includes expansion in one bioreactor and terminal differentiation of said isolated stem cells within a separate unconnected bioreactor system.
  • Another embodiment of the invention includes expansion within one bioreactor system connected to a separate bioreactor system to perform terminal differentiation of said isolated stem cells.
  • a portable Bioreactor based automated culture system produces universal donor O-negative red blood units using a renewable source of progenitor cells.
  • the Bioreactor Process Flow comprises one or more of the following steps not necessarily in the order listed.
  • insert expansion and differentiation chamber containing nanofiber culture surface In certain embodiments, infuse with expansion medium. In certain embodiments, stabilize temperature and with continuous, sterile gas exchange to the interior culture environment per optimized environmental values. In certain embodiments, infuse with CDl 33+ cells.
  • maintain environment periodically flushing to collect differentiated cells. In certain embodiments, flush every day. In certain embodiments, after sufficient expansion, flush expansion medium. In certain embodiments, infuse cells with detachment treatment (such as enzyme solution, etc.) to remove remaining cells. In certain embodiments, flush 70 -90% suspension volume into differentiation chamber, and re-suspend remaining 10% in expansion chamber with expansion medium. In certain embodiments, flush about 70, 80 or 90% suspension volume into differentiation chamber, and re-suspend the remaining portion in expansion chamber with expansion medium. In certain embodiments, suspend in differentiation medium. In certain embodiments, maintain differentiation chamber until sufficient cells are produced. In certain embodiments, flush and collect media containing detached, differentiated cells. In certain embodiments, the differentiated cells are RBCs.
  • the process is repeated 5 times. In certain embodiments, the process is repeated 5-12 times. In certain embodiments, the process is repeated 12 times. In certain embodiments, the process is repeated 6, 7, 8, 9, 10, or 1 1 times. In certain embodiments, the process is repeated indefinitely.
  • the bioreactor is a staged bioreactor system.
  • the combination of liquid suspension culture and 3D perfusion bioreactor modules enables construction of a system that is automated and packaged for use in the field.
  • the system uses selective adherence that makes use of the surface properties of hematopoietic cells at various stages of differentiation.
  • the entire system is scalable, in that suspension cultures can be adjusted in size for cell concentration and type, and the bioreactor modules can both be used in parallel, effecting unlimited capacity at a given stage, and also can be scaled up in size.
  • CDl 33+ cells Therapeutic methods of administration and uses for CDl 33+ cells are known to one of skill in the art and may be found, for example, in US Patent No. 7,470,538, US Published Application No. US-2004-0258670, and PCT No. WO 2008/085229, all of which are incorporated by reference herein.
  • the therapeutically effective amount of expanded CDl 33+ cells can range from the maximum number of cells that is safely received by the subject to the minimum number of cells necessary for either induction of new blood vessel formation in the ischemic tissue or for increasing blood flow to the ischemic tissue or for relieving blood loss.
  • the therapeutically effective amount of CD133+ cells is at least 1 x 10 4 per kg of body weight of the subject and, most generally, need not be more than 7 x 10 5 of each type of cell per kg.
  • CDl 33+ cells may be administered with hMSCs or other cells.
  • the ratio of CD133+ cells and hMSCs can vary from about 5:1 to about 1 :5. A ratio of about 1 :1 is preferable.
  • the hMSCs are autologous or HLA-compatible with the subject, the hMSCs can be isolated from other individuals or species or from genetically-engineered inbred donor strains, or from in vitro cell cultures.
  • the therapeutically effective amount of differentiated cells will vary by cell type and amount of loss suffered by those in need of administration. For example, enough RBCs should be administered to relieve the amount of blood loss in a patient suffering from an ailment resulting in lost blood.
  • the administration of blood cells are known to those of skill in the art.
  • the therapeutically effective amount of the cells of the disclosure can be suspended in a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier includes but is not limited to basal culture medium plus 1 % serum albumin, saline, buffered saline, dextrose, water, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention provides a use of cells of the disclosure, such as CDl 33+ cells, for the manufacture of a medicament to treat a subject in need thereof.
  • the medicament further comprises recombinant polypeptides, such as growth factors, chemokines or cytokines.
  • the medicaments comprise hMSCs or other cells.
  • the cells used to manufacture the medicaments may be isolated, expanded, derived, or enriched using any of the variations provided for the methods described herein.
  • the cell preparation or composition of the disclosure is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous, intra-arterial or intracardiac administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a local anesthetic to ameliorate any pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a cryopreserved concentrate in a hermetically sealed container such as an ampoule indicating the quantity of active agent.
  • composition When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Such methods include injection of the cells into a target site in a subject.
