WO2011119942A1 - Induction de cellules ips en utilisant des vecteurs épisomiques transitoires - Google Patents

Induction de cellules ips en utilisant des vecteurs épisomiques transitoires Download PDF

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WO2011119942A1
WO2011119942A1 PCT/US2011/029972 US2011029972W WO2011119942A1 WO 2011119942 A1 WO2011119942 A1 WO 2011119942A1 US 2011029972 W US2011029972 W US 2011029972W WO 2011119942 A1 WO2011119942 A1 WO 2011119942A1
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cells
population
hepatocytes
differentiated
ipsc
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Joel Schick
Robert Stull
H. Ralph Snodgrass
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Vistagen Therapeutics, Inc.
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors

Definitions

  • the field of this invention relates generally to inducible pluripotent stem cells and cell differentiation.
  • iPSC Pluripotent Stem Cell
  • Current strategies for inducible Pluripotent Stem Cell (iPSC) induction use integrating viral vectors to deliver the genes that promote reprogramming, yielding iPSC lines containing greater than 20 proviral integrants per clone (Takahashi, K. and Yamanaka, S., 2006 Cell 126:663-676).
  • the integration events and the resultant effects upon transgene expression complicate standardization of gene dose required to achieve reprogramming.
  • gene integration may result in oncogenic transformation (Okita, K. et al., 2007 Nature 448:313-317).
  • reprogramming genes is not essential (Takahashi, K. et al., 2007 Cell 131:1-12).
  • Other examples of iPSC induction using viral vectors to introduce reprogramming genes are provided by Meissner A. et al. (2007) Nat. Biotech. 25:1177-1181; Yu, J. et al. (2007) Science 318:1917-1920; Park, I.H. et al. (2008) Nature 451:141-146; Stadtfeld, M. et al. (2008) Sciencexpress, and U. S. Pat. Application Publication No. 2008/0233610.
  • An example of differentiation of iPSC induction using repeated plasmid transfection if provided by Okita, K. et al., (2008) Sciencexpress.
  • Non-viral approaches to the generation of iPSC include WO 2009/115295, U.S. Patent Application Publication US 2010/0003757 and Yu, J. et al. (2009) Science 324:797-801.
  • An example of differentiation of iPSC into insulin- secreting islet like cells is provided by Tateishi, K. et al., (2008) /. Biol. Chem.
  • the present invention provides methods of generating iPSC from
  • differentiated cells without the need for integration of reprogramming genes or for the use of viral vectors.
  • Reprogramming genes are expressed transiently thereby reducing risks posed by expressing proto-oncogenes such as c-myc.
  • a collection of genetically diverse iPSC may be generated.
  • Induced pluripotent cells of the invention can, in turn, be differentiated into cells of endoderm, mesoderm or ectoderm lineage.
  • the invention provides a method of inducing a population of pluripotent stem cells from a population of differentiated cells, said method comprising the steps of (i) introducing one or more episomal vectors comprising polynucleotides encoding reprogramming polypeptides into the population of differentiated cells, wherein the episomal vectors are maintained conditionally, (ii) incubating the population of cells of step (i) under conditions that are permissive for maintenance of the episomal vector, wherein the polynucleotides encoding reprogramming polypeptides are expressed thereby inducing the formation of a population of pluripotent stem cells from the population of differentiated cells, and, (iii) incubating the induced population of pluripotent stem cells of step (ii) under conditions that are non-permissive for maintenance of the episomal vector, wherein the episomal vector is diluted out or lost from the population of induced pluripotent stem cells due to cell division.
  • the episomal vector is
  • the episomal vector comprises an SV40 origin of replication and the differentiated cell comprises an SV40 large T antigen polypeptide.
  • the SV40 large T antigen polypeptide may be a temperature sensitive SV40 large T antigen; for example, but not limited to tsA357R-K.
  • nucleic acid encoding an SV40 large T antigen is introduced to the differentiated cell on an episomal vector.
  • the permissive condition for maintenance of the episomal vector is about 33-35 °C.
  • the non-permissive condition for maintenance of the episomal vector is about 39 °C.
  • the condition permissive for maintenance of the episomal vector is about 33-35 °C and the condition non-permissive for replication of the episomal vector is about 39 °C.
  • the differentiated cell comprises a polynucleotide encoding an SV40 large T antigen under the control of an inducible promoter; for example, but not limited to, a tetracycline regulatable promoter.
  • the condition permissive for replication of the episomal vector comprises induction of the regulatable promoter operably linked to the polynucleotide encoding the SV40 large T antigen.
