WO2009142717A2 - Methods and products for dedifferentiation of cells - Google Patents

Abstract

The invention relates to methods and compositions for dedifferentiating adult somatic cells, thereby generating more immature cells, including pluripotent stem cells.

Description

METHODS AND PRODUCTS FOR DEDIFFERENTIATION OF CELLS

Related Applications

This application claims priority under 35 U.S.C. §1 19(e) from U.S. provisional application serial number 61/054,430, filed May 19, 2008, the entire contents of which are incorporated by reference herein.

Field of the Invention

The invention relates to methods and compositions for producing immature cells, including induced pluripotent stem cells, from more mature cell types.

Government Support

This invention was made with U.S. government support under 5-R01-GM038627-23 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

Background of the Invention

Regenerative medicine relies on the ability to generate one or more cell lineages and/or complete tissues in vitro or in vivo in order to replace defective or non-existent cell lineages and/or tissues in vivo. Regenerative medicine therefore requires cells that are capable of generating these cell lineages and/or tissues. Such cells must have self-renewal capacity and, for most applications, be pluripotent. Self-renewal is the process through which a cell divides and thereby generates at least one cell that is identical to itself, both with respect to self-renewal and differentiative potential. Cells that have self-renewal potential are referred to as stem cells. These cells may be unipotent, multipotent, or pluripotent.

Unipotency refers to the ability of the cell to give rise to one lineage. Multipotency refers to the ability of the cell to give rise to two or more, but not all, lineages in the body. Pluripotency refers to the ability of the cell to differentiate into mesoderm, endoderm and ectoderm lineages in the body. Pluripotent cells may in some instances differentiate into all lineages of the body. Cells that are capable of self-renewal and are pluripotent are referred to as pluripotent stem cells.

Pluripotent stem cells occur naturally in the process of normal development. Identifying and isolating such cells in numbers that are clinically useful however is difficult. Thus, methods have been established for generating pluripotent stem cells from embryonic cells and tissues and recently from adult cells also. Examples include embryonic stem cells that are generated from the inner cell mass of blastocysts or blastomeres, and stem cells that are generated by transferring a nucleus from an adult somatic cell into an enucleated oocyte followed by chemically induced fertilization (so-called somatic cell nuclear transfer).

A major clinical hurdle to using stem cell based therapies to treat human disorder is the need for histocompatibility between the donor cells and the recipient subject. Ideally, the donor cells and the recipient are autologous. This situation however has been limited in the past to the use of donor cells derived from the recipient subject or from a genetically identical twin of the recipient subj ect.

It has recently been reported that pluripotent stem cells can be generated by reprogramming (or dedifferentiating) adult cells through the induced expression of particular early development gene combinations. Takahashi and Yamanaka reported that primary dermal fibroblasts can be reprogrammed by inducing expression of OCT4, SOX2, KLF4 and cMYC using retroviral transfection. (Takahashi and Yamanaka 2006 Cell 126:663.)

Thompson and co-workers reported a similar result using OCT4, SOX2, NANOG and LIN28. (Yu et al. Science 2007 318: 1017.) Jaenisch and co-workers also reported a similar finding. (Meissner et al. Nature Biotech 2007 25(10):l 177.) The immature cells generated by these reprogramming methods are referred to as induced pluripotent stem (iPS) cells, and have been identified through the endogenous expression of FBXl 5, OCT4 and/or NANOG. Since iPS cells are genetically identical to the recipient from whom they are derived, they represent a source of autologous cells for that recipient. However, the presently known methods used to generate iPS cells require retroviral transfection of some cancer inducing genes and therefore are not ideal for later clinical applications.

Summary of the Invention

The invention relates broadly to the ability to dedifferentiate mature cells into immature cells using hypoxic conditions. More specifically, the invention relates to the ability to produce immature cells, including pluripotent stem cells, from adult somatic cells by exposing such somatic cells to hypoxic conditions. The hypoxic condition may be a low oxygen gas environment in which the cells are placed and/or cultured, or it may be contact and/or exposure to an agent that mimics hypoxia (i.e., a hypoxia-mimicking agent). The invention thereby provides compositions and methods for generating pluripotent stem cells independent of the retroviral transfection methods of the prior art. Of particular clinical significance is the ability to generate "custom" stem cells that are autologous to a recipient subject without retroviral transfection, and without the introduction of exogenous genes, such as transcriptionally unregulated cancer-causing genes, into such stem cells. Thus, the invention provides, in one aspect, a method for dedifferentiating an isolated somatic cell from a subject comprising exposing an isolated somatic cell from a subject to a hypoxic condition for a time sufficient to dedifferentiate the isolated somatic cell, and isolating an immature cell derived from the isolated somatic cell.

The invention provides in another aspect a method for producing an induced pluripotent stem cell comprising exposing an isolated somatic cell to a hypoxic condition for a time sufficient to produce an induced pluripotent stem cell, and isolating an induced pluripotent stem cell derived from the isolated somatic cell.

Various embodiments apply equally to these and other aspects of the invention and these are recited below. In one embodiment, the isolated somatic cell is an isolated fibroblast. The isolated fibroblast may be an isolated dermal fibroblast, although it is not so limited.

In one embodiment, the isolated somatic cell is exposed to a hypoxic condition by culturing the isolated somatic cell in a hypoxic condition. In one embodiment, the hypoxic condition is a low oxygen gas environment. The low oxygen gas environment may be an oxygen level in a gas phase in contact with culture medium that is less than 15% oxygen or less than 10% oxygen. Alternatively, it may be less than 5% oxygen, less 4% oxygen, less than 3% oxygen, less than 2% oxygen, or less than 1% oxygen. In other embodiments, it may be about 0.05% to about 2% oxygen, about 0.1% to about 1.5% oxygen, or about 0.5% to about 1.5% oxygen, or about 1% oxygen. In one embodiment, the hypoxic condition is the presence of a hypoxia-mimicking agent. The hypoxia-mimicking agent may be desferoxamine, deferoxamine, cobalt chloride, S-nitroso-N-acetylcysteine, or 2,2'-dipyridyl, although it is not so limited.

