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WO2009140655A1 - Procédés de reprogrammation virale efficace de cellules somatiques dans des cellules pluripotentes du type cellules souches - Google Patents

Procédés de reprogrammation virale efficace de cellules somatiques dans des cellules pluripotentes du type cellules souches Download PDF

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WO2009140655A1
WO2009140655A1 PCT/US2009/044241 US2009044241W WO2009140655A1 WO 2009140655 A1 WO2009140655 A1 WO 2009140655A1 US 2009044241 W US2009044241 W US 2009044241W WO 2009140655 A1 WO2009140655 A1 WO 2009140655A1
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cells
cell
stem cell
promoters
viral
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Christian Kannemeier
Jane Pham
Carl Javier
John Sundsmo
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PrimeGen Biotech LLC
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to the field of reprogrammed cells referred to as induced pluripotent stem (iPS) cells.
  • reprogrammed cells are immunocompatible and will function in the appropriate post-natal cellular environment to yield functional cells after transplantation.
  • the disclosure relates generally to methods and kits for preparing cellular compositions for use in research, pharmaceutical drug development and transplantation in stem cell-based therapeutics; and, most particularly to adult stem cell-based therapeutics.
  • the disclosure provides compositions, methods and kits for reprogramming adult somatic tissue cells to become pluripotent stem cells that are similar to embryonic stem cells in their unlimited growth and differentiative capacities.
  • Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus.
  • cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number.
  • An example of multipotent cells is hematopoietic cells - blood cells that can develop into several types of blood cells, but cannot develop into brain cells.
  • At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells - cells that are considered to be permanently committed to a specific function.
  • Stem cells are a rare population of cells that can give rise to vast range of cells tissue types necessary for organ maintenance and function. These cells are defined as undifferentiated cells that have two fundamental characteristics; (i) they have the capacity of self-renewal, (ii) they also have the ability to differentiate into one or more specialized cell types with mature phenotypes.
  • Each group of stem cells has their own advantages and disadvantages for cellular regeneration therapy, specifically in their differentiation potential and ability to engraft and function de novo in the appropriate or targeted cellular environment.
  • ESC embryonic stem cells
  • ESC embryonic stem cells
  • pre-natal stem cells may be donated from spontaneous or elective abortions; tissues would otherwise be discarded; and, they are not created for research purposes.
  • MSC mesenchymal stem cells
  • somatic stem cells have genomes that have been altered by aging and cell division. Aging results in an accumulation of free radical insults, or oxidative damage, that can predispose the cell to forming neoplasms, reduce cell differentiation ability or induce apoptosis. Repeated cell division is directly related to telomere shortening which is the ultimate cellular clock that determines a cells functional life-span. Consequently, adult somatic stem cells have genomes that have sufficiently diverged from the physiological prime state found in embryonic and prenatal stem cells.
  • embryonic stem cells themselves may not be appropriate for direct transplant as they form teratomas after transplant, they are proposed as "universal donor" cells that can be differentiated into customized pluripotent, multipotent or committed cells that are appropriate for transplant. Additionally there are moral and ethical issues associated with the isolation of embryonic stem cells from human embryos.
  • Tissue cells had been believed to be "terminally differentiated", i.e., cells irrevocably committed to their fate and function as lung, liver or heart cells.
  • tissue culture i.e., cells irrevocably committed to their fate and function as lung, liver or heart cells.
  • certain adult mouse somatic tissue cells can be encouraged in tissue culture with growth factors or through genetic manipulation to expand their potency and become capable of forming several different kinds of tissue cells.
  • a number of these approaches suffer from the disadvantage that the resultant cells may potentially form tumors.
  • adult tissue cells are aged and subject to chromosomal oxidative and free radical damage and alterations such as telomere shortening. The latter genetic changes could substantially impact the future utility of such cells in patient therapies.
  • Alternatives are highly desirable.
  • stem cells in therapy has derived from bone marrow transplantation of hematopoietic stem cells in patients with cancers and autoimmune diseases.
  • the patient is treated with lethal levels of radiation and/or chemotherapy, i.e., to kill the cancer, and then the bone marrow and immune system, (destroyed by the cancer therapy), is reconstituted using either the patient's own bone marrow which has been rendered cancer-free in the laboratory (referred to as "autologous" for self derived bone marrow), or the bone marrow of a closely related donor (referred to as "allogeneic" for genetically closely related but not identical).
  • Stem cells in general have been reported to express low levels of transplantation antigens, i.e., genetically encoded by the major histocompatibility complex (MHC) and referred to as MHC class-l and class-ll antigens.
  • MHC major histocompatibility complex
  • Low level antigen expression on stem cells may be advantageous in limiting immune recognition and transplant rejection.
  • GVHD vascular endothelial growth factor
  • GVHD vascular endothelial growth factor
  • therapeutic alternatives would be highly desirable.
  • the plasma membrane bilipid layer of cells protects, sustains and preserves cells by retaining important macromolecules, sensing the environment, transporting needed nutrients and inhibiting access of all but small molecules. Transfer of information across the cell membrane is essential for development, function and survival.
  • Viruses recognize membrane receptors which they bind with specificity to gain entry into cells. Once inside, viruses can redirect host-cell protein synthesis with resultant preferential production of virus encoded proteins. Using this property, defective viruses have been genetically constructed to deliver instructions for manufacture of proteins of interest without production of viral proteins.
  • iPS induced pluripotent stem
  • iPS cells induced pluripotent stem cells
  • the iPS cells are very rare and were originally identified using drug selection markers.
  • two additional transcription factors can increase the number of iPS cell colonies, i.e., Nanog and Lin28.
  • the efficiency of reprogramming has been calculated by one investigator to be about 0.06%, i.e., 6 cells in 10,000. It has not been clear whether the low efficiency of iPS cell derivation is due to technical factors such as poor expression of viral constructs in the host cell; or alternatively, whether the low efficiency is due to an innate ability of a few rare cells in a culture to be reprogrammed; or alternatively, whether a sequential stoichiastic process is required in which different transcription factors must be expressed at specific times during the reprogramming process.
  • Sequential stepwise lentivirus infection does not yield iPS cells (disclosed herein) indicating that all 3, 4, 5 or 6 different lentivirus constructs need to infect a single cells within a finite time interval to induce reprogramming. There is a great need for a reliable, routine method for virus-induced derivation of iPS cells.
  • compositions, methods and kits are provided for reprogramming adult somatic cells to produce induced pluripotent stem (iPS) cells that express embryonic stem cell (ESC) markers; and, are capable of unlimited growth and are capable of forming any cell in the body.
  • the methods involve viral vectors containing in serial array a stem cell responsive promoter element, a pluripotent stem cell transcription factor element, a stem cell non- responsive promoter element and a reporter element.
  • a viral vector for transduction of a somatic cell to induce a pluripotent stem cell comprising in serial array: a stem cell responsive promoter element, a pluripotent stem cell transcription factor element, a stem cell non-responsive promoter element and a reporter element.
  • the pluripotent stem cell transcription factor element comprises more than 80% of a nucleotide sequence selected from the group of coding regions for genes consisting of Oct-4, Sox-2/3, Klf4, Nanog, c-myc, Lim, Rybp, Zfp219, SaIW, Requiem, Arid 3b, P66 ⁇ , Rex-1 , Nad , Sp1 , HDAC2, NF45, Cdk1 and EWS.
  • the gene coding regions occur in nature and are further selected from mammals, birds, reptiles, boney fishes, cartilagenous fishes, amphibians, marsupials, insects, protozoans and invertebrates.
  • the stem cell responsive promoter element is down-regulated in a stem cell.
  • the stem cell responsive promoter element is selected from the group of promoters consisting of CMV promoters, HSV-1 promoters, HSV-2 promoters, cardiac sodium-calcium exchanger (NCX1 ) promoters, muscle MyoD promoters, connective tissue collagenase A2 (col A2) promoters, ubiquitous phosphoglycerate kinase (PGK) promoters, cardiomyocyte atrial natriuretic factor (ANF) promoters, cardiac ventricular myosin light chain (MLC2v) promoters, the type Il alveolar epithelial cell (AT-2)-specific promoters and human surfactant protein C (SP-C) promoters.
  • CMV promoters CMV promoters
  • HSV-1 promoters HSV-1 promoters
  • HSV-2 promoters cardiac sodium-calcium exchanger
  • NCX1 cardiac sodium-calcium exchanger
  • muscle MyoD promoters muscle MyoD promoters
  • PGK connective tissue collagen
  • the stem cell non-responsive promoter element is selected from the group of promoters consisting of EF1 alpha promoters, Ubiquitin-C promoters, retroviral LTR promoters, actin promoters, ATP synthase promoters, microglobulin promoters and IRES promoters.