  • the SV40 large T antigen is induced by tetracycline or a derivative of tetracycline.
  • nucleic acid encoding an SV40 large T antigen is introduced to the differentiated cell on an episomal plasmid.
  • the episomal vector comprises a polyoma virus origin of replication.
  • the differentiated cell comprises a polynucleotide encoding a polyoma large T antigen polypeptide.
  • the episomal vector comprises a polyoma virus origin of replication and the cell comprises a polynucleotide encoding a polyoma large T antigen.
  • the polyoma large T antigen polypeptide is a ts-a polypeptide or a ts25 polypeptide.
  • the polynucleotide encoding the polyoma large T antigen is under the control of an inducible promoter.
  • the episomal vector comprises a BKV virus origin of replication.
  • the differentiated cell comprises a polynucleotide encoding a BKV large T antigen polypeptide.
  • the episomal vector comprises a polyoma virus origin of replication and the cell comprises a polynucleotide encoding a BKV large T antigen.
  • the polynucleotide encoding the BKV large T antigen is under the control of an inducible promoter.
  • the episomal vector comprises a polyoma virus origin of replication.
  • the differentiated cell comprises a polynucleotide encoding a polyoma large T antigen polypeptide.
  • the episomal vector comprises a polyoma virus origin of replication and the cell comprises a polynucleotide encoding a polyoma large T antigen.
  • the polyoma large T antigen polypeptide is a ts-a polypeptide or a ts25 polypeptide.
  • the polynucleotide encoding the polyoma large T antigen is under the control of an inducible promoter.
  • the episomal vector comprises a bovine papilloma virus (BPV) origin of replication.
  • the episomal vector comprises the BVP MO and/or MME sequences.
  • the differentiated cell comprises polynucleotides encoding BPV El and/or E2 polypeptides.
  • the episomal vector comprises a BPV origin of replication, including the MO and/or MME sequences, and the cell comprises polynucleotides encoding BPV El and/or E2 polypeptides.
  • polynucleotides encoding BPV El and/or E2 polypeptides are under the control of one or more inducible promoters.
  • the episomal vector comprises a
  • the differentiated cell comprises a polynucleotide encoding an EBNA1 polypeptide.
  • the episomal vector comprises an EBV origin of replication and the cell comprises a polynucleotide encoding an EBNA1 polypeptide under the control of an inducible promoter.
  • the reprogramming polypeptides include, but are not limited to, one or more of the following: OCT3/4, SOX2, KLF4 and MYC.
  • the polynucleotides encoding reprogramming polypeptides are operably linked to one or more regulatory elements.
  • the one or more regulatory elements are inducible regulatory elements.
  • the more than one polynucleotides encoding reprogramming polypeptides may be on separate episomal vectors.
  • the separate episomal vectors comprising polynucleotides encoding reprogramming polypeptides may be introduced into the differentiated cells in approximately equal amounts.
  • the separate episomal vectors comprising polynucleotides encoding reprogramming polypeptides may be introduced into the differentiated cells in different amounts.
  • one or more of the episomal vectors further comprise nucleic acid encoding a modulator of vector replication; e.g. SV40 large T antigen.
  • the differentiated cell is a fibroblast.
  • Episomal vectors may be introduced into the differentiated cells by methods known in the art.
  • episomal vectors may be introduced to cells by electroporation or lipophilic transfection.
  • the invention provides induced pluripotent stem cells prepared by (i) introducing one or more episomal vectors comprising polynucleotides encoding reprogramming polypeptides into the population of differentiated cells, wherein the episomal vectors are maintained conditionally, (ii) incubating the population of cells of step (i) under conditions that are permissive for maintenance of the episomal vector, wherein the polynucleotides encoding reprogramming polypeptides are expressed thereby inducing the formation of a population of pluripotent stem cells from the population of differentiated cells, and, (iii) incubating the induced population of pluripotent stem cells of step (ii) under conditions that are non-permissive for maintenance of the episomal vector, wherein the episomal vector is diluted out from the population of induced pluripotent stem cells.
  • the differentiated cell comprises nucleic acid encoding a polypeptide that modulates replication of the episom
  • the invention provides methods of differentiation of the population of induced pluripotent stem cells.
  • the population of induced pluripotent stem cells may be differentiated into endoderm cells, ectoderm cells or mesoderm cells or any combination of endoderm cells, ectoderm cells and mesoderm cells.
  • the invention provides populations of differentiated cells prepared from a population of induced pluripotent cells by the methods of the invention.
  • the invention provides compositions comprising a population of differentiated cells prepared by the methods of the invention.