In one embodiment, the isolated somatic cell is exposed to the hypoxic condition for at least 3 days, at least 6 days, or at least 9 days. In another embodiment, the isolated somatic cell is exposed to the hypoxic condition for at least 12 days, at least 15 days, at least 18 days, or at least 21 days. In yet another embodiment, the isolated somatic cell is exposed to the hypoxic condition for about 5 days to about 22 days. In still another embodiment, the isolated somatic cell is exposed to the hypoxic condition for about 6 days to about 21 days. In still another embodiment, the isolated somatic cell is exposed to the hypoxic condition for about 9 days to about 21 days.

In one embodiment, the immature cell expresses OCT4. In another embodiment, the immature cell expresses OCT4 and KLF4. In one embodiment, the isolated somatic cell is a normal cell. In another embodiment, the isolated somatic cell is a cancer cell. In important embodiments, the isolated somatic cell is a human cell.

The invention provides in a further aspect a composition comprising a population of immature cells or a population of induced pluripotent stem cells produced according to any of the foregoing methods. In some embodiments the population is derived from a normal cell, while in others it is derived from a cancer cell. The population is preferably clonal.

The invention provides in still another aspect a method for producing a differentiated cell population comprising culturing the immature cells or induced pluripotent stem cells generated according to any of the foregoing methods for a time and under conditions sufficient to produce differentiated cells of one or more lineages, isolating the differentiated cells.

The invention provides in still another aspect a composition comprising a differentiated cell population produced according to any of the foregoing methods.

In another aspect, the invention provides an induced pluripotent stem cell that does not contain exogenous nucleic acid. In one embodiment, the exogenous nucleic acid is OCT4, SOX2, KLF4, cMYC, LIN28, or NANOG. The invention further provides compositions comprising such cells including pharmaceutical compositions. The invention also provides cultures comprising such cells. The invention provides a method for producing a differentiated cell population comprising culturing these induced pluripotent stem cells for a time and under conditions sufficient to produce differentiated cells of one or more lineages, isolating the differentiated cells.

In another aspect, the invention provides an induced pluripotent stem cell that does not contain a retroviral nucleic acid. The invention further provides compositions comprising such cells including pharmaceutical compositions. The invention also provides cultures comprising such cells. The invention provides a method for producing a differentiated cell population comprising culturing these induced pluripotent stem cells for a time and under conditions sufficient to produce differentiated cells of one or more lineages, isolating the differentiated cells. These and other embodiments of the invention will be described in greater detail herein.

Each of the limitations of the invention can encompass various embodiments of the invention. It is therefore anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and/or the arrangement of components set forth in the following description or illustrated in the Figures. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Brief Description of the Figures

FIG. 1. mRNA levels of transcription factors in panel cells after 0, 3, 6 and 9 days (last 4 columns) of culture under hypoxic conditions (i.e., a gas phase having 1 % oxygen in these experiments), as compared to panel cells grown for the same time under normoxic conditions. FIGs. 2A and 2B. BJ cells cultured under normoxic conditions for 20 days maintain their replicative potential and fibroblast morphology (A) while BJ cells cultured under hypoxic conditions (i.e., a gas phase having 1 % oxygen in these experiments) and but then returned to normoxic conditions adopt a rounded morphology and lose contact inhibition (B).

FIG. 3. A schematic of one embodiment of the dedifferentiation process of the invention.

It is to be understood that the Figures are not required to enable the claimed invention.

Detailed Description of the Invention

The invention provides in part methods for generating immature cells from more mature cells independent of exogenous gene expression. More specifically, the invention is based in part on the surprising and unexpected discovery that adult somatic cells can be reprogrammed into more immature cell types, including pluripotent stem cells, through exposure to hypoxia or hypoxic conditions. The immature cell types retain the same genetic makeup as the starting cell population and thus are ultimately autologous (i.e., genetically identical) to the somatic cell donor. These immature cell types may be stem cells, and such stem cells may be unipotent, multipotent or pluripotent. As used herein, immature cells may be referred to as precursors or precursor cells, and a subset of such precursor cells are stem cells.

The invention therefore embraces the methods for generating immature cells from more mature cells, compositions for generating immature cells from more mature cells, compositions of the immature cells generated according to the invention, and methods of use for such cells, including therapeutic and screening methods. The invention is based, in part, on the observation that adult somatic cells can be reprogrammed into immature cells by exposing such cells to hypoxia or hypoxic conditions (e.g., using agents that mimic hypoxia). As used herein, hypoxia means reduced oxygen level relative to normal oxygen level. This level may be expressed in a number of ways, as discussed in greater detail herein. A hypoxic condition is therefore a condition in which oxygen level is reduced relative to normal oxygen level. Air is normally about 20% oxygen. Cells are usually cultured in incubators having a 95% air and 5% CO₂ mixture, resulting in about 19% oxygen. These oxygen levels are considered normal, as used herein. Thus, a below normal oxygen level is one that is less than 20% if the cells are exposed to air, or it is less than 19% if the cells are in an incubator that normally provides the 95%/5% gas phase described above.