  • the viral vector nucleic acid backbone comprises a portion of a replication defective virus selected from the group consisting of RNA and DNA viruses.
  • the viral vector nucleic acid backbone is further selected from the group retroviral vectors, lentiviral vectors, adenoviral vectors, HIV viral vectors, SIV viral vectors, HTLV viral vectors, FIV viral vectors and SV40 viral vectors.
  • composition for reprogramming a somatic cell to an induced pluripotent stem cell comprising two or more of the disclosed viral vectors.
  • a method for reprogramming somatic cells to induced pluripotent stem cells using viral transduction with a first and a second viral vector comprising the steps of: determining the number of expression units of the first and the second viral vector; mixing equal numbers of expression units of the first and the second viral vectors thereby establishing a reprogramming mixture; and exposing the somatic cells to the reprogramming mixture under conditions effective for viral vector mediated transduction and at a modulus of infection greater than 1.0.
  • a method for reprogramming somatic cells to induce pluripotent stem cells using viral transduction with the viral vectors comprising the steps of: optimizing a transduction efficiency for a first pluripotent stem cell transcription factor element present in a first viral vector preparation and for a second pluripotent stem cell transcription factor element present in a second viral vector preparation, each preparation comprising a plurality of viral vector particles, by: introducing different numbers of the first and the second viral vector particles into each of a plurality of different test cell cultures; determining a number of test cells in each of the different test cell cultures that are positive for the reporter element; and identifying an optimal number of first viral particles and an optimal number of second viral particles for achieving the highest number of test cells positive for each of the reporter elements, thereby optimizing the transduction efficiency of the first and the second pluripotent stem cell transcription factor elements; mixing the first optimal number of viral particles with the second optimal number of viral particles to produce a reprogramming composition; adding the steps of: optimizing a transduction efficiency for a first pluripot
  • the somatic cells are selected from the group consisting of cells derived from the integument, muscle, heart, kidney, liver, pancreas, lung, nervous system, reticuloendothelial system, vascular system, gastrointestinal system and urogenital system.
  • the viral vector comprises an RNA virus or a DNA virus.
  • kits for reprogramming adult somatic cells to induce pluripotent stem cells comprising: a plurality of recombinant viral vector particles; a diluent; and, instructions for using the recombinant viral vector particles to induce adult somatic cells to become pluripotent stem cells, wherein the viral vector has in serial array a stem cell responsive promoter element, a pluripotent stem cell transcription factor element, a stem cell non-responsive promoter element and a reporter element.
  • Fig. 1 depicts in schematic fashion Lentivirus plasmid maps used in constructing the instant bicistronic viral vectors, as follows: namely, Fig. 1A depicts the plasmid map of the CMV-Oct-4-EF1-GFP construct; Fig. 1 B depicts the plasmid map of the CMV-Sox2-EF1- GFP construct; Fig. 1 C depicts the plasmid map of the CMV-Klf4-EF1-GFP construct; Fig. 1 D depicts the plasmid map of the CMV-c-myc-EF1-GFP construct; and, Fig. 1 E depicts the plasmid map of the CMV-Nanog-EF1-GFP construct.
  • Fig. 2 depicts in a graphic fashion the results of flow cytometric analysis of human embryonic fibroblasts transduced with individual instant viral bicistronic vectors to determine the number of expression units in individual viral vector preparations wherein the y axis represents the fluorescence intensity and the x axis represents the number of cells having that particular fluorescence intensity, as follows: namely, Fig. 2A depicts Oct-4 transduced cells; Fig. 2B depicts Sox-2 transduced cells; Fig. 2C depicts Klf4 transduced cells; Fig. 2D depicts Nanog transduced cells; Fig. 2E depicts c-myc transduced cells; and Fig. 2F depicts a negative control (untreated) that was used to set the negative cut-off gate for the fluorescence sorting of the cells in Figures 2A-2E.
  • FIG. 3 depicts flow cytometric analysis of human embryonic fibroblasts transduced with five (5) viral bicistronic vectors, wherein the y axis represents the fluorescence intensity and the x axis represents the number of cells having that particular fluorescence intensity, using the compositions and methods as illustrated in the Examples section.
  • Fig. 4 shows images of iPS cell colonies at day 18 resulting from the use of the compositions and methods as illustrated in the Examples section.
  • Fig. 5 shows images of iPS cell colonies at day 25 resulting from the use of the compositions and methods as illustrated in the Examples section.
  • Bicistronic Reprogramming Virus As used herein in reference to the instant viral vectors, "bicistronic reprogramming virus", comprise in serial array a stem cell responsive promoter element, a pluripotent stem cell transcription factor element, a stem cell non- responsive promoter element and a reporter element. Representative examples of bicistronic reprogramming virus are provided in the Examples section below.
  • Cell division cycle refers to the cell cycle process of preparing for and executing mitosis to duplicate a cell's genetic information and to form a daughter cell.
  • Those skilled in the art recognize methods for determining the status of a cell within the cell cycle, e.g., for determining the stage in the cell cycle as being G 0 , G 1 , G 2 or M, as well as, determining that a cell has undergone DNA duplication and cell division to form a daughter cell.
  • Cell Reprogramming dose is intended to refer to the amount of pluripotent stem cell transcription factor DNA, RNA or protein that, when delivered into a somatic cells, is effective to (a) induce colony formation; (b) unlimited growth; and, (c) ability to differentiate into any cell type in the mammalian body.
  • Cell surface marker means that the subject cell has on its cellular plasma membrane a protein, an enzyme or a carbohydrate capable of binding to an antibody and/or digesting an enzyme substrate.
  • the subject proteins, enzymes and carbohydrates are recognized in the art to serve as identifying characteristics of particular types of cells, i.e., serving as "markers" to identify particular cell types.
  • compositions which are considered to be permanently epigenetically modified to fulfill a specific function in a tissue. Committed cells are also referred to as “terminally differentiated cells.”
  • Continuous cell culture As used herein, “continuous cell culture” refers to cells in the subject tissue culture that can be passaged on a regular basis continuously in the laboratory, i.e., an immortalized cell line.
  • Dedifferentiation refers to a process of cellular change resulting in an increase in a range of possible cellular functions from a narrow range of specialized functions to a broader range of possible cellular functions, e.g. from a single committed specific function to multiple different possible functions. Dedifferentiation leads to a less committed cell type.
  • Delivery Particle is intended to refer to a particle capable of delivering one or more transcription factor DNAs, RNAs, proteins or protein complexes into a somatic cell in a manner effective to induce intrinsic reprogramming.
  • delivery particles include carbon nanotubes such as single walled and multiwalled nanotubes; polysaccharide particles such as chitin, chitosan, polydextrin, cyclodextrin and agarose beads; magnetic particles; and the like.
  • the instant delivery particle has a size of less than about 5nm in diameter and less than about 300nm in length; more preferably, the instant delivery particle has a size of less than about 3nm in diameter and less than about 350nm in length; and, most preferably, the instant delivery particle has a size of less than about 1 nm in diameter and less than about 200nm in length.
  • Differentiation refers to a process of systematic developmental changes, with accompanying epigenetic changes that occur in cells as they acquire the capacity to perform particular specialized functions in tissues. In cells, differentiation leads to a more committed cell.
  • Embryo refers to an animal in the early stages of growth and differentiation that are characterized implantation and gastrulation, where the three germ layers are defined and established and by differentiation of the germs layers into the respective organs and organ systems.
  • the three germ layers are the endoderm, ectoderm and mesoderm.
  • Embryonic Stem Cell As used herein, “embryonic stem cell”, abbreviated “ESC”, refers to any cell that is totipotent and derived from a developing embryo that has reached the developmental stage to have attached to the uterine wall. In this context embryonic stem cell and pre-embryonic stem cell are equivalent terms.
  • Embryonic stem cell-like (ESC-like) cells are totipotent cells not directly isolated from an embryo. ESC-like cells can be derived from precursor mesenchymal stem cells (PMSC) that have been dedifferentiated in accordance with the teachings of the present disclosure, i.e., yielding pluripotent precursor mesenchymal stem cells.
  • PMSC precursor mesenchymal stem cells
  • Epigenetic is intended to refer to the physical changes that are imposed in a cell upon chromosomes and genes wherein the changes affect the functions of the DNA and genes in the chromosomes and which do not alter the nucleotide sequence of the DNA in the genes.
  • Representative examples of the subject epigenetic changes include covalent chemical modifications of DNA such as methylation and acetylation, as well as, non-covalent and non-chemical modifications of DNA by virtue of DNA super-coiling and association with chromosomal proteins like histones.