  • the invention provides populations of the hepatocytes prepared from iPS cells prepared by (i) introducing one or more episomal vectors comprising polynucleotides encoding reprogramming polypeptides into the population of differentiated cells, wherein the episomal vectors are maintained conditionally, (ii) incubating the population of cells of step (i) under conditions that are permissive for maintenance of the episomal vector, wherein the polynucleotides encoding reprogramming polypeptides are expressed thereby inducing the formation of a population of pluripotent stem cells from the population of differentiated cells, and, (iii) incubating the induced population of pluripotent stem cells of step (ii) under conditions that are non-permissive for maintenance of the episomal vector, wherein the episomal vector is diluted out from the population of induced pluripotent stem cells.
  • the differentiated cell comprises nucleic acid encoding a polypeptide that modulates replication of the episomal vector.
  • the hepatocytes are differentiated from induced pluripotent stem cells comprising a known CYP3A4 allele.
  • the invention provides a population of hepatocytes derived from iPS cells.
  • the population of hepatocytes is differentiated from induced pluripotent stem cells comprising a known CYP3A4 allele.
  • the invention provides a panel of hepatocytes comprising populations of hepatocytes of derived from iPS cells.
  • the panel of hepatocytes comprises populations of hepatocytes with different CYP3A4 alleles. In some cases, the panel of hepatocytes comprising populations of hepatocytes derived from iPS cells comprises one or more populations of hepatocytes with different drug metabolism alleles.
  • Examples of drug metabolism alleles include, but are not limited to, CYP2C9*2, CYP2C9*2, CYP2C9*3; CYP2C19*2, CYP2C19*3; CYP2E1*5; CYP2D6*2, CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*8, CYP2D6*10, CYP2D6*14, CYP2D6*lxN, CYP2D6*2xN;
  • the invention provides methods for screening compounds for toxicity, comprising contacting a population of hepatocytes derived from iPS cells with the compound and determining the effect of the compound on phenotypic or metabolic changes to the cells.
  • methods of screening compounds for toxicity comprise contacting one or more panels of hepatocytes derived from iPS cells and, determining any phenotypic or metabolic changes in the population of hepatocytes that result from being combined with the compound.
  • the panel of hepatocytes comprising populations of hepatocytes derived from iPS cells comprises one or more populations of hepatocytes with different drug metabolism alleles. Examples of drug metabolism alleles include, but are not limited to, CYP2C9*2, CYP2C9*2, CYP2C9*3; CYP2C19*2,
  • the invention provides methods for screening the metabolism of a compound, comprising contacting a population of hepatocytes derived from iPS cells with the compound and determining the metabolic changes to the compound.
  • methods of screening compounds the metabolism of a compound comprise contacting one or more panels of hepatocytes derived from iPS cells and, determining the metabolic changes to the compound.
  • the panel of hepatocytes comprising populations of hepatocytes derived from iPS cells comprises one or more populations of hepatocytes with different drug metabolism alleles. Examples of drug metabolism alleles include, but are not limited to, CYP2C9*2, CYP2C9*2, CYP2C9*3; CYP2C19*2,
  • the invention provides methods for screening compounds for their ability to modulate hepatocyte cell function, comprising contacting a population of hepatocytes derived from iPS cells with the compound and determining any effect of the compound on phenotypic or metabolic changes to the population of hepatocytes that result from being combined with the compound.
  • methods of screening compounds for their ability to modulate hepatocyte cell function comprise contacting one or more panels of hepatocytes derived from iPS cells and, determining any effect of the compound on phenotypic or metabolic changes to the population of hepatocytes that result from being combined with the compound.
  • the panel of hepatocytes comprising populations of hepatocytes derived from iPS cells comprises one or more populations of hepatocytes with different drug metabolism alleles.
  • drug metabolism alleles include, but are not limited to, CYP2C9*2, CYP2C9*2, CYP2C9*3; CYP2C19*2, CYP2C19*3; CYP2E1*5; CYP2D6*2, CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*8, CYP2D6*10, CYP2D6*14, CYP2D6*lxN, CYP2D6*2xN;
  • Figure 1 shows transfection efficiency of NHDF using the Amaxa ® nucleofectin system.
  • Panel A demonstrates FITC and Panel B demonstrates phase contrast photographs of the same field of view.
  • Figure 2 shows TAg facilitates the persistence of episomal constructs.
  • Cells were photographed using FITC (panels A, D, G and J), TR1TC (panels B, E, H and K), or phase contrast (panels C, F, I and L) of the same field of view.