  • Cells may be inserted into a delivery device which facilitates introduction by injection or implantation into the subjects.
  • delivery devices may include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject.
  • the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location.
  • cells are formulated for administration into a blood vessel via a catheter (where the term "catheter” is intended to include any of the various tube-like systems for delivery of substances to a blood vessel).
  • the cells may be prepared for delivery in a variety of different forms.
  • the cells may be suspended in a solution or gel.
  • Cells may be mixed with a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid, and will often be isotonic.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Modes of administration of the cells of the disclosure include but are not limited to systemic intracardiac, intracoronary, intravenous or intra-arterial injection and injection directly into the tissue at the intended site of activity.
  • the preparation can be administered by any convenient route, for example by infusion or bolus injection and can be administered together with other biologically active agents. Administration may be systemic.
  • the site of administration may be close to or nearest the intended site of activity.
  • a systemic administration such as intravenous administration, is preferred.
  • endothelial generating cells such as CDl 33+ cells and the hMSCs will, when administered, migrate or home to the ischemic tissue in response to chemotactic factors produced due to the injury.
  • the expanded CDl 33+ cells are co-administered simultaneously with the hMSCs.
  • the hMSCs are administered before or after the injection of the endothelial generating cells.
  • Administration of the mesenchymal stem cells/stromal cells may be carried out using the same mode or different modes of administration.
  • CD 133+ cells can be administered by intracoronary injection, while stromal cells might be administered intravenously.
  • Ischemic tissue that can be treated by the methods of the invention include, but are not limited to, limb ischemia, myocardial ischemia (especially chronic myocardial ischemia), ischemic cardiomyopathy, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, intestinal ischemia, and the like.
  • a recombinant polypeptide or a drug is administered to the subject in combination with the administration of cells.
  • the polypeptide or drug may be administered to the subject before, concurrently, or after the administration of the cells.
  • the recombinant polypeptide or drug promotes angiogenesis, vasculogenesis, or both.
  • the recombinant polypeptide or drug promotes the proliferation or differentiation of the CDl 33+ cells, of the mesenchymal stem cells, or of both.
  • the recombinant polypeptide is VEGF, BFGF, SDF, CXCR-4 or CXCR-5, or a fragment thereof which retains a therapeutic activity to the ischemic tissue.
  • the invention methods are useful for therapeutic vasculogenesis.
  • Administration of cells of the disclosure according to invention methods can be used as a sole treatment or as an adjunct to surgical and/or medical treatment modalities.
  • the methods described herein for treatment of myocardial ischemia can be used in conjunction with coronary artery bypass grafting or percutaneous coronary interventions.
  • the methods described herein are particularly useful for subjects that have incomplete revascularization of the ischemic area after surgical treatments and, therefore, have areas of ischemic but viable myocardium.
  • Subjects that can significantly benefit from the therapeutic vasculogenesis according to the methods of the invention are those who have large areas of viable myocardium jeopardized by the impaired perfusion supplied by vessels that are poor targets for revascularization techniques.
  • the therapeutic vasculogenesis according to the methods of the invention can particularly benefit subjects with chronic myocardial ischemia.
  • the cells of the disclosure are infused into a coronary artery, preferably a coronary artery supplying the area of myocardial ischemia.
  • a coronary artery supplying the area of myocardial ischemia.
  • the selected coronary artery for infusion is preferably an epicardial vessel that provides collateral flow to the ischemic myocardium in the distribution of the totally occluded vessel.
  • the therapeutically effective amount of the cells of the disclosure is a maximum number of cells that is safely received by the subject.
  • the injection route is intracoronary, and hMSCs in culture become larger than those originally isolated, the maximum dose should take into consideration the size of the vessels into which the cells are infused, so that the vessels do not become congested or plugged.
  • the minimum number of cells necessary for induction of new blood vessel formation in the ischemic myocardium can be determined empirically, without undue experimentation, by dose escalation studies. For example, such a dose escalation could begin with approximately 10 4 /kg body weight of CD 133+ cells alone, or in combination with approximately 10 4 /kg hMSCs.
  • Effective amounts of cells of the disclosure sufficient to cause the desired neovascularization can be done based on animal data using routine computational methods.
  • the effective amount is about 1.5x10 5 CDl 33+ cells per kg body mass to about 3x10 5 per kg body mass.
  • the effective amount is about 3x10 5 per kg body mass to about 4.5x10 5 CDl 33+ cells per kg body mass.
  • the effective amount is about 4.5xlO 5 per kg body mass to about 5.5xlO 5 CD133+ cells per kg body mass.
  • the effective amount is about 5.5x10 5 per kg body mass to about 7x10 5 CD 133+ cells per kg body mass.