  • Representative examples of the way the subject changes affect the functions of the genes in cells include increasing or decreasing the levels of RNAs, and thereby protein products, produced by certain genes and/or changing the way that transcription factors bind at gene region sites termed "promoters”.
  • Epigenetic imprinting is intended to refer to the epigenetic changes imposed upon a DNA in the process of development and differentiation of a cell into a tissue. For instance, the changes imposed upon the DNA in a cell during development of a neural crest cell into a spinal cord or a brain cell, or development of a cardiomyocyte into cardiac muscle cell, or a keratinocye into a skin cell, or a myocyte into a skeletal muscle cell.
  • Expanding is intended to refer to the process for increasing the number of cells in a tissue culture of intrinsically reprogrammed somatic cells.
  • Representative methods for increasing the numbers of reprogrammed cells include tissue culture (a) in media containing one or more growth factors; (b) conditioned media, e.g., "conditioned” by adding the subject media to cultures of embryonic stem cells; and/or (c) in the presence of "feeder” cells, e.g., mouse embryonic fibroblasts (MEFs) producing growth factors and extracellular matrix supportive of stem cell growth.
  • feeder e.g., mouse embryonic fibroblasts (MEFs) producing growth factors and extracellular matrix supportive of stem cell growth.
  • the process of expanding cell numbers can be accomplished e.g., in tissue culture, in a bioreactor or in a cell-compatible implant.
  • the process involves reprogramming the somatic cells in vitro or in vivo and isolating and collecting the reprogrammed somatic cells into an implant material for return to the patient.
  • the host incubates the reprogrammed cells inside the implant material, the implant material keeps the reprogrammed cells from differentiating back into somatic cells and the size of the subject implant material determines the size of the therapeutic unit dose administered to the patient.
  • expression when in reference to a gene, refers to the combined processes of DNA transcription into RNA and/or RNA translation into protein.
  • Extrinsic differentiation refers to the process of introducing one or more reprogramming agents into the outside environment of a cell to effect a change in the cell from a less committed state to a more committed state.
  • Representative examples of differentiation-inducing agents include tissue specific growth factors, their analogs, derivatives and chemical mimetics thereof.
  • Extrinsic reprogramming refers to the process of inducing an epigenetic genomic change in a somatic cell by introducing one or more extrinsic reprogramming agents into the outside environment of a somatic cell, wherein the epigenetic genomic change in the cell effects a change in the functional properties of the cell as evidenced by a change in the cell from a more committed state to a less committed state.
  • extrinsic reprogramming agents include stem cell growth factors such as LIF, bFGF, EGF and the like, as well as, analogues, derivatives and chemical mimetics thereof.
  • Representative examples of methods for effecting extrinsic reprogramming include introducing growth factor ligands into cell culture media, i.e., wherein the growth factor ligand binds to a cell surface receptor and triggers one or more signal transduction process that ultimately induce the epigenetic change in the cell.
  • Germ Line Stem Cells refers to the conserved and protected multipotent, pluripotent and totipotent cells in the reproductive organs that insure the propagation of the species, i.e., ovarian and testicular germ line stem cells.
  • homogeneous refers to cells that are uniformly distributed within the non-cellular components of the composition, e.g., uniformly distributed within a solution, an emulsion, a gel or a biodelivery matrix.
  • Intrinsic differentiation refers to the process of introducing one or more differentiation-inducing agents into a cell to effect an epigenetic change in the cell from a less committed state to a more committed state.
  • Representative examples of differentiation-inducing agents include tissue specific transcription factors like Myo-D, their analogs, derivatives and chemical mimetics thereof.
  • Representative examples of methods for inducing intrinsic differentiation include introducing a single walled nanotube (SWNT) into a cell that carries with it the Myo-D transcription factor thereby effecting a change in the commitment of the cell from a multipotent state to a muscle cell state, i.e., as illustrated in the Examples section.
  • SWNT single walled nanotube
  • iPS cell refers to an adult somatic cell that has been processed using intrinsic reprogramming methods, to effect an epigenetic change from a "committed” and/or “terminally differentiated” state to a less committed state, e.g., a multipotent or "pluripotent” state.
  • That an adult somatic cell has been therapeutically reprogrammed in an intrinsic reprogramming process to an iPS cell is determined by assessing the expression of ESC stem cell markers, i.e., cell surface markers, mRNA markers or RT-PCR markers; or, assessing the potential for stable continuous growth in tissue culture passage; or, assessing the pluripotent differentiative functional capacity of the cells, i.e., to form cell types derived from the ectoderm (e.g., skin), mesoderm (e.g., organs) and endoderm (e.g., linings of the body cavities and blood vessels).
  • ectoderm e.g., skin
  • mesoderm e.g., organs
  • endoderm e.g., linings of the body cavities and blood vessels.
  • Representative examples of ESC stem cell mRNA and RT-PCR and immunohistochemical markers include: Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 Stellar, Alkaline Phosphatase and Rex-1.
  • Representative examples of ESC stem cell surface markers include: CD44, SSEA-4, CD105, CD166, CD90 and CD49f. Representative examples of means for assessing pluripotent differentiative capacity of cells are illustrated in the Examples section.
  • Infection is intended to mean attachment, binding and entry of a replication competent virus into a cell.
  • Intrinsic Reprogramming refers to the process of introducing an intrinsic reprogramming agent into a somatic cell to induce an epigenetic genomic change in the cell that effects a change in the functional properties of the cell as evidenced by a change in the cell from a more committed state to a less committed state.
  • Representative examples of intrinsic reprogramming agents include pluripotent stem cell transcription factors, as well as, analogues, derivatives and chemical mimetics thereof.
  • methods for effecting intrinsic reprogramming include introducing one or more pluripotent stem cell transcription factors into a cell, e.g., by introducing a viral vector into a cell that encodes the pluripotent stem cell transcription factors. .
  • Maturation refers to a process of cellular change toward a more committed state. Representative examples that such a process may be ongoing in an immature cell include evidence for biosynthesis of proteins such as enzymes and extracellular proteins present in the more committed cell type.
  • Multipotent refers to stem cells that can give rise to several other cell types, but those cell types are limited in number.
  • An example of a multipotent stem cell is a hematopoietic stem cell - e.g., a bone marrow stem cell that, while committed to develop into lineages of blood cells such as red and white blood cells, is lacking in the capacity to develop into other types of tissue cells, such as brain cells.
  • Multipotent adult progenitor cells refers to multipotent cells isolated from the bone marrow which have the potential to differentiate into mesenchymal, endothelial and endodermal lineage cells.
  • Passage refers to the process of splitting a growing cell culture into multiple different containers, e.g., one container into three containers (1 :3 passage condition), so that the growth of the cells can continue in a new non-crowded space.
  • Continuous cell cultures can be passaged in a routine manner indefinitely under the same passage conditions.
  • Terminal cell cultures e.g., of differentiated tissue cells, growth more slowly with time in tissue culture, i.e., requiring fewer and fewer passages and splitting to fewer and fewer containers.
  • Post-natal Stem Cell refers to any cell that is multipotent and derived from a multi-cellular organism after birth.
  • Pluripotent refers to cells that can give rise to any cell type except the cells of the placenta or other supporting cells of the uterus.
  • Pluripotent Stem Cell Culture refers to a tissue culture preparation of cells obtained from an animal and serially passaged by splitting the growing cells into containers more than 20 times, preferably more than 30 times, more preferably greater than 60 times and most preferably greater than 100 times.
  • Pluripotent Stem Cell Transcription Factor refers to a transcription factor expressed in a pluripotent stem cell and functionally involved in inducing or maintaining the epigenetic genomic state conducive to unlimited growth and differentiation of the pluripotent stem cell; and/or, directly involved in the unlimited growth potential of the pluripotent stem cell; and/or, involved in maintaining the capacity of the pluripotent stem cell to differentiate into a cell of an ectodermal, mesodermal or endodermal lineage.
  • pluripotent stem cell transcription factors include Oct-4, Sox-2, Klf-4, Nanog, c-myc, Rybp, Zfp219, Sall4, Requiem, Arid 3b, P66 ⁇ , Rex-1 , Nad , Nanog, Sp1 , HDAC2, NF45, Cdk1 and EWS.
  • Embodiments disclosed herein provide methods for reprogramming cells in primary somatic cell cultures with pluripotent stem cell transcription factor DNAs, RNAs and proteins.
  • Pre-natal Stem Cell As used herein, “pre-natal stem cell” refers to a cell that is multipotent and derived from a developing multi-cellular fetus that is no longer in early or mid-stage organogenesis.