  • Figure 3 is a graph showing growth of NHDF at 32°C. The least fit trend lines are shown for cells cultured at 32°C (closed squares) and 37°C (open circles).
  • FIG. 4 demonstrates characterization of hepatocyte-like cultures derived from hES.
  • Panel A is a graph representing FACs analysis of definitive endoderm formation at day 6 as assessed by CXCR4/cKlT expression.
  • Panel B is a graph showing normalized ALB transcript levels in hES derived cultures (T32) versus fetal liver (FL) and adult liver (AL) samples at day 32.
  • Panels C, D and E are photographs showing immuno staining of day 32 cells for AFP (panel C), a phase contrast control (panel D), and for ALB with DAPI counterstain (panel E).
  • Figure 5 shows cells two days (5A) and eight days (5B) following transfection of reprogramming genes and CMV-Timer.
  • Cells were photographed using phase contrast (left panels), FITC (middle panels), and TRITC (right panels) of the same field of view.
  • 1 indicates cells transfected with 1 ⁇ g TAg plasmid; 1 ⁇ g mixture of SOX2, OCT3/4, KLF4 and c-MYC episomal plasmids; and 0.1 ⁇ g CMV-Timer.
  • 2 indicates cells transfected with 2 ⁇ g TAg plasmid; 1 ⁇ g mixture of SOX2, OCT3/4, KLF4 and c-MYC episomal plasmids; and 0.1 ⁇ g CMV-Timer.
  • 3 indicates cells transfected with 0.1 ⁇ g TAg plasmid; 1 ⁇ g mixture of SOX2, OCT3/4, KLF4 and c-MYC episomal plasmids; and 0.1 ⁇ g CMV-Timer.
  • 4 indicates cells transfected with pcDNA6.2 CAT control.
  • the invention is based, in part, on the discovery that iPSC may be generated from a population of differentiated cells by the transient expression of reprogramming genes delivered to the differentiated cells on episomal (i.e. nonintegrating) vectors.
  • episomal vectors encoding reprogramming genes comprise a Simian Vacuolating virus 40 (SV40) origin (SV40 ori) of replication and the population of differentiated cells comprises a temperature sensitive SV40 large T antigen (TAg). At the permissive temperature, the episomal vectors replicate and are maintained in the SV40 origin (SV40 ori) of replication and the population of differentiated cells comprises a temperature sensitive SV40 large T antigen (TAg). At the permissive temperature, the episomal vectors replicate and are maintained in the SV40 origin (SV40 ori) of replication and the population of differentiated cells comprises a temperature sensitive SV40 large T antigen (TAg). At the permissive temperature, the episomal vectors replicate and are maintained in the SV40 origin (SV
  • the differentiated cell thereby allowing expression of the reprogramming genes and the induction of pluripotency.
  • the temperature is shifted to the non-permissive temperature.
  • the episomal vectors no longer replicate and are no longer maintained in the cell population.
  • the episomal vectors are diluted out of the population of induced pluripotent cells resulting in a population of iPSC which does not contain recombinant reprogramming genes.
  • episomal vectors encoding reprogramming genes comprise an SV40 origin of replication and the population of differentiated cells comprises a polynucleotide encoding SV40 TAg under the control of an inducible promoter; for example the tetracycline (tet) inducible promoter.
  • an inducible promoter for example the tetracycline (tet) inducible promoter.
  • SV40 TAg is expressed and the episomal vectors replicate and are maintained in the population of differentiated cells, thereby allowing expression of the reprogramming genes and the induction of pluripotency. After pluripotency is induced in the population of cells, the activator of SV40 TAg expression is removed from the culture.
  • the episomal vectors no longer replicate and are no longer maintained in the cell population. Ultimately the episomal vectors are diluted out of the population of induced pluripotent cells resulting in a population of iPSC which does not contain recombinant reprogramming genes.
  • the invention provides methods of screening factors for their ability to differentiate iPSC into different lineages of cells. Test factors are contacted with iPSC generated by transient expression of reprogramming genes and any phenotypic or metabolic changes in the cell that result from being combined with the compound are determined. Genetic markers for different lineages of cells; for example, mesoderm, endoderm and ectoderm, may be used to determine the role of the factor in specific cell differentiation.
  • the invention provides methods of determining factors involved in endoderm development.
  • the invention provides methods of determining factors involved in mesoderm development.
  • the invention provides methods of determining factors involved in ectoderm development.
  • the invention provides methods of generating populations of iPSC from differentiated cells by the transient expression of reprogramming genes.
  • reprogramming genes include, but are not limited to, oct3/4, sox2, klf4 and c-myc.