  • the effective amount is about 7x10 5 per kg body mass to about 1 x 10 6 CDl 33+ cells per kg body mass. In another embodiment the effective amount is about 1 x 10 6 per kg body mass to about 1.5x10 6 CDl 33+ cells per kg body mass. In one embodiment the effective amount of human CD 133+ cells is between about 1.5xlO 6 and 4.5xlO 6 CD133+ cells per kg of the subject's body mass and In a preferred embodiment the effective amount is about 5xlO 5 CD133+ cells per kg of the subject's body mass.
  • the composition comprising the CD 133+ cells is introduced into a vessel of the subject without substantially altering the arterial pressure. In other embodiments, the composition is introduced into a vessel by blocking arterial flow for an amount of time, such as from 5 seconds to two minutes, such that the injected cells can pool and adhere to the vessel. In one embodiment, a balloon catheter is used to allow pressure driven administration.
  • One aspect of the invention further provides a pharmaceutical formulation, comprising: (a) CDl 33+ cells enriched from umbilical cord blood and expanded; (b) optionally mesenchymal stem cells containing surface antigens identified by monoclonal antibodies SH2, SH3 or SH4 enriched from bone marrow; and (c) a pharmaceutically acceptable carrier.
  • the formulation comprises from 10 4 to 10 9 CDl 33+ cells.
  • the composition comprises from 10 4 to 10 9 mesenchymal stem cells.
  • the formulation is prepared for administration by a catheter.
  • the CDl 33+ cells are CD133+/CD34+.
  • Example 1 Using biofunctional nanofiber scaffold that can at least partially mimic the bone marrow (BM) stem cell niche for efficient expansion of human umbilical cord blood (UCB) derived hemangioblasts, we transfected expanded (-225 fold) stem cell population with pro- angiogenic growth factors, and evaluated the neovascularization potential.
  • BM bone marrow
  • URB umbilical cord blood
  • Flow cytometric analysis revealed that expanded cells retained their progenitor stem cell characteristics. These expanded cells express higher levels of CXCR4 and LFA-I molecules, are directly linked to cellular homing and adhesion as compared to freshly selected UCB selected cells.
  • Functional analysis revealed that these cells can uptake efficiently AcLDL, migrate in a transwell membrane plate, and can differentiate into endothelial or smooth muscle phenotype.
  • hind limb ischemic model was generated and tested with modified stem cell therapy.
  • Expanded cells transfected with angiogenic factors efficiently augmented blood flow and neovascularization than that of expanded cells or freshly isolated cells or media only treated animals. These expanded stem cells are expected to become a major adjuvant for cell-based therapy.
  • HSCs human hematopoietic stem/progenitor cells
  • Cord blood was processed following the similar protocol published earlier (Das H Immunity and Blood) The hepa ⁇ mzed cord blood was diluted with PBS and carefully layered over 10 ml of Ficoll After 30 min cent ⁇ fugation in a swinging bucket rotor at 14000 rpm, the upper layer was aspirated and the mononuclear cell layer was collected Following labeling with magnetic bead conjugated anti-CD133 (CDl 33) monoclonal antibody (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), two cell separation cycles were run using the AUTO-MACS cell sorter (Miltenyi Biotec) according to the manufacturer's protocol and reagents After the separation, the purity of the cell product was determined by flow cytometry
  • the PAAc-grafted PES nanofiber mesh and films were further conjugated with ethylene diamine (EtDA) using a 2-step carbodiimide cross-linking method. Briefly, each scaffold was first gently shaken in 2 mL acetonitrile containing 50 mm N- hydroxysuccinimide (NHS) and 50 mm dicyclohexylcarbodiimide (DCC). After 6 h, the reaction solution was carefully aspirated and each scaffold was immediately immersed into 2 mL acetonitrile containing 0.03 mmol EtDA.
  • NHS N- hydroxysuccinimide
  • DCC dicyclohexylcarbodiimide
  • Fiber diameters were measured by analyzing representative scanning electron microscopy (SEM) mages of nanofibers using NIH ImageJ software (rsb.info.nih.gov/ij/). At least 250 measurements were recorded for each analysis.
  • SEM representative scanning electron microscopy
  • Human umbilical cord blood CDl 33+ sorted cells were isolated from fresh cord obtained from CWRU Hospital as mentioned earlier.
  • Purified recombinant human stem cell factor (SCF), Flt-3 ligand (FH3), TPO and IL-3 were purchased from Peprotech Inc. (Rocky Hill, NJ, USA).
  • the StemSpan SFEM medium was purchased from StemCell Technologies (Vancouver, BC, Canada). Different substrates were secured at the bottoms of wells of a 24- well aminated tissue culture plate.