  • Primary Culture As used herein, “primary culture” refers to a tissue culture preparation of cells obtained from an animal and serially passaged by splitting the growing cells into containers fewer than 100 times, preferably fewer than 60 times, more preferably fewer than 30 times, and most preferably fewer than 20 times.
  • promoter is used to refer to elements that are generally located in the 5' region of genes, which bind transcription regulatory factors, and which binding alters the function of the gene, e.g., increasing or decreasing the amount of an RNA produced by the gene.
  • Regenerate When used in regard to the instant therapeutic methods, “regenerate” is intended to refer to the process of rebuilding the structural cellular and extracellular elements of a diseased and/or aged tissue so that it is returned to a structure that is less-diseased and more normal and/or youthful.
  • Rejuvenate When used in regard to the instant therapeutic methods, "rejuvenate” is intended to refer to the process of rendering an aged tissue more youthful and vibrant.
  • reporter cell line is intended to refer to a plurality of reprogrammed somatic cells capable of unlimited self-renewal, constructed by instrinsic reprogramming of a normal or a diseased somatic cell, and containing one or more marker genetic elements.
  • reporter cell lines are disclosed in the examples section below, e.g., human testicular cells containing an RFP (red fluorescent protein) marker gene under the control of an Oct-4 promoter.
  • Reprogamming refers to the epigenetic genomic changes that result in a committed cell being induced to enter a less committed state. Representative examples include epigenetic changes sufficient to induce terminally differentiated somatic cell to exhibit functional properties of a Multipotent or a Pluripotent cell. Induced pluripotent stem (iPS) cells are one example of reprogrammed cells wherein adult somatic cells are modified by introduction of transcription factors to become ESC-like.
  • iPS Induced pluripotent stem
  • Restore When used in regard to the instant therapeutic methods, "restore” is intended to refer to the process of bringing the function of a tissue from a diseased or aged state back to a more normal and/or youthful state.
  • RT-PCR marker As used herein with regard to a cell in a cell culture of RPSC, "RT-PCR marker" means that the subject cell has in its cellular cytoplasm an RNA that can be copied and amplified using a polymerase chain reaction (PCR) methodology.
  • PCR polymerase chain reaction
  • the subject RNAs are recognized in the art to serve as identifying characteristics of particular types of cells, i.e., serving as "markers" to identify particular cell types.
  • Somatic Cell refers to any cell in a tissue in the mammalian body except gametes and their precursors. Representative examples include fibroblasts, epithelial cells, retinal pigment epithelial cells, lung epithelial cells, kidney proximal tubule cells.
  • Somatic Stem Cells refers to diploid multipotent or pluripotent stem cells resident in a tissue in the mammalian body. Somatic stem cells are not totipotent stem cells and many are now understood not to be pluripotent. Representative examples include neural stem cells, kidney stem cells, muscle satellite stem cells, cartilage satellite stem cells and the like.
  • substantially purified means that, with regard to the cells in the composition, fewer than 25% are of a type other than RPSC; preferably, fewer than 15% are of a type other than RPSC; more preferably, fewer than 10% are of a type other than RPSC; and, most preferably, fewer than 5% are of a type other than RPSC.
  • Therapeutic Unit Dose When used in reference to reprogrammed cells, "therapeutic unit dose” is intended to refer to that number of cells that is effective to regenerate, restore or rejuvenate a tissue to its natural non-diseased and/or non-aged state,
  • Totipotent refers to cells that have an epigenetic genomic state that allows them to differentiate into any cell type in any tissue of a mammalian body including the placenta. Without reprogramming, native human embryonic cells only have totipotent properties during the first few divisions after fertilization of an ovum (egg).
  • Transaction is intended to refer to the process of delivering 3, 4, 5 or 6 lentiviral vectors simultaneously into a cell in a manner effective to induce intrinsic reprogramming of a somatic cell.
  • Representative examples of transaction processes are disclosed in applicant's co-pending provisional patent applications 60/953,395, filed August 1 , 2007; 60/974,395, filed September 2, 2007; and, 61/024,836, filed January 1 , 2008, incorporated herein by reference, including compositions, methods and uses of particles for delivery, e.g. single and multi-walled nanotubes (SWNT), chitosan particles, cyclodextrin particles and the like.
  • SWNT single and multi-walled nanotubes
  • the instant transaction process delivers a Cell Reprogramming Dose of one or more pluripotent stem cell transcription factor lentiviral vectors into the cell in a manner effective to induce up-regulated expression of one or more genes having a promoter region that binds Oct-4, Sox-2, Klf-4, Nanog, c-myc, Rybp, Zfp219, SaIW, Requiem, Arid 3b, P66 ⁇ , Rex-1 , Nad , Sp1 , HDAC2, NF45, Cdk1 or EWS as well as proteins associated therewith in pluripotent stem cell transcription factor complexes.
  • Transcription Factor Complex is intended to refer to the natural unassisted association of multiple different transcription factor proteins into an aggregate by virtue of the innate propensities of the different transcription factor proteins for one another.
  • the Oct-4 transcription factor complex is one example of the self-association of the Oct-4 protein with other proteins including e.g. Rybp, Zfp219, Sall4, Requiem, Arid 3b, P66 ⁇ , Rex-1 , Nad , Nanog, Sp1 , HDAC2, NF45, Cdk1 and EWS.
  • Transduction is intended to mean recombinant replication-defective viral vector mediated introduction of genetic information into a cell.
  • Transfection is intended to mean plasmid DNA mediated introduction of genetic information into a cell.
  • Optimal Dilution As used herein in reference to transduction of a bicistronic reprogramming virus into cells, "optimal dilution", is intended to mean that solution concentration of viral vector which is effective to introduce a gene of interest into about 30% to 60% of cells; preferably, greater than 40% host cells; more preferably greater than about 50% of cells; and, most preferably, greater than about 60% of cells.
  • Reporter is intended to mean a gene which when expressed inside a cell results in production of a signal generating compound.
  • signal generating compounds include fluorescent, luminescent and paramagnetic compounds, as well as, enzymes and enzyme co-factors.
  • reporter include green and red fluorescent proteins and enzymes such as luciferase, alkaline phosphatase (AP), beta-galactosidase (lacZ), glucuronidase and the like.
  • Transduction Mixture As used herein in reference to introduction at the same time of three (3), four (4), five (5) or six (6) viral vectors into a cell, "transduction mixture”, means a mixture of the instant bicistronic viral vectors
  • stem cell responsive promoter means a promoter that is down-regulated in an induced pluripotent stem cell.
  • promoters regulated in this manner include viral promoters, e.g., the human cytomegalovirus (CMV) promoters such as the CMV immediate-early enhancer, as well as, HSV-1 promoters and HSV-2 promoters; and, promoters expressed in differentiated tissues, e.g., the cardiac sodium-calcium exchanger (NCX1 ) promoter, the muscle MyoD promoter, the connective tissue collagenase A2 (col A2) promoter, the ubiquitous phosphoglycerate kinase (PGK) promoter, the cardiomyocyte atrial natriuretic factor (ANF) promoter, the cardiac ventricular myosin light chain (MLC2v) promoter, the type Il alveolar epithelial cell (AT-2)-specific promoter and the
  • Non-Stem Cell Responsive Promoter means a promoter that is not down-regulated in an induced pluripotent stem cell.
  • Representative examples of non- stem cell responsive promoters include EF1 alpha promoter, the Ubiquitin-C promoter, the retroviral LTR promoter, the actin promoter, the ATP synthase promoter, the microglobulin promoter and the IRES promoter.
  • Viral vector means a recombinant replication incompetent virus capable of expressing a gene of interest in a cell.
  • Representative examples of viral vectors include recombinant RNA and DNA viruses, e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, HIV viral vectors, SIV viral vectors, HTLV viral vectors, FIV viral vectors and SV40 viral vectors.
  • the present disclosure provides bicistronic reprogramming viral vectors in which a first stem cell responsive promoter drives downstream production of a pluripotent stem cell transcription factor and a second non-stem cell responsive promoter drives a reporter such as green fluorescent protein (GFP) or red fluorescent protein (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the disclosure provides expression-based methods for optimizing the efficiency of delivery of 3, 4, 5 or 6 different Antiviruses into a single human somatic cell to effect simultaneous high level expression of 3, 4, 5 or 6 genes in a manner effective to produce epigenetic reprogramming and induction of iPS cells. The method allows simultaneous optimization of both transduction and expression efficiency.
  • the method determines the number of viral particles needed to produce the maximal number of cells expressing the virus construct for each individual lentiviral vector (of the 3, 4, 5 or 6).