Hypoxic conditions may be induced in a variety of ways. In a first instance, hypoxic conditions may be induced by reducing the oxygen content of the gas with which the cells are in contact. For example, if the cells are being cultured in an incubator, the gas phase within that incubator is adjusted to be less than 19% oxygen. Depending on the embodiment, the gas phase may be less than 18% oxygen, less than 17% oxygen, less than 16% oxygen, less than 15% oxygen, less than 14% oxygen, less than 13% oxygen, less than 12% oxygen, less than 11 % oxygen, less than 10% oxygen, less than 9% oxygen, less than 8% oxygen, less than 7% oxygen, less than 6% oxygen, less than 5% oxygen, less than 4% oxygen, less than 3% oxygen, less than 2% oxygen, less than 1% oxygen, less than 0.9% oxygen, less than 0.8% oxygen, less than 0.7% oxygen, less than 0.6% oxygen, less than 0.5% oxygen, less than 0.4% oxygen, less than 0.3% oxygen, less than 0.2% oxygen, less than 0.1% oxygen, less than 0.09% oxygen, less than 0.08% oxygen, less than 0.07% oxygen, less than 0.06% oxygen, or about 0.05% oxygen. The Examples demonstrate the ability to dedifferentiate adult fibroblasts in culture in about 1% oxygen and in about 0.1% oxygen. The oxygen level in the gas phase in contact with the culture media may also be expressed as a range, and in this respect includes about 0.05% to about 5% oxygen, about 0.1% to about 3% oxygen, about 0.5% to about 2% oxygen, and about 1% to about 1.5% oxygen. In some embodiments, the gas phase is about 1% oxygen. When used in this regard herein, the term about means +/- 0.5%.

Oxygen levels may also be referred to in terms of partial pressure (as measured in mmHg). Oxygen control devices may express oxygen levels as a percentage. pθ_2gas can be determined based on knowledge of a percent oxygen measurement using the following formula:

pθ₂ = (% oxygen) x (760 - 47)

In this equation, 760 is the atmospheric pressure and 47 is the vapor pressure of water at 37⁰C. Gas phase oxygen concentrations of 1%, 5%, 20%, and 40% correspond to pθ_2gas of 7 mmHg, 36 mmHg, 142 mmHg, and 285 mmHg. Atmospheric pθ₂ is therefore about 142 mmHg.

In many cases, during cell culture in bioreactors, the % oxygen is actually given as % of air saturation. The equation to convert between the two is

% absolute = % air saturation x 0.2 Cell culture is typically performed in a humidified environment consisting of 95% air and 5% CO₂ and this results in a pθ_2gas of about 142 mmHg. In the context of the invention, low pθ₂ refers to pθ₂ that is less than atmospheric partial pressure. More specifically, in some embodiments, low pθ₂ refers to a pθ₂ that is less than 142 mmHg. Low pθ₂ therefore may be a pθ₂ that is less than 140 mmHg, less than 135 mmHg, less than 130 mmHg, less than 125 mmHg, less than 120 mmHg, less than 1 15 mmHg, less than 1 10 mmHg, less than 105 mmHg, less than 100 mmHg, less than 95 mmHg, less than 90 mmHg, less than 85 mmHg, less than 80 mmHg, less than 75 mmHg, less than 70 mmHg, less than 65 mmHg, less than 60 mmHg, less than 55 mmHg, less than 50 mmHg, less than 45 mmHg, less than 40 mmHg, less than 35 mmHg, less than 30 mmHg, less than 25 mmHg, less than 20 mmHg, less than 15 mmHg, less than 14 mmHg, less than 13 mmHg, less than 12 mmHg, less than 1 1 mmHg, less than 10 mmHg, less than 9 mmHg, less than 8 mmHg, less than 7 mmHg, less than 6 mmHg, less than 5 mmHg, less than 4 mmHg, less than 3 mmHg, less than 2 mmHg, or less than 1 mmHg. In some instances it is about or less than 7 mmHg. In some instances it is about or less than 1 mmHg. pθ_2gas can be regulated during culture using manual and automated devices. Examples of commercially available automated devices include but are not limited to OxyCycler C42 from BioSpherix (Redfield, NY) and MC0-5M from Sanyo (Bensenville, IL).

Hypoxic conditions can also result from contacting the cells (or the media in which the cells exist) with agents that mimic hypoxia. Such agents are referred to herein as hypoxia-mimicking agents. These agents may be but are not limited to chemical compounds. Some of these agents induce changes in a cell that would normally result following exposure to a hypoxic environment. Agents that reduce histone demethylase activity such as iron chelators and inhibitors of alpha-ketoglutarate can be used as hypoxia-mimicking agents. Inhibitors of other co-factors of histone demethylases can also be used as hypoxia-mimicking agents. Examples of hypoxia-mimicking agents include but are not limited to desferoxamine, cobalt chloride, S-nitroso-N-acetylcysteine, and 2,2'-dipyridyl.

The adult somatic cells of the invention are any non-germ cell from a non-embryonic, non-fetal subject. The cell may be a normal cell or it may be a cancer cell (e.g., isolated from a biopsy). Preferably, the cell is one that is easily isolated from the subject. Dermal fibroblast cells therefore are preferred in some instances. Other adult somatic cells include but are not limited to oral fibroblasts, foreskin fibroblasts, breast fibroblasts, and the like . Methods for harvesting such cells are known in the art and the invention is not limited in this regard.

The adult somatic cells are isolated. This means that they are harvested and separated from the environment in which they normally exist. Thus, if the adult somatic cells are primary dermal fibroblasts, they are isolated when they are harvested from a subject and exist in vitro. The subjects from whom adult somatic cells can be harvested include human subjects, companion animals such as dogs, cats, ferrets, and the like, agricultural animals such as cows, pigs, horses, sheep, ostriches, and the like, zoo animals such as bears, zebra, giraffes, lions and other wild cat species, and the like, aquatic species such as fish, dolphins, whales, sharks, and the like, and research animals such as mice, rats, rabbits, monkeys, and the like.