  • CD 133+ cells Eight hundred CD 133+ cells were seeded onto each scaffold in 0.6 mL StemSpanTM serum-free expansion medium, which consists of 1% BSA, 0.01 mg/mL recombinant human insulin, 0.2 mg/mL human transferrin, 0.1 mm 2- mercaptoethanol and 2 mm 1-glutamine in Iscove's MDM, supplemented with 0.04 mg/mL low-density lipoprotein (Athens Research and Technology Inc., USA), 100 ng/mL SCF, 100 ng/mL FH3, 50 ng/mL TPO and 20 ng/mL IL-3. Cells were cultured at 37 °C in an atmosphere containing 5% CO2 for 10 days without medium change.
  • StemSpanTM serum-free expansion medium which consists of 1% BSA, 0.01 mg/mL recombinant human insulin, 0.2 mg/mL human transferrin, 0.1 mm 2- mercaptoethanol and 2 mm 1-glutamine in
  • TCPS tissue culture polystyrene surface
  • Fluorescently labeled antibodies for CD34 and other cell surface markers were purchased from BD Biosciences (USA). Fluorescently labeled antibodies for CD41 were purchased from Dako (USA).
  • the cell samples were incubated at 4 0 C for >30 min in 2% FBS Hanks' buffer in the presence of various antibody combinations. After antibody staining, the cells were washed twice using Hanks' buffer and fixed in 1 % paraformaldehyde. Cells were analyzed by triple-color flow cytometry on a FACSCalibur analyzer (BD Biosciences). Relevant isotype controls were also included to confirm specificity and for compensation setting. At least 20,000 events were acquired.
  • the Milan-Mulhouse gating method was used for cell enumeration [24], where a double gating (CD34+CD45+) strategy was used to identify the primitive hematopoietic progenitor cell population in the ex vivo expansion cultures.
  • the CD34 marker is generally expressed by primitive hematopoietic progenitor cells, while CD45 marker is expressed on all cells of hematopoietic origin with the exception of red blood cells and their immediate precursors.
  • Freshly isolated human CD 133+ MACS sorted cells or nano-fiber expanded cells were transfected with either GFP containing vector (pmaxGFP) or VIP vectors (VEGF IRIS and PDGF in pAMFG vector, Generous gift from Dr. Blau, Stanford University, CA) using Amaxa Inc., human CD34 cell nucleofactor kit, following manufacturer's protocol.
  • pmaxGFP GFP containing vector
  • VIP vectors VEGF IRIS and PDGF in pAMFG vector, Generous gift from Dr. Blau, Stanford University, CA
  • Amaxa Inc. human CD34 cell nucleofactor kit
  • 1 -3x10 6 cells were transfected with 2-4 ug of plasmid DNA in lOOul of CD34 cell nucleofactor solution and using programs: U-008/ U-001. After transfection cells were either cultured with DMEM, EBM-2 or SMBM complete media for further studies.
  • ELISA assay was performed by using Quantikine human PDGF-BB ELISA kit from R&D Systems, Minneapolis, MN, following manufacturer's protocol.
  • DiI-Ac-LDL uptake was performed following standard protocol. In brief, after expansion of CD 133+ cells on nanofiber for 10 days cells were plated in glass bottom chamber slides for another 10 days with RPMl 1640 complete media with changes of media in every 3 1 day. Aspirated culture media and washed cells with PBS. Added serum free RPMI 1640 containing l Oug/ml DiI-Ac-LDL and incubated for 4h at 37°C. Aspirated media and washed cells twice with PBS to remove free DiI-Ac-LDL. Fixed cells with 3% formalin in PBS for 10 min followed by washing with PBS. Slides were mounted with Vectashield includes DAPI. Visualized under fluorescent microscope and digital photographs were taken.
  • mice Male SCID/NOD mice (7 weeks old) were purchased from Jackson laboratory (Barharbor ME). Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital, the proximal left femoral artery was ligated at 2 points 3mm apart, and the artery between the ligatures was excised. Three groups of mice were made, and each group (6-9 mice) was injected with either media alone or 5x10 5 freshly isolated CD 133+ cells or 10-day nano-f ⁇ ber-expanded cells via intra- ventricular delivery in 300 ul volume. At various time points, such as days 1, 7, 14 and 28, mice were assessed for functional recovery and blood flow. At the end of the study at day 28, mice were sacrificed and both gastrocnemius muscles were excised.
  • mice were assessed for functional recovery and blood flow of the study.