  • the number of cells expressing reporter is determined by cytofluorimetric counting (illustrated in the Examples section, below), or e.g., by microscopically counting the number of individual cells that are positive for expression of the reporter at different dilutions of a viral stock solution and then multiplying the dilution factor times the number of positive cells.
  • the number of viral expression units per milliliter in the stock solution can be calculated.
  • the instant method compensates for any possible innate differences in expression of different recombinant viral vectors.
  • the disclosure provides that for treatment of adult somatic cells to induce iPS cells, the following applies: namely, [0094] a) Equal numbers of the respective different expression units are used to construct a mixture of the 3, 4, 5 or 6 different viral vectors in which each virus is, at the same time, present at the number of units optimal for expression of its own particular virus construct and in numbers of units equal to all of the other virus constructs in the mixture;
  • the adult somatic cells are counted and the number of expression units for each of the 3, 4, 5, or 6 viral vectors in the mixture, referred to as the modulus of infection (MOI), is at least equal to the number of adult somatic cells, i.e., an MOI greater than 1.0; preferably, the MOI is greater than 1.25; more preferably, the MOI is greater than 1.5; and, most preferably, the MOI is greater than 2;
  • MOI modulus of infection
  • the resultant combined transduction efficiency can be determined within 48 hours of infection, i.e., by counting the number of cells expressing the reporter; and,
  • the method allows sorting of the individual viral vector transduced cells, by sorting for cells expressing the reporter, thereby enriching for cells that have received the necessary reprogramming instructions.
  • the number of cells expressing the reporter is determined at different dilutions of a virus stock solution about 48 hrs after the viral vectors are transduced into the cells.
  • the virus dilution giving about 30% to about 60% of the total cells reporter positive is considered the optimal dilution; preferably, greater than about 40% of the total cells are reporter positive; more preferably, greater than about 50% of the total cells are reporter positive; and, most preferably, greater than about 60% of the total cells are reporter positive.
  • the process is repeated for each different lentivirus construct to determine each different optimal dilution.
  • a transduction mixture of lentiviral vectors is then prepared by mixing each of the different 3, 4, 5 or 6 Antiviruses to achieve its respective different final optimal dilution.
  • the transduction mixture when added to cells, induces expression of the reporter in about 60% to about 98% of the total cells after about 48 hrs in tissue culture; preferably, greater than about 60% of the total cells are reporter positive; more preferably, greater than about 75% of the total cells are reporter positive; and, most preferably, greater than about 90% of the total cells are reporter positive.
  • compositions offering advantages in that the design of the instant bicistronic lentiviral vectors provides a method for shutting down the expression of the viral vector gene in the transduced cell when it becomes an iPS cell, i.e., by using a stem cell responsive promoter that is active in a somatic cell and down-regulated in a stem cell.
  • This has the advantage of enabling a shutdown of expression from the stem cell responsive promoter once the cell is re-programmed and endogenous pluripotent genes are up-regulated; and, while the reporter construct (e.g. GFP) still expresses from the stem cell non-responsive promoter. Therefore, the resulting transduced cells, as well as the iPS cells, are easily identifiable by reporter expression.
  • the present disclosure provides bicistronic lentiviral vectors in which the stem cell responsive promoter is a promoter selected from a promoter that is expressed in a differentiated tissue cell type.
  • the stem cell responsive promoter is a promoter selected from a promoter that is expressed in a differentiated tissue cell type.
  • Representative non-limiting examples include promoters expressed in differentiated tissues, e.g., the cardiac sodium-calcium exchanger (NCX1 ) promoter, the muscle MyoD promoter, the connective tissue collagenase A2 (col A2) promoter, the ubiquitous phosphoglycerate kinase (PGK) promoter, the cardiomyocyte atrial natriuretic factor (ANF) promoter, the cardiac ventricular myosin light chain (MLC2v) promoter, the type Il alveolar epithelial cell (AT-2)-specific promoter and the human surfactant protein C (SP-C) promoter.
  • the cardiac sodium-calcium exchanger e.g., the cardiac
  • the instant differentiated tissue promoter when an adult somatic cell is reprogrammed to achieve an iPS cell status the instant differentiated tissue promoter is down-regulated. If the resultant culture of iPS cells begins to spontaneously differentiates back into the cell type from which they originated, the promoter regains activity and pluripotent gene expression is reestablished, i.e., driving the cells back toward the iPS cell phenotype.
  • This composition and method has the advantage of trapping cells in an iPS cell state and preventing their spontaneous differentiation, i.e., a highly useful feature for maintaining long-term cultures of iPS cells for research purposes and uses in screening to identify useful new pharmaceutical compounds.
  • the present disclosure provides biologically useful pluripotent therapeutically reprogrammed adult somatic cells and methods for their preparation.
  • the instant cells have pluripotent growth and differentiative capacities similar to embryonic stem cells (that is, ESC- like).
  • therapeutically reprogrammed cells can be prepared for use in autologous therapies, i.e., where the cells are collected, reprogrammed and returned to the subject.
  • autologous therapies i.e., where the cells are collected, reprogrammed and returned to the subject.
  • the instant therapeutically reprogrammed cells are immunologically identical to the host and therefore suitable for therapeutic applications.
  • Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus.
  • cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number.
  • An example of multipotent cells is hematopoietic cells - blood cells that can develop into several types of blood cells, but cannot develop into brain cells.
  • At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells - cells that are considered to be permanently committed to a specific function.
  • Embryonic stem cells are cells derived from the inner cell mass of the pre- implantation blastocyst-stage embryo and have the greatest differentiation potential, being capable of giving rise to cells found in all three germ layers of the embryo proper. From a practical standpoint, embryonic stem cells are an artifact of cell culture since, in their natural epiblast environment, they only exist transiently during embryogenesis. Manipulation of embryonic stem cells in vitro has lead to the generation and differentiation of a wide range of cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerves, skeletal muscle, chondrocytes, adipocytes, liver and pancreatic islets. Growing embryonic stem cells in co-culture with mature cells can influence and initiate the differentiation of the embryonic stem cells to a particular lineage.
  • an embryo and a fetus are distinguished based on the developmental stage in relation to organogenesis.
  • the pre-embryonic stage refers to a period in which the pre-embryo is undergoing the initial stages of cleavage.
  • Early embryogenesis is marked by implantation and gastrulation, wherein the three germ layers are defined and established.
  • Late embryogenesis is defined by the differentiation of the germ layer derivatives into formation of respective organs and organ systems.
  • the transition of embryo to fetus is defined by the development of most major organs and organ systems, followed by rapid pre-natal growth.
  • Embryogenesis is the developmental process wherein an oocyte fertilized by a sperm begins to divide and undergoes the first round of embryogenesis where cleavage and blastulation occur. During the second round, implantation, gastrulation and early organogenesis takes place. The third round is characterized by organogenesis and the last round of embryogenesis, wherein the embryo is no longer termed an embryo, but a fetus, is when pre-natal growth and development occurs.
  • the first two tissue lineages arising from the morulae post-cleavage and compaction are the trophectoderm and the primitive endoderm, which make major contributions to the placenta and the extraembryonic yolk sac. Shortly after compaction and prior to implanting the epiblast or primitive ectoderm begins to develop.
  • the epiblast provides the cells that give rise to the embryo proper. Blastulation is complete upon the development of the epiblast stem cell niche wherein pluripotent cells are housed and directed to perform various developmental tasks during development, at which time the embryo emerges from the zona pellucida and implants to the uterine wall. Implantation is followed by gastrulation and early organogenesis. By the end of the first round of organogenesis, all three germ layers will have been formed; ectoderm, mesoderm and definitive endoderm and basic body plan and organ primordia are established.
  • embryogenesis is marked by extensive organ development at which time completion marks the transformation of the developing embryo into a developing fetus which is characterized by pre-natal growth and a final round of organ development.
  • the gestation period is ended by birth, at which time the organism has all the required organs, tissues and cellular niches to function normally and survive post-natally.
  • the process of embryogenesis is used to describe the global process of embryo development as it occurs, but on a cellular level embryogenesis can be described and/or demonstrated by cell maturation.
  • Pre-natal stem cells have been isolated from the pre-natal bone marrow (hematopoietic stem cells), pre-natal brain (neural stem cells) and amniotic fluid (pluripotent amniotic stem cells). In addition, stem cells have been described in both adult male and prenatal tissues. Pre-natal stem cells serve multiple roles during the process of organogenesis and pre-natal development, and ultimately become part of the somatic stem cell reserve.
  • Maturation is a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation and/or dedifferentiation.
  • a cell, or group of cells interacts with its cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches and these niches, or microenvironments, house stem cells that direct and regulate organogenesis. At the time of birth, maturation has progressed such that cells and appropriate cellular niches are present for the organism to function and survive post- natally. Developmental processes are highly conserved amongst the different species allowing maturation or differentiation systems from one mammalian species to be extended to other mammalian species in the laboratory.