The immature or precursor cells generated according to the invention are defined as cells which are functionally or genetically more immature than their parent cells (i.e., the adult somatic cell which gave rise to the immature or precursor cell). Functional immaturity is defined, according to the invention, as an increased level of self-renewal and/or more differentiative potential. Self-renewal may be deduced based on an increase in proliferative potential. This may be observed as the capacity to proliferate in cells that had been quiescent and possibly senescent. This may also be observed as a shorter doubling time. Such increases in proliferative potential however should not be confused with malignant transformation in these cells which is usually associated the loss of contact inhibition or factor independent growth. An increase in differentiative potential refers to the ability of the cell to differentiate into a different lineage from that of the mature cell from which it derived. The generation of an immature cell may therefore be shown by the generation of a cell that can differentiate into multiple lineages as demonstrated in in vitro (e.g., embryoid body (EB) formation) or in vivo (e.g., teratoma formation) assays described in Yu et al. Science 2007 318:1917, Park et al. Nature 2008 451(7175): 141 -6, and Takahashi et al. Cell 2006 126:663. Genetic immaturity is defined, according to the invention, as the expression of early markers, optionally concomitant with the loss of expression of more mature markers. Early markers when used in this context refer to genes that are expressed in stem cells or genes that are used to induce stem cells from more mature cells. These markers include OCT4, SOX2, NANOG, KLF4, LIN28, hTERT, SSEAl, and alkaline phosphatase. The expression of these markers may be deduced at the mRNA level (e.g., by Northern or RT-PCR) or at the protein level (e.g., by enzyme assay, by Western analysis, by FACS analysis). The generation of an immature cell may therefore be shown by the generation of a cell that expresses at least one and preferably more of any of these early markers.

Human iPS cells are defined, according to the invention, as immature cells that resemble human embryonic stem (hES) cells in a number of respects. Morphologically, iPS cells are small round translucent cells that preferably grow in vitro in colonies that are themselves characterized as tightly packed and sharp-edged. Genetically, iPS cells express markers of pluripotency such as OCT4 and NANOG, cell surface markers such as SSEA3, SSEA4, Tra-1-60, and Tra-1-80, and the intracellular enzyme alkaline phosphatase. These cells have a normal karyotype. Their cell cycle profile can be characterized by a short Gl phase, similar to that of hES cells.

As used herein, the terms "dedifferentiating" or "reprogramming" are used interchangeably to refer to the process of generating a relatively more immature cell from a relatively more mature cell. Also as used herein, the terms "generating" and "producing" are used interchangeably.

Dedifferentiation may occur through culture of mature cells in hypoxic conditions. The media components are generally dictated by the growth requirements of the mature cell used as the starting cell. As an example, if the mature cell is a fibroblast, then the culture conditions may be DMEM (or an equivalent thereof), fetal bovine serum (e.g., 5-10%), nonessential amino acids, and optionally antibiotics. It will be understood that if the hypoxic condition results from the use of hypoxia-mimicking agents, the cultures may also contain such agents. Thus, in accordance with the invention, human dermal fibroblasts are initially plated on a tissue culture solid support (e.g., a tissue culture dish or multiwell dish) in media as described herein. The oxygen level can be reduced immediately regardless of whether the cells are attached to the solid support. The cells are cultured for a period of time sufficient to dedifferentiate them into immature or precursor cells, including iPS cells. As described herein, the presence of such immature or precursor cells can be determined functionally (e.g., in a differentiation assay) or genetically (e.g., for mRNA analysis of early gene markers). This period of time may be at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, or longer. In some instances, the culture period is about 6 to about 24 days, about 9 to about 21 days, about 12 to about 18 days, or about 15 days. In some instances, the culture period is about 9 days. In other instances, it is about 21 days. When used in this context, the term about means +/- 0.5 days.

In some instances during the culture period it may be preferable to seed the cells onto feeder layers such as fibroblast feeder layers. Preferably the feeder layers consist of cells from the same species as the somatic cells being dedifferentiated. As an example, dedifferentiation of a human somatic cell population preferably employs a human feeder cell layer such as a human fibroblast feeder layer. In other instances, however, it is possible to use feeder layers from another species. As an example, dedifferentiation of a human somatic cell population may employ a mouse embryonic fibroblast (MEF) feeder layer. In instances where the mature somatic cell population is fibroblasts, such transfer may not be necessary since some of the fibroblasts will remain mature and will act as the feeder cells themselves. The feeder cells may in some cases be mitotically inactivated in order to prevent consumption of nutrients by the feeder cells at the expense of the somatic cells and their immature progeny. Mitotic inactivation means that the cells are treated in a manner that prevents them from dividing further but that is not necessarily cytotoxic to the cells. Mitotic inactivation of feeder cells can be accomplished by ultraviolet (UV), X-, or gamma- irradiation (e.g., at 10- 50 Gy), or by chemical means such as senescence inducing drugs (e.g., mitomycin C, toyocamycin, tertbutylhydroperoxide (t-BHP) and hydrogen peroxide (H₂O₂)). In alternative embodiments, laminin-coated dishes are used instead of feeder cells.