  • Ten-day expanded cells were either cultured with EGM2 medium (Cambrex) or SMBM medium for another 14 days to a chamber slide (Labtek, Nunc International Inc) changing media in every 3' days. All cultures were performed in quadruplicate, incubated at 37 0 C in 5% CO 2 and 95% humidity, and scored after 14 days of culture by light microscopy.
  • vWF F-actin von Willebrand factor
  • paraffin embedded tissue sections were incubated with monoclonal mouse anti-human nuclear antigen antibody (MAB 1281 ; 1 :50; Chemicon) or monoclonal mouse anti-HLA-class 1 antibody W6/32 (Dako M0736).
  • MAB 1281 monoclonal mouse anti-human nuclear antigen antibody
  • W6/32 monoclonal mouse anti-HLA-class 1 antibody W6/32 (Dako M0736).
  • sections were stained with anti-mouse CD31 antibody (Chemicon). After blocking in Envision blocking buffer (DAKO), sections were placed in primary antibody overnight at 4 °C. On the following day, the sections were rinsed and then incubated with secondary antibody (FITC/PE-conjugated secondary goat anti-mouse IgG.).
  • mice Five hundred thousand GFP vector or empty vector transfected nanofiber expanded stem cells were injected into each of the hind limb ischemic mice via intra-myocardial delivery. After 36h of injection mice were sacrificed and organs were harvested.
  • Progenitor stem cells have capacity for pluripotent differentiation. It is important to verify that our expanded cells retain pluripotency similar to freshly isolated UCB-derived ACl 33+ cells. In this assay, we assessed the ability of 10-day expanded cells to differentiate to the smooth muscle or endothelial lineage via sub culturing with "differentiation-media" for an additional 14 days on chamber slides. Differentiated cells could be identified by their morphology on light microscopy with readily discernible differences between the endothelial and smooth-muscle lineages. Cells cultured with smooth muscle differentiation media were visibly more spread out on the plate than that of endothelial media cultured cells, which were more elongated in shape.
  • Example 7 Transfection of expanded stem cells with VEGF and PDGF
  • VIP single coupled vector
  • VEGF and PDGF obtained from Dr. Blau, Stanford University, CA
  • ELISA immuno-histochemical staining for PDGF
  • ELISA was performed from the culture supernatants both 24h and 48h post-transfection.
  • Example 8 Evaluation of biological effects in vivo using genetically modified stem cell therapy
  • neovascularization was much more prominent and significantly increased in the expanded cells compare to media-alone groups and most prominent in genetically modified expanded cells verified either by immunostaining or total capillary counts. Taken together these data demonstrate that genetically modified cells with angiogenic factors were significantly potent than that of unmodified cells.
  • Example 9 Detection of injected stem cells in the ischemic region in our hind limb ischemic mouse model.
  • nanofibers can serve as an effective substrate to promote the expansion of functional HSCs that retain properties of their freshly-derived counterparts.
  • Expanded cells are more potent in mediating neovascularization in a hind limb ischemic model compare to freshly isolated CDl 33+ cells.
  • expanded cells genetically modified with proangiogenic VlP vector showed dramatic improvement in neovascularization.
  • our expanded cells maintain the potential to differentiate into endothelial or smooth muscle lineages and can migrate to distant ischemic zones to provide local beneficial effects.
  • Example 10 Optimizing NANEX nanofiber culturing conditions to determine optimal progenitor cell source (CDl 33+ or CD34+) for self renewal and differentiation.
  • Applicants disclose technology that will provide a continuous manufacturing system to enable the production of fresh universal donor red blood units within an automated self-contained unit using a self-renewing starting population of progenitor cells.
  • An effective development of manufacturing capabilities to grow universal donor 'O- negative" red blood units will allow direct application in acute care settings of dramatic blood loss, without violating the FDA's concerns involving patient consent. This solution is both technologically attainable and desirable from a clinical and regulatory standpoint.
  • Applicants describe methods to isolate and expand early hematopoietic stem and progenitor cells using a nanofiber-based culture system.
  • This system causes erythroid differentiation and produces mature red cells using a combination of a 3D modular perfusion bioreactor and liquid culture.
  • the resulting red cells may be stored and analyzed to validate their characteristics.
  • CDl 33 is generally considered a marker for more primitive cells. It is co-expressed on the majority of CD34+ cells. Our preliminary studies showed that the aminated nanofibers promoted efficient expansion of UCB derived CD 133+ and CD34+ HSCs, suggesting that functionalized PES nanofibers are superior substrate for ex vivo HSC expansion.1 ,2
  • cells will be harvested as described earlier 1 ,2 and second ex vivo expansion culture to evaluate the fold expansion, proliferation, phenotype maintenance and differentiation of CD133+/CD34+ cells, one may identify the best culture condition for efficient and robust expansion outcomes in serum-free culture, and evaluate the differentiation potential of nanofiber- expanded CD133+/CD34+ cells in vitro.