  • a single stem cell clone can contribute to generations of lineages such as lymphoid and myeloid cells for more than a year and therefore have the potential to spread mutations if the stem cell is damaged.
  • the body responds to a compromised stem cell by inducing apoptosis thereby removing it from the pool and preventing potentially dysfunctional or tumorigenic properties.
  • Apoptosis removes compromised cells from the population, but it also decreases the number of stem cells that are available for the future. Therefore, as an organism ages, the number of stem cells decrease. In addition to the loss of the stem cell pool, there is evidence that aging decreases the efficiency of the homing mechanism of stem cells.
  • Telomeres are the physical ends of chromosomes that contain highly conserved, tandem repeated DNA sequences.
  • Telomeres are involved in the replication and stability of linear DNA molecules and serve as counting mechanism in cells; with each round of cell division the length of the telomeres shortens and at a pre-determined threshold, a signal is activated to initiate cellular senescence.
  • Stem cells and somatic cells produce telomerase, which inhibits shortening of telomeres, but their telomeres still progressively shorten during aging and cellular stress.
  • stem cells can be differentiated into particular cell types in vitro and shown to have the potential to be multipotent by engrafting into various tissues and transit across germ layers and as such have been the subject of much research for cellular therapy.
  • immune rejection is the limiting factor for cellular therapy.
  • the recipient individual's phenotype and the phenotype of the donor will determine if a cell or organ transplant will be tolerated or rejected by the immune system.
  • the present disclosure provides methods and compositions for providing functional immunocompatible stem cells for cellular regenerative/reparative therapy.
  • the disclosure provides cellular compositions of therapeutically reprogrammed adult pluripotent somatic cells (RPSC) in which greater than 5% of cells present express at least one ESC stem cell marker selected from the group consisting of Oct-4, Nanog, SSEA- 3, SSEA-4, TRA-1-60 and Rex-1 ; preferably, greater than 10% of the cells express at least one of these ESC stem cell markers; more preferably, greater than 50% of the cells express at least one of these ESC stem cell markers; and, most preferably, greater than 75% of the cells express at least one of these ESC stem cell markers.
  • the instant cellular compositions are stable continuous cell cultures of RPSC; suspensions of cells; and, biodelivery devices containing cells e.g. prepared for therapeutic use in subjects in need thereof.
  • the instant RPSC are derived by therapeutically reprogramming adult somatic cells derived from humans, domesticated animals, wild mammals, birds and boney fishes.
  • adult somatic cell for derivation of the instant RPSC is of course at the discretion of the physician and patient and will vary depending upon at least the medical condition, age, location where the treatment is to be administered and chromosomal status, e.g., the extent of age-related DNA damage.
  • adult somatic cells useful in the instant methods include ectodermal cells such as fibroblasts and epithelial cells; mesodermal organ cells such as bone marrow cells, CD34 + peripheral blood stem cells, cardiomyocytes, myocytes, vascular smooth muscle cells, hepatocytes and renal cells; and, endodermal endothelial cells such as vascular endothelial cells.
  • germ line stem cells such as those described in Applicants' co-pending patent applications 11/488,362, filed July 17, 2006; 11/694,687, filed March 30, 2007; 60/954,496, filed August 7, 2007; and, 61/026,502, filed February 6, 2008 are preferred cells for production of RPSC.
  • Certain embodiments provide methods for producing RPSC involving the steps of obtaining a somatic cell sample from an adult or pre-natal subject; therapeutically reprogramming the adult somatic cells in the cell sample using an intrinsic reprogramming method that introduces pluripotent stem cell transcription factor genes into a cell; and, verifying that the adult somatic cells are RPSC by testing for the expression of an ESC stem cell marker.
  • ESC stem cell markers are provided.
  • the instant viral vectors are provided.
  • methods are provided for transduction of cells by delivery of pluripotent stem cell transcription factor DNAs into cells in a manner effective to induce reprogramming of somatic cells.
  • reporter cell lines and processes for constructing such cell lines find a variety of uses in medicine including screening for pharmaceutical compounds that alter gene expression.
  • Representative examples of reporter cell lines include, e.g., human testicular cells containing an RFP (red fluorescent protein) marker gene under the control of an Oct-4 promoter.
  • Other examples of reporter cell lines include intrinsically reprogrammed somatic cells containing markers for up- regulation of apoptotic genes including e.g., calpain and cdk5/p25; alteration of oxygen homeostasis, e.g.
  • HIF-1 changed mitochondrial function, e.g., PGC-1 ; cytoprotection, e.g., ALDH1A1 , ALDH1A7, BIRC5/surviving, GST M5, GST A2, GST P1 , NAD(P) quinine reductase (NQO1 ) and Nrf2; adipocyte/fat development, e.g., SRC-3; induction of immune tolerance, e.g., FoxP3; and, induction of immune T-helper cells, e.g., STAT6 or GAT A-3.
  • cytoprotection e.g., ALDH1A1 , ALDH1A7, BIRC5/surviving, GST M5, GST A2, GST P1 , NAD(P) quinine reductase (NQO1 ) and Nrf2
  • adipocyte/fat development e.g., SRC-3
  • induction of immune tolerance e.g., FoxP3
  • methods for treating a subject in need of regenerative, restorative or rejuvenative stem cell therapy with autologous iPS cells that obviate problems of transplant rejection and graft versus host disease.
  • the method involves collecting a tissue sample from the subject; isolating somatic cells from the tissue; reprogramming the isolated somatic cells according to the methods disclosed herein to produce multipotent or pluripotent stem cells; expanding the numbers of the reprogrammed cells to produce a therapeutic unit dose; and, (a) if the aim of the therapy is to provide a stem cell therapy, then returning the cells to the subject, or alternatively, (b) if the aim of the therapy is to provide a differentiated cell therapy, then differentiating the reprogrammed stem cells back into a somatic cell before returning the cells to the subject.
  • the instant therapeutic method solves a significant problem inherent in tissue transplantation therapies: namely, in most cases because somatic cells are terminally differentiated, they cannot be successfully propagated in tissue culture under conditions that will enable production of a therapeutic unit dose. As a result, it is at present common to transplant patients with cells derived from another individual, e.g., cadaveric cells or cord blood cells. Reprogramming somatic cells restores their potential for unlimited growth without producing cancerous cells. While not wishing to be tied to any particular mechanism(s), it is presently believed that the instant intrinsic reprogramming methods preserve the epigenetic imprinting of the original tissue of origin. For example, skin cells that are intrinsically reprogrammed "remember" via their epigenetic imprinting that they are skin cells and not cancer cells.
  • Treatments for age-related macular degeneration (both the wet and dry forms), involving collecting retinal pigment epithelial (RPE) cells from the eye of a patient with the disease; reprogramming the RPE cells according to the disclosed methods; expanding the cells to produce a therapeutic unit dose; and (a) if stem cell therapy is the objective, delivering the therapeutic unit dose of reprogrammed cells to the patient, or alternatively, (b) if differentiated cell therapy is the objective, then re-differentiating the reprogrammed cells back into RPE before delivery to the patient;
  • RPE retinal pigment epithelial
  • IDDM Type-1 insulin-dependent diabetes mellitus
  • Type-2 diabetes involving collecting islet cells ( ⁇ , ⁇ , ⁇ and the like) from the pancreas of a new- onset patient, intrinsically reprogramming the islet cells according to the instant methods, expanding the reprogrammed cells to produce a therapeutic unit dose and (a) if the objective in the therapy is to provide a stem cell therapy, then delivering the therapeutic unit dose of the reprogrammed cells to a tissue location in the patient, or alternatively, (b) if the objective in the therapy is to provide a differentiated cell therapy, then differentiating the reprogrammed islet cells back into specialized islet cells, e.g.
  • tissue location in the patient may be the same or different from the origin of the tissue sample.
  • somatic cells may be collected from the pancreas and returned e.g. to sites in the liver, skin or kidney capsule;
  • Treatments for non-union bone fractures involving collecting osteocytes and osteoblasts from a patient; intrinsically reprogramming the cells according to the instant methods; expanding the cells to produce a therapeutic unit dose; and (a) if the objective is stem cell therapy, delivering the therapeutic unit dose of the reprogrammed cells to the patient, or alternatively, (b) if the objective is differentiated cell therapy, differentiating the reprogrammed cells back into osteocytes and osteoblasts before delivery of the therapeutic unit dose to the patient.