The immature or precursor cells produced by the methods of the invention may also be differentiated in vitro, partially or completely. Differentiation protocols are known in the art and include those described in U.S. Patent Nos. 7,326,572 (endoderm differentiation),

7,282,366 (hepatocyte differentiation), 7,250,294 (neural differentiation), 7,033,831 (islet cell differentiation), and in published PCT application WO2008/156708. Some of these methods require the formation of three dimensional masses of cells, akin to embryoid bodies (EB) albeit without a requirement of pre-implantation embryo derived stem cells. In one embodiment, dermal fibroblasts are dedifferentiated into more immature cells which are then differentiated into neural cells.

Immature or precursors cells may be cultured in the presence of one or more factors and/or chemicals in order to drive differentiation down one or more lineages. A variety of these factors are known in the art. For example, members of the BMP family of factors have been used to differentiate stem cells such as embryonic stem cells. These include the use of BMP -4 and BMP-7 to generate endoderm-like differentiation. (Xu et al. Nat Biotechnol 20:1261-1264, 2002; Pera et al. J Cell Sci 1 17:1269-1280, 2004.) Activin A can be used to differentiate pluripotent stem cells such as embryonic stem cells into definitive endoderm using monolayers or three dimensional (e.g., EB) culture systems. (D'Amour et al. Nat Biotechnol 23:1534-1541, 2005.) Nervous system cells have been observed as a result of culture with epidermal growth factor and fibroblast growth factor (resulting in the generation of neurospheres that comprise neural stem cells), subsequent removal of these factors (resulting in the generation of astrocyte-like cells) or supplementation with nerve growth factor (resulting in the generation of neurons and glial cells). (Kim et al. Nature 418:50-6, 2002; Lee et al. Nat Biotechnol 18:675-9, 2000.) Dopaminergic neurons, useful in

Parkinson's disease, may be formed through culture or contact with FGF20 and FGF2. Hepatic cell differentiation may be induced through contact and/or culture with an insulin, dexamethasone, and collagen type I (via EB formation) combination; a sodium butyrate and DMSO combination; an FGF4, HGF and collagen type I combination; an aFGF, HGF, oncostatin M, dexamethasone and collagen type I combination; and a bFGF, variant HGF, DMSO and dexamethasone combination in the presence of poly-amino-urethane coated non- woven polytetrafluoroethylene fabric. (Shirahashi et al. Cell Transplant 13: 197-21 1 , 2004; Rambhatla et al. Cell Transplant 12: 1-1 1, 2003; Schwartz et al. Stem Cells Dev 14:643-655, 2005; Baharvand et al. Int J Dev Biol 50:645-652, 2006; Soto-Gutierrez et al. Cell Transplant 15:335-341 , 2006.) Hepatic differentiation may also occur spontaneously. (Lavon et al. Differentiation 72:230-238, 2004.) Cardiomyocyte differentiation can occur spontaneously or through modulation of oxygen levels. (Kehat et al. J Clin Invest 108:407-441, 2001.) Pancreatic differentiation, including differentiation towards beta-islet cells, can be induced using Activin A, retinoic acid, FGF2 and FGFlO, betacellulin, HGF, Exendin 4, DKKl and DKK3. (Gu et al. Mech Dev 120:35-43, 2003; Grapin-Botton et al. Trends Genet 16: 124- 130, 2000; D'Amour et al. Nat Biotechnol 23: 1534-1541, 2005a; D'Amour et al. published US application US2005-0266554A1.) Endothelial differentiation may be induced in the presence of ECM proteins such as collagen type IV, optionally in the presence of VEGF and bFGF. (Gerecht-Nir et al. Lab Invest 83: 181 1-1820, 2003.) Further reference may be made to published PCT application WO2009/007852 for a review of various differentiative procedures known in the art and applicable to the differentiation of the immature and precursor cells of the invention. Such teachings, and in particular those found on pages 57-61 (under the subheading "Cell Differentiation") of WO2009/007852, are incorporated by reference herein.

The differentiative potential of immature or precursor cells may also be analyzed using in vivo techniques such as teratoma generation (e.g., following subcutaneous or intramuscular injection of the cells, for easy access, observation and harvest of the teratoma). (Keller et al. Genes Dev 19: 1 129-1 155, 2005; Spagnoli et al. Curr Opin Genet Different

16:469-475, 2006.) As an example, human immature or precursor cells generated according to the invention may be introduced into immuno-comprised mice (e.g., SCID mice) and the resulting teratoma may be analyzed for the presence of endoderm, mesoderm and ectoderm lineages and/or markers. The presence of endodermal, mesodermal and ectodermal lineages can be determined via immunohistochemical staining, microscopy (e.g., transmission electron microscopy, TEM), RT-PCR, and the like. Further reference can be made to Human Embryonic Stem Cells: The Practical Handbook. Eds. Sullivan, Cowan and Eggan, Harvard University, John Wiley & Sons (Publishers), 2007 for a more thorough discussion of these techniques.

Thus, the immature cells and/or their differentiated progeny, for example as generated according to the foregoing methods, may be used in a variety of in vitro and in vivo methods including but not limited to therapeutic or cosmetic applications, and in vitro screening methods.

The immature cells and/or their differentiated progeny may be provided as pharmaceutical compositions that are sterile and appropriate for in vivo use, together with a pharmaceutically acceptable carrier. The cells may be provided as a frozen aliquot of cells, or a culture of cells, in some embodiments including feeder cells also. In some instances the immature cells and/or their differentiated progeny will be a clonal population. These cells may further be included in a kit that additionally comprises at a minimum instructions for use of the cells, and optionally comprises one or more other agents whether active (such as for example a hypoxia-mimicking agent) or inactive. The cells may be used alone or together with another agent, whether active or inactive, including but not limited to a differentiating agent, a scaffold, a matrix, and the like.

As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.