  • Culture variables to be altered include media components, temperature, incubation time, and percentage carbon dioxide.
  • Mononuclear cells may be isolated by Ficoll density gradient centrifugation as described earlier.1 ,2 Briefly, cells may be incubated with CD133+/CD34+ (terminally differentiated endothelial cell as a control) specific magnetic beads per manufactures instructions (AutoMACS, Miltenyi). CD133+/CD34+ cells selected from UCB may be enumerated and characterized by flow cytometry. Selected cells may be characterized by specific surface markers, including CD 133, CD34, CD31 , CXCR2/CXCR1 (IL-8 receptors), and KDR (VEGFR2) to determine purity and surface characteristics of cells after bead selection3.
  • CD133+/CD34+ terminal differentiated endothelial cell as a control
  • CD133+/CD34+ cells selected from UCB may be enumerated and characterized by flow cytometry. Selected cells may be characterized by specific surface markers, including CD 133, CD34, CD31 , CXCR2/CXCR1 (IL-8
  • Recombinant human stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO) and interleukin-3 (IL-3) may be purchased from StemCell Technologies (Vancouver, BC, Canada). Media used for cell expansion (StemSpan SFEM Medium), colony forming cell assay (MethoCult GF+ H4435) and LTC-IC assay (MyeloCult H5100) may also be purchased from StemCell Technologies. CD133+/CD34+ cell culture. Different nanofiber meshes (diameter of 12 mm) may be fixed in the wells of a 24-well tissue culture plate.
  • CD133+/CD34+ cells may be seeded onto each scaffold in 0.6 mL StemSpanTM serum-free expansion medium supplemented with 40 ⁇ g/mL LDL, 100 ng/mL SCF, 100 ng/mL Flt3, 50 ng/mL TPO and 20 ng/mL IL-3.
  • Cells may be cultured at 37°C in an incubator for 3 to 10 days without medium change. Similar culture may also be performed on tissue culture polystyrene surface (TCPS), which serves as a positive control in this study. Cells may be harvested after 10 days of expansion. All substrates may be washed once with non-trypsin cell dissociation solution and twice with 2% FBS Hanks' buffer at 5-10 min intervals between each wash.
  • TCPS tissue culture polystyrene surface
  • the cell suspensions may then be concentrated through centrifugation at 500 ⁇ g for 10 min. Aliquots of the concentrated cells may then be used for cell counting by a hematocytometer and flow cytometry analyses.
  • Nanex 133/34 cells may be characterized by flow cytometric analysis for change in surface phenotype. Briefly, cell surface markers may be blocked with FCR Blocking Reagent (1 :5; Miltenyi Biotec) and incubated for 20 min at 4 °C with the following antibodies: anti-CD34-PE, and anti- CD133/2 FlTC (all from Miltenyi Biotec). CDl 17 labeling may be performed using anti- CDl 17 antibody (clone YB5.B8; 1 : 100; BD Panningen, BD Bioscience, San Diego, CA, USA). Isotype controls may be purchased from BD Pharmingen.
  • cells may be washed with MACS sorting buffer and analyzed using a FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Dead cells may be excluded via propidium iodide staining. Data analysis may be performed with BD CELLQuest software.
  • Nanofiber-expanded UCB CD133+/CD34+ cells should maintain functional integrity.
  • Example 1 1 - Ex vivo Expansion of Cord Blood CD133+/CD34++ cells- Arteriocyte's 4500-fold cell factory.
  • the nanofiber system yields 4500 fold expansion of total cells ( TC population) and 805 fold CD34+ populations of progenitor cells in a 10-day culture in preliminary studies of human UCB CDl 33+ and CD34+ cells cultured using the NANEX nanofiber system.
  • the expanded CDl 33+ and CD34+ cells successfully reconstitute hematopoiesis in NOD/SCID mice at efficiencies higher than that of standard suspension culture on tissue culture plates.l , 2,28,40
  • nanofiber-based cell expansion technology offers a mechanism for HSC phenotype maintenance/self-renewal via nanofiber-mediated adhesion.
  • Cells expanded on this nanofiber niche consist of two distinct populations of cells: those which adhere to nanofibers (adherent fraction accounting for -46% of total expanded cells) and those that release into suspension cells (suspension population accounting for -54% of total expanded cells).
  • Cell phenotypic analysis showed that the adherent population expressed significantly higher percentage of progenitor markers (CD34+CD45+, 43.8%), compared with the non-adherent cell population (21.9%, p ⁇ 0.05).