  • the instant intrinsic reprogramming methods yield efficiencies for five transcription factor reprogramming at greater than about 1% efficiency, preferably greater than about 5% efficiency and most preferably greater than about 10% efficiency. This high efficiency enables, for the first time, autologous stem cell therapies using reprogrammed adult somatic cells.
  • the route of delivery according to the instant methods is determined by the disease and the site where treatment is required.
  • it may prove desirable to apply the instant cellular compositions at the local site e.g., by placing a needle into the tissue at that site or by placing a timed-release implant or patch); while in a more acute disease clinical setting it may prove desirable to administer the instant cellular compositions systemically.
  • the instant cellular compositions may be delivered by intravenous, intraperitoneal, intramuscular, subcutaneous and intradermal injection, as well as, by intranasal and intrabronchial instillation (e.g., with a nebulizer), transdermal delivery (e.g., with a lipid-soluble carrier in a skin patch), or gastrointestinal delivery (e.g., with a capsule or tablet).
  • intranasal and intrabronchial instillation e.g., with a nebulizer
  • transdermal delivery e.g., with a lipid-soluble carrier in a skin patch
  • gastrointestinal delivery e.g., with a capsule or tablet.
  • the preferred therapeutic cellular compositions for inocula and dosage will vary with the clinical indication.
  • the inocula may typically be prepared from a frozen cell preparation, e.g.
  • a physiologically acceptable diluent such as saline, phosphate-buffered saline or tissue culture medium.
  • a physiologically acceptable diluent such as saline, phosphate-buffered saline or tissue culture medium.
  • the instant cellular compositions may to be administered alone or in combination with one or more pharmaceutically acceptable carriers, e.g. in either single or multiple doses.
  • suitable pharmaceutical carriers may include inert biodelivery gels or biodegradable semi-solid matrices, as well as, diluents or fillers, sterile aqueous solutions and various nontoxic solvents.
  • the subject pharmaceutically acceptable carriers generally perform three functions: namely, (1 ) to maintain and preserve the cells in the instant cellular composition; (2) to retain the cells at a tissue site in need of regeneration, restoration or rejuvenation; and, (3) to improve the ease of handling of the instant composition by a practitioner, e.g., to improve the properties of an injectable composition or the handling of a surgical implant.
  • compositions formed by combining an instant cellular composition with a pharmaceutically acceptable carrier may be administered according to the instant methods in a variety of dosage forms such as syrups, injectable solutions, and the like.
  • the subject pharmaceutical carriers can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like.
  • additional ingredients such as flavorings, binders, excipients, and the like.
  • capsules might additionally include additives such as lactose or milk sugar and/or polyethylene glycols as cellular preservatives.
  • solutions may be prepared in sesame or peanut oil or in aqueous polypropylene glycol, as well as sterile aqueous isotonic saline solutions.
  • the subject aqueous solution is preferably suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • Such aqueous solutions of instant cellular composition may be particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal injection.
  • the subject sterile aqueous media employed are obtainable by standard techniques well known to those skilled in the art.
  • it may prove desirable to stabilize a instant cellular composition e.g.
  • Improving the shelf-life stability of cell compositions may be accomplished by adding excipients such as: a) hydrophobic agents (e.g., glycerol); b) non-linked sugars (e.g., sucrose, mannose, sorbitol, rhamnose, xylose); c) non-linked complex carbohydrates (e.g., lactose); and/or d) bacteriostatic agents or antibiotics.
  • excipients such as: a) hydrophobic agents (e.g., glycerol); b) non-linked sugars (e.g., sucrose, mannose, sorbitol, rhamnose, xylose); c) non-linked complex carbohydrates (e.g., lactose); and/or d) bacteriostatic agents or antibiotics.
  • the preferred pharmaceutical compositions for inocula and dosage for use in the instant methods will vary with the clinical indication.
  • the inocula may typically be prepared from a concentrated cell solution by the practicing physician at the time of treatment, e.g., by thawing and then diluting a concentrated frozen cell suspension in a storage solution into a physiologically acceptable diluent such as phosphate-buffered saline or tissue culture medium.
  • a physiologically acceptable diluent such as phosphate-buffered saline or tissue culture medium.
  • the effective amount of the instant cellular composition per unit dose depends, among other things, on the body weight, physiology, and chosen inoculation regimen.
  • a unit dose of the instant cellular composition refers to the number of cells in the subject suspension. Generally, the number of cells administered to a subject in need thereof according to the practice of the disclosed methods will be in the range of about 10 5 /site to about 10 9 /site. Single unit dosage forms and multi-use dosage forms are considered within the scope of the disclosure, as disclosed further below.
  • the instant cellular composition may be provided in an emollient cream or gel.
  • non-toxic cell-preservative emollient pharmaceutically acceptable carriers include cell-oil-in-water and cell-water-in-oil emulsions, i.e., as are known to those skilled in the pharmaceutical arts.
  • the disclosed provides different routes for delivery of the instant cellular compositions as may be suitable for use in the different disease states and sites where treatment is required.
  • routes for delivery of the instant cellular compositions as may be suitable for use in the different disease states and sites where treatment is required.
  • topical, intrathecal, intramuscular or intra-rectal application it may prove desirable to apply the subject cells in a cell-preservative salve, ointment or emollient pharmaceutical composition at the local site, or to place an impregnated bandage or a dermal timed-release lipid-soluble patch.
  • intra-rectal application it may prove desirable to apply the instant cellular compositions, e.g. in a suppository.
  • pulmonary airway restoration, regeneration and rejuvenation it may prove desirable to administer the instant cellular compositions by intranasal or intrabronchial instillation (e.g., as pharmaceutical compositions suitable for use in a nebulizer).
  • intranasal or intrabronchial instillation e.g., as pharmaceutical compositions suitable for use in a nebulizer.
  • gastrointestinal regenerative medicine it may prove desirable to administer the instant cellular compositions by gastrointestinal delivery (e.g., with a capsule, gel, trouch or suppository).
  • suppositories for urethral and vaginal use in regenerative medical treatments of infertility and the like.
  • the subject pharmaceutical compositions are administered via suppository taking advantage of the migratory capacity of instant cells, e.g., migration between the cells in the epithelial lining cells in the rectum, into the interstitial tissues and into the blood stream in a timed- release type manner.
  • the instant methods i.e., employing the instant cellular compositions make it feasible to administer therapy in a multi-dosage form, e.g. via an implantable mini- pump (such as used for delivery of insulin in patients with Type 1 insulin-dependent diabetes mellitus).
  • an implantable mini- pump such as used for delivery of insulin in patients with Type 1 insulin-dependent diabetes mellitus
  • the method may involve administration of an intravenous bolus injection or perfusion of the instant cellular compositions, or may involve administration during (or after) surgery, or a prophylactic administration.
  • the instant administration may involve a combination therapy, e.g., the instant cellular composition and a second drug, e.g., an anti-coagulant, anti-infective or antihypertensive agent.
  • the route of delivery of the subject preparations determined by the particular disease.
  • topical application it may be useful to apply the instant cellular compositions at the local site (e.g., by injection, while for other indications the preparations may be delivered by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, and intradermal injection, as well as, by transdermal delivery (e.g., with a lipid-soluble carrier in a skin patch placed on the skin), or even by oral and/or gastrointestinal delivery (e.g., with a capsule, tablet or suppository).
  • therapeutically reprogrammed pluripotent adult somatic cells are provided.
  • Therapeutic reprogramming refers to a dedifferentiate process wherein an adult somatic cell or multipotent stem cell, i.e., a cell committed to forming certain tissue cell lines, is exposed intracellular ⁇ to Oct-4 complex proteins, supra, according the teachings of the present disclosure to yield an RPSC, i.e., an ESC-like pluripotent cell capable of forming any body cell.
  • Embodiments disclosed herein provide RPSC cellular compositions that contain greater than 75% of cells expressing one or more pluripotent stem cell marker such as Oct- 4, nanog, SSEA- 3/4, TRA-1-60 and Rex-1 ; preferably, greater than 80% of cells express one or more pluripotent stem cell markers; more preferably, greater than 90% of cells express one or more pluripotent stem cell markers; and, most preferably, greater than 95% of cells express one or more pluripotent stem cell markers.
  • pluripotent stem cell marker such as Oct- 4, nanog, SSEA- 3/4, TRA-1-60 and Rex-1 .
  • Embodiments disclosed herein provide RPSC cellular compositions where pluripotency is confirmed by requiring that the cells have been passaged more than 10 times since their isolation from pre-natal bone marrow; preferably, the cells have been passaged more than 12 to 14 times since isolation; more preferably, the cells have been passaged more than 15 to 16 times since isolation; and, most preferably, the cells have been passaged more than 17 to 18 times since isolation.