The immature cells and/or their differentiated progeny may be formulated for local or systemic administration including as part of an implant. The invention further contemplates methods for screening agents (or compounds, as the terms are used interchangeably herein) for toxicity and in some embodiments therapeutic efficacy. The readouts from such in vitro assays are correlative of the in vivo toxicity or efficacy such agents would exhibit in human subjects. Thus, the effect of the agent on the differentiated cells generated according to the invention in vitro is a form of surrogate marker or readout for how the agent will function in vivo in a human subject. Importantly, since the cells generated according to the invention are autologous to a subject, it is possible to personalize a particular medical treatment based on the response (or lack thereof) of the immature cells and/or their differentiated progeny to the agent being tested. As an example, the invention contemplates dedifferentiating somatic cells from subjects having or at risk of developing a condition into immature cells such as iPS cells, and then differentiating these immature cells into the lineages that are affected by the particular condition. The final differentiated cell population may then be used directly (for example, in a transplant) or they may be used as a screening tool to identify therapies for the subject. As a more specific example, dermal fibroblasts from a subject having Parkinson's disease are dedifferentiated into iPS cells and the iPS cells are then differentiated into neural cells. The neural cells so generated can be transplanted into the donor subject or they can be used to screen for therapies useful to treat such donor. This therapeutic or screening process can be applied to any number of conditions including other neurodegenerative conditions (e.g.,

Alzheimer's, ALS, and the like), hematopoietic malignancies (e.g., leukemia, lymphoma and the like, particularly where such malignancies are the result of contact or exposure to carcinogens such as radiation), degenerative liver conditions, and the like.

The immature cells generated by the invention can also be used to screen for agents that promote or drive self-renewal or differentiation into particular lineages. Agents that promote self-renewal would allow for greater numbers of precursor cells to be generated and could be applied to any precursor population (including primary precursor and stem cell populations) and not just those generated according to the methods described herein. Agents that promote differentiation into particular lineages would be useful for preparing cells of particular lineages, to be used in turn therapeutically or for screening purposes.

In some more common embodiments, such testing will focus on the toxicity of agents in particular differentiated progeny. Accordingly, in these assays, the readout would be cell death (or conversely cell viability). These in vitro assays may employ suspensions of differentiated cells, adherent populations of differentiated cells, or three dimensional structures comprised of differentiated cells (e.g., in vitro organ tissues, matrices and architectures).

In another embodiment, the immature cells generated according to the invention may be differentiated into liver cells and thereby used to generate an ex vivo liver that can be used to assay the liver toxicity or liver metabolism of one or more agents. These various agents include those already used clinically (e.g., in the case of establishing a personalized treatment for a particular subject), as well as those in development (e.g., in the case of identifying a novel agent for the treatment of a disorder). These agents may be provided in an isolated form but are not so limited. They may be provided as library members, such as for example small molecules present in a library to be screened according to the invention. The agents may be naturally occurring or non-naturally occurring.

Drugs that can be tested according to these methods particularly for whether they are toxic to cells include but are not limited to adrenergic agent; adrenocortical steroid; adrenocortical suppressant; aldosterone antagonist; anabolic; analeptic; analgesic; androgen; anesthesia, adjunct to; anesthetic; anorectic; anterior pituitary suppressant; anti-acne agent; anti-adrenergic; anti-allergic; anti-androgen; anti-anemic; anti-anginal; anti-arthritic; antiasthmatic; anti-atherosclerotic; anticholelithic; anticholelithogenic; anticholinergic; anticoagulant; anticoccidal; anticonvulsant; antidepressant; antidiabetic; antidiarrheal; antidiuretic; anti-emetic; anti-epileptic; anti-estrogen; antifibrinolytic; antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine; antihyperlipidemia; antihyperlipoproteinemic; antihypertensive; antihypotensive; anti-inflammatory; antikeratinizing agent; antimigraine; antimitotic; antimycotic, antinauseant, antineoplastic, antineutropenic, antiparkinsonian; antiperistaltic, antipneumocystic; antiproliferative; antiprostatic hypertrophy; antipruritic; antipsychotic; antirheumatic; antiseborrheic; antisecretory; antispasmodic; antithrombotic; antitussive; anti-ulcerative; anti-urolithic; benign prostatic hyperplasia therapy agent; blood glucose regulator; bone resorption inhibitor; bronchodilator; carbonic anhydrase inhibitor; cardiac depressant; cardioprotectant; cardiotonic; cardiovascular agent; choleretic; cholinergic; cholinergic agonist; cholinesterase deactivator; coccidiostat; cognition adjuvant; cognition enhancer; depressant; diagnostic aid; diuretic; dopaminergic agent; ectoparasiticide; emetic; enzyme inhibitor; estrogen; fibrinolytic; free oxygen radical scavenger; gastrointestinal motility effector; glucocorticoid; gonad-stimulating principle; hair growth stimulant; hemostatic; histamine H2 receptor antagonists; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive; immunomodulator; immunoregulator; immunostimulant; immunosuppressant; impotence therapy adjunct; keratolytic; LHRH agonist; liver disorder treatment; luteolysin; mental performance enhancer; mood regulator; mucolytic; mucosal protective agent; mydriatic; nasal decongestant; neuromuscular blocking agent; neuroprotective; NMDA antagonist; non- hormonal sterol derivative; oxytocic; plasminogen activator; platelet activating factor antagonist; platelet aggregation inhibitor; post-stroke and post-head trauma treatment; progestin; prostaglandin; prostate growth inhibitor; prothyro tropin; psychotropic; pulmonary surface; relaxant; repartitioning agent; scabicide; sclerosing agent; sedative; sedative- hypnotic; selective adenosine Al antagonist; serotonin antagonist; serotonin inhibitor; serotonin receptor antagonist; steroid; symptomatic multiple sclerosis; thyroid hormone; thyroid inhibitor; thyromimetic; tranquilizer; amyotrophic lateral sclerosis agent; cerebral ischemia agent; Paget' s disease agent; unstable angina agent; uricosuric; vasoconstrictor; vasodilator; wound healing agent; xanthine oxidase inhibitor. Those of ordinary skill in the art will know or be able to identify agents that fall within any of these categories, particularly with reference to the Physician's Desk Reference.