  • suspension cell population expressed higher percentage of erythroid marker (46.2% of CD71 high, 35.7% of GIyA) than adherent population (14.1 % of CD71 high, 12.4% of GIyA, /? ⁇ 0.05).
  • the overall percentage of erythroid-committed cells was 21.5%.
  • CFU assay also indicated significant commitment of the suspension cells towards the erythroid lineage compared to the adherent cells.
  • Example 12 Purification, functional characterization and ex vivo expansion of UCB derived CDl 33+ or CD34+ cells on nanofibers.
  • Human umbilical cord blood CDl 33+ / CD34+ sorted cells were isolated from fresh cord obtained from CWRU Hospital. Cells were cultured at 37 °C in an atmosphere containing 5% CO2 for 10 days without medium change as described earlier41. Similar cultures were also performed on tissue culture polystyrene surface (TCPS), which serve as a positive control in this study. Cells were harvested after 10 days of expansion and washed once with non-trypsin cell dissociation solution and twice with 2% FBS Hanks' buffer at 5- 10 min intervals between each wash. Aliquots of the concentrated cells were then used for cell counting by a hematocytometer, flow cytometry analysis, as well as for further studies.
  • tissue culture polystyrene surface TCPS
  • Example 13 Purification and surface phenotvping of Nanex 133+ cells.
  • CD34 marker is generally expressed by primitive hematopoietic progenitor cells
  • CD45 marker is expressed on all cells of hematopoietic origin with the exception of red blood cells and their immediate precursors.
  • Table 1 Characterization of nanofiber scaffold expanded cells. Flow cytometric analysis was performed for the characterization of nanofiber scaffold expanded cells at day
  • the table is a representative of the three independent analyses of the expanded cells.
  • Nanex 133+ expanded cells were transwell plated and migratory capacity was assessed in the presence or absence of stromal derived factor SDF).
  • Example 14 In vitro functional evaluation of Nanexl33+ expanded cells.
  • Example 15 In vivo evaluation of Nanex 133+ expanded cells.
  • Nanex 133+ cells were delivered via intra-myocardial delivery or tail vein injection to assess engraftment of transplanted Nanex 133+ cells. This model is directly applicable to serial tail vein sampling for functional assessment of the Nanex expanded erythrocytes.
  • the surface phenotyping of differentiated erythrocyte progenitor cells may be carried using CD34, CD 13, CD 15, CD 19, CD38, CD45 antibodies and GIyA using flow cytometry.
  • the Milan-Mulhouse gating method may be used for cell enumeration, where a double gating (CD34+CD45+) strategy was used to identify the primitive hematopoietic progenitor cell population in the ex vivo expansion cultures.
  • the CD34 marker is generally expressed by primitive hematopoietic progenitor cells, while CD45 marker is expressed on all cells of hematopoietic origin with the exception of red blood cells and their immediate precursors.
  • the morphological characteristics of differentiated cells may be studied by May- Gru ' nwald-Giemsa reagent staining and analyses, whereas enucleated cells may be monitored for standard hematological variables including the MCV (fl), Mean Cell Hemoglobin (MCHC) (%) and MCH (pg/cell) using an XE2100 automat (Sysmex, Roche Diagnostics).
  • Cells may be labeled with unconjugated or fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies.
  • Antibodies to CD71 (Dako) and to CD45, CD36 and CD34 (Immunotech) may be used for phenotyping and cells may be stained with the vital nucleic acid dye LDS-751. Analyses may be done on FACSCalibur flow cytometer (Becton Dickinson) using Cell Quest software.
  • BFU-E, CFU-E and CFU-GM progenitors may be assayed as previously described.12, 13 to assess the differentiation stage of erythrocyte progenitor population after di fferenti ati on .
  • Deformability measurements The differentiation of expanded reticulocytes into mature RBCs may be tested by deformability.
  • the reticulocytes obtained on day 15 of culture may be separated from nucleated cells by passage through a deleukocyting filter (Leucolab LCG2, Macopharma) and the enucleated cells may be examined by ektacytometry, a laser diffraction method.
  • ektacytometer Technicon, Bayer
  • cells may be suspended in 4% polyvinylpyrrolidone solution then exposed to an increasing osmotic gradient (from 60 to 450 mosM). The change in their laser diffraction pattern may be recorded.
  • the photometric measurement produces a signal termed the deformability index (Dl). Analysis of the DI curve provides a measure of the dynamic deformability of the cell membrane as a function of the osmolality at a constant applied shear stress of 170 dynes/cm.
  • Digitonin (0.2%) may be added to erythrocytes obtained after leukocyte depletion and hemoglobin may be quantified by spectrophotometry using Drabkin's reagent.