  • the cells have undergone more than 20 cell division cycles since their isolation from pre-natal bone marrow; preferably, the cells have undergone greater than 30 cell division cycles since isolation; more preferably, the cells have undergone greater than 40 cell division cycles since isolation; and, most preferably, the cells have undergone greater than 50 cell division cycles since isolation.
  • the instant RPSC cellular compositions are precursors in production of differentiated tissue cells (DTC) such as adipocytes, chondrocytes, neural cells, epithelial cells, muscle cells, cardiomyocytes, pancreatic islet cells, osteocytes, lung parenchymal cells, liver hepatocytes and renal epithelial and proximal tubule cells.
  • DTC differentiated tissue cells
  • Embodiments of the present disclosure provide methods for producing DTC compositions, e.g., by culturing the instant cellular compositions under defined conditions in a differentiation media that is suitable and sufficient for the induction and growth of specific different types of DTCs.
  • That a instant cellular composition has differentiated into a DTC may be determined by testing the staining reaction of the cells or testing for the presence of a cell surface marker or an RT- PCR marker.
  • Representative examples of staining tests for determining that a instant cellular composition has differentiated into a DTC include Oil Red O staining for adipocytes, Alcian Blue staining for chondrocytes and Alizarin Red S staining for osteocytes.
  • cell surface markers for determining that a reprogrammed cell according to the disclosed methodd has differentiated into a DTC include tub-Ill, Map2, Nestin, 04, GaIC and GFAP for certain neural cells; tub-Ill, Map2, Nestin, 04, GaIC and GFAP for other types of neural cells; and, troponin, connexin 43 and cardiac-actin for cardiomyocytes.
  • methods for autologous cell therapy i.e., a process where a practitioner collects adult somatic cells from a subject; a laboratory or a machine therapeutically reprograms the cells in the sample ex vivo to product RPSC; and, the cells are then administered therapeutically to the same subject.
  • Autologous RPSC do not express "foreign” histocompatibility antigens; are recognized as “self” by the immune system of the subject; are not subject to transplant rejection; and, do not mediate graft versus host disease (GVHD).
  • GVHD mediate graft versus host disease
  • the present therapeutic reprogrammed pluripotent adult somatic cells with ESC-like cell have plasticity and may be used as a cellular replacement therapy in different disease/trauma states including e.g. treatments of Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, sickle cell anemia, thalasemia, cystic fibrosis, fibromyalgia, Type-1 diabetes, nonunion bone fractures, cosmetic and reconstructive surgery for skin, cartilage and bone, myocardial infarct, stroke, spinal cord injury, traumatic injury, and restoring, regenerating and rejuvenating damaged and aged tissues.
  • Parkinson's disease Huntington's disease
  • multiple sclerosis multiple sclerosis
  • Alzheimer's disease e.g., Alzheimer's disease, sickle cell anemia, thalasemia, cystic fibrosis, fibromyalgia
  • Type-1 diabetes non
  • pluripotent stem cell transcription factors present in embryonic cells
  • pluripotent stem cell transcription factors need to be produced inside the cells so that they can exert their effects inside the nucleus.
  • independent investigators have expressed four (or five) different pluripotent stem cell transcription factors in fibroblasts using viral transduction and expression systems, but these methods have disadvantages in that the transduction efficiencies are very low, i.e., in one report fewer than 6 cells in 10,000 may receive all of the needed five transcription factors.
  • the reprogramming methods for somatic cells are also very labor intensive requiring 30-40 days tissue culture containing the appropriate growth factors and on feeder layers of fetal cells. Many 30 day cultures presently fail to yield iPS cells. As a result, it is at present very difficult to routinely induce iPS cells and equally difficult to identify them in culture.
  • an optimized viral transduction method was developed.
  • each of the respective different ligated plasmid DNAs was transformed into Stbl3 bacteria (invitrogen). Each of the bacterial cultures were grown overnight at 37°C and each of the resultant plasmids was individually isolated from the respective bacterial lysates using a midi-prep procedure (Invitrogen).
  • Replication Defective Viral Vector For mammalian cell production of replication defective lentivirus, 293FT cells (Invitrogen) were grown in D-MEM medium containing Penicillin/Streptomycin, Sodium-pyruvate, non essential amino acids (all Gibco) and 10% FBS (Hyclone); and, each of five cultures was transfected with each of the respective different lentiviral plasmid DNAs prepared above. The day before transfection each of the different recipient 293FT cell cultures was split and re-seeded at 6x10 6 cells/10 cm dish.
  • each respective viral vector For precipitation of each respective viral vector, a measured volume (about 10%) of the different harvested 293FT cell culture medium was mixed 1 part to 5 parts PEG (1 :5) with PEG-it precipitation solution (Systems Biosciences). The resultant 1 :5 diluted PEG viral medium solution was stored at 4°C overnight. The remaining 90% of the viral vector supernatant was stored at 4°C and processed according to Example 2, below. Each different precipitated viral vector in the respective different PEG medium was collected by centrifugation at 1500xg for 30 min at 4°C. and resuspended in a measured volume of D- MEM to produce the respective different viral vector test solution.
  • EU the number of expression units
  • the total EU in the remaining 90% of the viral vector supernatant, (set aside in Example 1 ) was calculated, (i.e., about 5x10 5 EU per ml, total volume of 20ml), and then the viral vectors were precipitated with PEG as described above, and resuspended in a measured volume of D-MEM to produce the respective different viral vector stock solutions.
  • MOI modulaton of infection
  • the five (5) transcription factor transduced cells were cultured in D-MEM growth medium for 6 days and split as necessary. Subsequently the cells were transferred onto mitomycin-C inactivated mouse embryonic feeder (MEF) cells at a density of 5x10 4 cells/ 10 cm dish. After 10-18 days in culture on the mouse feeder layer colonies of green cells were observed (Fig. 4) which stained positive for SSEA-4 (Fig. 4, day 18; Fig. 5, day 25), i.e., a stem cell and iPS cell marker. The SSEA-4 positive cells were manually picked onto a fresh MEF feeder layer and continuously grown as a cell line of rapidly growing cells with embryonic stem cell-like properties.
  • MEF mitomycin-C inactivated mouse embryonic feeder

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Abstract

L'invention concerne des compositions cellulaires, des cultures cellulaires continues stables, des lignes de cellules marqueuses, des préparations pharmaceutiques, des vecteurs viraux codant pour des facteurs de transcription de cellules souches pluripotentes et des procédés relatifs à ceux-ci, tous ces éléments étant relatifs à la reprogrammation de cellules somatiques pour induire des cellules souches pluripotentes.
PCT/US2009/044241 2008-05-15 2009-05-15 Procédés de reprogrammation virale efficace de cellules somatiques dans des cellules pluripotentes du type cellules souches Ceased WO2009140655A1 (fr)

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WO2011109837A3 (fr) * 2010-03-05 2011-12-15 Yupo Ma Procédés et compositions pour le traitement du diabète au moyen de cellules pancréatiques de type bêta dérivées d'ips
WO2012112603A1 (fr) * 2011-02-14 2012-08-23 University Of Utah Research Foundations Génération de cellules ips et procédés associés
CN104195177A (zh) * 2014-08-05 2014-12-10 南京大学 一种显著提高鱼类基因组编辑效率的方法
US9228204B2 (en) 2011-02-14 2016-01-05 University Of Utah Research Foundation Constructs for making induced pluripotent stem cells
GB2564437A (en) * 2017-07-10 2019-01-16 Univ Brunel Method of testing a gene therapy vector

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011109837A3 (fr) * 2010-03-05 2011-12-15 Yupo Ma Procédés et compositions pour le traitement du diabète au moyen de cellules pancréatiques de type bêta dérivées d'ips
CN103037939A (zh) * 2010-03-05 2013-04-10 Y.马 用iPS衍生的胰贝塔样细胞治疗糖尿病的方法和组合物
WO2012112603A1 (fr) * 2011-02-14 2012-08-23 University Of Utah Research Foundations Génération de cellules ips et procédés associés
US9228204B2 (en) 2011-02-14 2016-01-05 University Of Utah Research Foundation Constructs for making induced pluripotent stem cells
CN104195177A (zh) * 2014-08-05 2014-12-10 南京大学 一种显著提高鱼类基因组编辑效率的方法
GB2564437A (en) * 2017-07-10 2019-01-16 Univ Brunel Method of testing a gene therapy vector
GB2564437B (en) * 2017-07-10 2020-11-04 Univ Brunel Method of testing a gene therapy vector using induced pluripotent stem cells
US11834722B2 (en) 2017-07-10 2023-12-05 Brunel University London Method of testing a gene therapy vector

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