The following Examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

Examples Summary We have developed a novel and unexpected process to dedifferentiate primary human foreskin (BJ) fibroblasts and pancreatic ductal cancer (panel) cells, as judged by both the expression of markers of pluripotency, namely OCT4, KLF4, SOX2 (and PDXl in the case of panel cells), and the change in cellular morphology from that of fibroblast morphology to that of less differentiated cells, including induced pluripotent stem (iPS) cells. This process involves exposing the cells (e.g., in a culturing step) to hypoxia or hypoxic conditions.

Importantly, the method does not involve the integration or transfer of exogenous DNA into the recipient cell. We have achieved levels of expression of otherwise silenced endogenous genes encoding reprogramming transcription factors, e.g., OCT4, comparable to those measured in virally engineered iPS cells. The hypoxic conditions are believed to affect cellular physiology thereby resulting in full reprogramming of somatic cells such as skin cells into iPS cells. All cells derive their energy ultimately from atmospheric oxygen, which is used as a fuel for oxidative metabolism in mammalian cells. Small molecules that affect the way that cells perceive their energy status induce a global switch of metabolism from an oxidative to a glycolytic state, in part by upregulating protein levels of key transcriptional regulators such as HIFl -alpha and HIF2- alpha. This switch results in the down regulation of mitochondrial citric-acid cycle metabolites such as alpha-ketoglutarate. Both iron and alpha-ketoglutarate are important cofactors of histone demethylases and other protein hydroxylases, which are enzymes critical for modulating the epigenetic state of cells. Highly proliferative cells like stem cells have a highly glycolytic metabolism.

We reasoned that directed alterations of chromatin and glycolytic metabolism using hypoxic conditions would drive the dedifferentiation of differentiated cells like skin fibroblasts, into dedifferentiated or iPS cells. We have discovered that these conditions achieve the dedifferentiation process without the use of exogenous DNA of any kind. Once initiated, we also note that we can accelerate this process by using agents that change the activity of chromatin-modifying enzymes. Examples include 5-aza-deoxycytidine, 5- azacytidine, 5-aza-2'deoxycytidine (also known as Decitabine in Europe), 5, 6-dihydro-5- azacytidine, 5, 6-dihydro-5-aza-2'deoxycytidine, 5-fluorocytidine, 5-fluoro-2'deoxycytidine, and short oligonucleotides containing 5-aza-2'deoxycytosine, 5, 6-dihydro-5-aza- 2'deoxycytosine, and 5-fluoro-2'deoxycytosine.

Briefly, in a representative experiment, human dermal fibroblasts were initially seeded in 6 well tissue culture dishes at a density of 15000 cells per well. The oxygen level was reduced to either 0.1% or 1% at the beginning of the culture period, without any intervening culture period necessary to allow the fibroblasts to attach to the dish surface. The media on the cells was changed about every 3 days. The cells started to become contact inhibited by about 6 days at which point they were harvested and split (e.g., the cells were diluted about 10 fold). The culture period was 21 days, although cells were harvested from the culture starting at day 6 and at various times thereafter for analysis. 800,000 cells are lysed and RNA is prepared using the Qiagen RNeasy Kit according to manufacturer's instructions. Quantitative PCR was carried out using Applied Biosystems High Capacity cDNA Reverse Transcription Kit (for generating cDNA from isolated RNA from cells) according to manufacturer's instructions. Primers used in the quantitative PCR methods are as described by Yamanaka et al. Cell. 2007 Nov 30;131(5):861-72 and are shown below.

hOCT3/4-S1165 GAC AGG GGG AGG GGA GGA GCT AGG (SEQ ID NO:1) hOCT3/4-AS1283 CTT CCC TCC AAC CAG TTG CCC CAA AC (SEQ ID NO: 2) hSOX2-S1430 GGG AAA TGG GAG GGG TGC AAA AGA GG (SEQ ID NO: 3) hSOX2-AS1555 TTG CGT GAG TGT GGA TGG GAT TGG TG ( SEQ ID NO : 4 ) h-NANOG CAG CCC CGA TTC TTC CAC CAG TCC C

(SEQ ID NO: 5) h-NANOG CGG AAG ATT CCC AGT CGG GTT CAC C

(SEQ ID NO: 6) hMYC-S253 GCG TCC TGG GAA GGG AGA TCC GGA GC (SEQ ID NO: 7) hMYC-AS555 TTG AGG GGC ATC GTC GCG GGA GGC TG (SEQ ID NO: 8)

Using quantitative PCR, we observed increased expression of OCT 4, KLF4 and cMYC in the pancreatic ductal carcinoma panel cells grown in a gas phase of about 1% oxygen. (FIG. 1.) In addition, there was an increase in mRNA levels of telomerase QiTERT) and pancreas progenitor marker (PDXl) after the 20-day treatment (data not shown). OCT4 expression was elevated as early as day 6 of culture indicating that immature cells may be present in such cultures at least this early. Similar results were observed when the cells were exposed to 0.1% oxygen.