  • Glucose-6-phosphate dehydrogenase and pyruvate kinase activities may be determined by measurement of the rate of increase in NADPH absorbance at 340 nm45, using a Synchron CX4 Beckman spectrophotometer and reagents from Randox Laboratories and Roche Diagnostics, respectively. Results will be expressed in units per gram of hemoglobin.
  • Hemoglobin analyses The percentage of the various hemoglobin fractions may be measured by CE-HPLC using a Bio-Rad Variant II Hb analyzer (Bio-Rad Laboratories). Globin chain composition may be determined by RP-HPLC as previously described.
  • Methemoglobin functional properties The Methemoglobin fraction may be determined spectrophotometrically in the near UV region (350-450 urn). Spectra may also be measured for samples equilibrated under pure CO, and after addition of potassium cyanide (final concentration of 200 mM) to the buffered solution of hemoglobin. The total heme concentration will be determined from the maximum absorption of the CO spectrum at 420 nm after addition of 200 mM potassium dithionite. Oxygen equilibrium curves may be measured by a continuous method using a double-wavelength spectrophotometer (Hemox analyzer, TCS). RBCs may be suspended in 50 mM bis-Tris buffer containing 140 mM NaCl at 37 1C and pH 7.4.
  • hemoglobin binding properties may be studied by flash photolysis of solutions in 1-mm optical cuvettes. Briefly, the kinetics of the rebinding of CO to intracellular hemoglobin tetramers may be analyzed at 436 nm after photolysis with a 10- ns pulse at 532 nm as described previously.
  • CB derived CDl 33+ cells express HLA class I and II surface molecules and elicit allogeneic T-cell proliferation by immune competent adult mononuclear cells. It is important to validate the loss of 133+/34+ expression of these cells as they differentiate.
  • the separation process described herein should sufficiently fractionate out the immunologically active progenitor cells from the terminally differentiated universal donor erythrocytes.
  • Example 17 - Culturing System Capability Improved the self-renewing progenitor cell nano fiber based culturing system yielded about 5000 fold expansion in 20 days. Total cell expansion after reculture resulted in about 50,000 fold expansion in 27 days (Figure 24). Applicants have differentiated over 80+% of nanofiber expanded cells to erythrocyte phenotype in 21/23 days span in liquid culture ( Figure 38).
  • Nanofiber Expanded CDl 33+ cells were seeded at 5xl O 4 /ml at DO and subsequently at various concentrations in defined medium for RBC production according to UPMC protocol comprising Stem Cell Growth Factor 1 , Erythropoietin and Interleukin 3 for varying lengths of time. Because of a high CDl 17 expression SCF was maintained up to Day 16 (Protocol B) and compared to the standard protocol (Protocol A) ( Figure 29).
  • Protocol B resulted in greater total expansion and produced an increased number of RBCs but the percentage of RBCs of total cells was similar (Figure 38).
  • Cytology was analyzed at day 0, 6, 9, 12, 16, 19, 21 , and 23 ( Figures 12 and 30-37). Further analysis of cytology, phenotype, expansion and progenitors were conducted on days 12, 16, 21 and 23 (Tables 2-6). Functional assays were performed on differentiated RBCs as described in Examples 15 and 16. Deformability, enzyme content, photodissociation, oxygen binding, erythrophagocytosis, and in vivo fate of Nanex expanded and differentiated RBCs were similar to control RBCs ( Figures 13-17).
  • Meiselman HJ, Kohn DB. An in vitro model of human red blood cell production from hematopoietic progenitor cells. Blood 91, 2664-71 (1998).
  • Integrin alpha 4 beta 1 and glycoprotein IV are expressed on circulating reticulocytes in sickle cell anemia. . Blood 82, 3548-3555 (1993).

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Abstract

L'invention concerne, entre autres choses, des procédés et des systèmes pour multiplier des cellules CDl 33+. L'invention concerne en outre des procédés et des systèmes pour augmenter le flux sanguin vers un tissu artificiel chez un sujet le nécessitant, par exemple vers un myocarde artificiel. L'invention concerne en outre des procédés et des systèmes pour induire la différentiation des cellules CD 133+ multipliées. L'invention concerne aussi des procédés et des systèmes pour traiter un sujet le nécessitant avec des cellules différenciées. L'invention concerne également des procédés et des systèmes optimisés pour produire des globules rouges à partir de cellules CD 133+.
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US11806440B2 (en) 2017-02-02 2023-11-07 Nfs Ip Holdings, Llc Methods of improving bone-soft tissue healing using electrospun fibers
US11576927B2 (en) 2018-12-11 2023-02-14 Nanofiber Solutions, Llc Methods of treating chronic wounds using electrospun fibers
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