The fibroblast BJ cells when exposed to 1% oxygen initially retained their fibroblast- like morphology and replicative potential as compared to normoxic conditioned cells that were grown in parallel. After incubating in the presence of 1% oxygen and then returning the cells to untreated (i.e., normoxic) conditions, the cells adopted a rounded morphology and grew in colonies similar to pre-iPS cells. They also adopted a more glycolytic nature as judged by increased lactate production. Representative images of cells and colonies are provided as FIG. 2. Similar results were observed when the cells were exposed to 0.1% oxygen. After 6 days of culture under hypoxic conditions, BJ cells expressed approximately 5 times more OCT4 and SOX2 than untreated (i.e., normoxic) BJ cells. BJ cells express relatively high basal levels of KLF4 normally. No changes were observed in cMYC levels upon incubating BJ cells under these hypoxic conditions, although increased cMYC levels were observed in panel cells so treated. (FIG. 1.) Equivalents

It should be understood that the preceding is merely a detailed description of certain embodiments. It therefore should be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention, and with no more than routine experimentation. It is intended to encompass all such modifications and equivalents within the scope of the appended claims.

All references, patents and patent applications that are recited in this application are incorporated by reference herein in their entirety.

What is claimed is:

Claims

CIaims

1. A method for dedifferentiating an isolated somatic cell from a subject comprising exposing an isolated somatic cell from a subject to a hypoxic condition for a time sufficient to dedifferentiate the isolated somatic cell, and isolating an immature cell derived from the isolated somatic cell.

2. A method for producing an induced pluripotent stem cell comprising exposing an isolated somatic cell to a hypoxic condition for a time sufficient to produce an induced pluripotent stem cell, and isolating an induced pluripotent stem cell derived from the isolated somatic cell.

3. The method of claim 1 or 2, wherein the isolated somatic cell is an isolated fibroblast.

4. The method of claim 3, wherein the isolated fibroblast is an isolated dermal fibroblast.

5. The method of any one of claims 1-4, wherein the isolated somatic cell is exposed to a hypoxic condition by culturing the isolated somatic cell in a hypoxic condition.

6. The method of any one of claims 1-5, wherein the hypoxic condition is a low oxygen gas environment.

7. The method of claim 6, wherein the low oxygen gas environment is less than 15% oxygen, or less than 10% oxygen.

8. The method of claim 6, wherein the low oxygen gas environment is less than 5% oxygen, less than 4% oxygen, less than 3% oxygen, less than 2% oxygen, or less than 1% oxygen.

9. The method of claim 6, wherein the low oxygen gas environment is about 0.05% to about 2% oxygen, about 0.1% to about 1.5% oxygen, or about 0.5% to about 1.5% oxygen, or about 1% oxygen.

10. The method of any one of claims 1-9, wherein the hypoxic condition is the presence of a hypoxia-mimicking agent.

1 1. The method of claim 10, wherein the hypoxia-mimicking agent is desferoxamine, deferoxamine, cobalt chloride, S-nitroso-N-acetylcysteine, or 2,2'-dipyridyl.

12. The method of any one of claims 1-11, wherein the isolated somatic cell is exposed to the hypoxic condition for at least 3 days, at least 6 days, or at least 9 days.

13. The method of any one of claims 1-11, wherein the isolated somatic cell is exposed to the hypoxic condition for at least 12 days, at least 15 days, at least 18 days, or at least 21 days.

14. The method of any one of claims 1-1 1, wherein the isolated somatic cell is exposed to the hypoxic condition for about 5 days to about 22 days.

15. The method of any one of claims 1-11, wherein the isolated somatic cell is exposed to the hypoxic condition for about 6 days to about 21 days.

16. The method of any one of claims 1-11, wherein the isolated somatic cell is exposed to the hypoxic condition for about 9 days to about 21 days.

17. The method of any one of claims 1-16, wherein the immature cell expresses OCT4.

18. The method of any one of claims 1-16, wherein the immature cell expresses OCT4 and KLF4.

19. The method of any one of claims 1-18, wherein the isolated somatic cell is a normal cell.

20. The method of any one of claims 1-18, wherein the isolated somatic cell is a cancer cell.

21. The method of any one of claims 1-20, wherein the isolated somatic cell is a human cell.

22. A composition comprising a population of immature cells produced according to the method of any one of claims

1-21.

23. A composition comprising a population of human induced pluripotent stem cells produced according to the method of any one of claims 1-21.

24. The composition of claim 22 or 23, wherein the population is derived from a normal cell.

25. The composition of claim 22 or 23, wherein the population is derived from a cancer cell.

26. The composition of claim 22 or 23, wherein the population is a clonal population.

27. A method for producing a differentiated cell population comprising culturing the immature cells of any one of claims 1-21 for a time and under conditions sufficient to produce differentiated cells of one or more lineages, isolating the differentiated cells.

28. A method for producing a differentiated cell population comprising culturing the induced pluripotent stem cells of any one of claims 1-21 for a time and under conditions sufficient to produce differentiated cells of one or more lineages, isolating the differentiated cells.

29. A composition comprising a differentiated cell population produced according to the method of claim 27 or 28.

30. An induced pluripotent stem cell that does not contain exogenous nucleic acid.

31. An induced pluripotent stem cell that does not contain a retroviral nucleic acid.

32. The induced pluripotent stem cell of claim 30, wherein the exogenous nucleic acid is OCT4, SOX2, KLF4, cMYC, LIN28, or NANOG.

33. A composition comprising the induced pluripotent stem cell of any one of claims 30-

32.

34. A culture comprising the induced pluripotent stem cell of any one of claims 30-32.

35. A method for producing a differentiated cell population comprising culturing the induced pluripotent stem cells of any one of claims 30-32 for a time and under conditions sufficient to produce differentiated cells of one or more lineages, isolating the differentiated cells.