  • polynucleotides expressing reprogramming genes are introduced to the population of differentiated cells on one or more episomal vectors.
  • the episomal vectors comprise an SV40 origin of replication.
  • reprogramming genes are under the control of one or more regulatory elements which permit expression of the reprogramming genes in the population of differentiated cells.
  • the regulatory elements controlling expression of one or more of the reprogramming genes are inducible regulatory elements.
  • the invention provides methods of altering the levels of expression of reprogramming genes relative to one another. As such, the efficiency of induction of pluripotency in a cell population may be optimized.
  • each reprogramming gene is provided on a separate episomal vector. The relative ratio of each vector delivered to a population of differentiated cells can be altered to optimize
  • the relative expression of reprogramming genes in a population of differentiated cells can be controlled by the use of different regulatory elements with different expression levels to optimize reprogramming of the population of differentiated cells to a population of iPSC.
  • Populations of iPSC may be subsequently differentiated into populations of more mature phenotype.
  • a population of iPSC of the invention may be differentiated into a population of mesoderm cells or derivatives of mesoderm cells.
  • a population of iPSC of the invention may be differentiated into a population of ectoderm cells or derivatives of ectoderm cells.
  • a population of iPSC of the invention may be differentiated into a population of endoderm cells or derivatives of endoderm cells.
  • the endoderm cells may be further differentiated to hepatocytes or hepatocyte-like cells.
  • iPSC are generated from dermal fibroblasts and are subsequently differentiated into hepatocytes or hepatocyte-like cells.
  • the invention provides methods of generating iPSC from differentiated cells of known genotypes.
  • the invention provides methods of generating populations of iPSC from populations of differentiated cells comprising known alleles of genes involved in drug metabolism. These populations of iPSC, with known drug metabolism alleles, can be used to generate populations of hepatocytes or hepatocyte-like cells which express known drug metabolism alleles.
  • the invention provides panels of hepatocytes or hepatocyte-like cells derived from iPSC with known genotypes of drug metabolism genes.
  • populations of hepatocytes or hepatocyte-like cells derived from populations of iPSC with known genotypes of drug metabolism genes are used for drug discovery, metabolism and toxicity studies.
  • the term “totipotent cell” refers to a cell capable of developing into all lineages of cells.
  • the term “population of totipotent cells” refers to a composition of cells capable of developing into all lineages of cells.
  • the term “pluripotent cell” refers to a cell capable of developing into a variety (albeit not all) lineages.
  • a “population of pluripotent cells” refers to a composition of cells capable of developing into less than all cell lineages. As such, a totipotent cell or composition of cells is less developed than a pluripotent cell or composition of cells.
  • “Multipotent cells” are more differentiated relative to pluripotent cells, but are not terminally differentiated.
  • the terms “develop,” “differentiate,” and “mature” all refer to the progression of a cell from the stage of having the potential to differentiate into at least two different cellular lineages to becoming a specialized cell. Such terms can be used interchangeably for the purposes of the present application.
  • a “differentiated cell” is a mature cell that has undergone progressive developmental changes to a more specialized form or function. Cell differentiation is the process a cell undergoes as it matures to an overtly specialized cell type. Differentiated cells have distinct characteristics, perform specific functions, and are less likely to divide than their less differentiated counterparts.
  • An "undifferentiated" cell for example, an immature, embryonic, iPSC or primitive cell, typically has a non-specific appearance.
  • a "dedifferentiated" cell is a cell that has changed from a more differentiated state to a less differentiated state.
  • a dedifferentiated cell may be a cell that has changed from a mature state to a multipotent or pluripotent state.
  • Episomal vectors are eukaryotic expression vectors which are maintained and replicate extrachromasomally whenever the necessary trans-acting factors are provided. Such vectors are advantageous under conditions where one does not desire integration of introduced polynucleotides into the host cell chromosomes.
  • Examples of episomal vectors include, but are not limited to vectors based on sequences from DNA viruses such as SV40, polyoma virus, Epstein-Barr virus, BK virus, and bovine papilloma virus (Craenenbroeck, K. et al. (2000) Eur. J. Biochem. 267:5665-5678).
  • episomal vectors have an SV40 origin of replication. Such vectors may be maintained and replicated in the presence of SV40 TAg.
  • a "regulatory sequence” refers to any or all of the DNA sequences that controls gene expression.
  • regulatory sequences include promoters, positive regulatory elements such as enhancers or DNA-binding sites for transcriptional activators, negative regulatory elements such as DNA-binding sites for a transcriptional repressors and insulators. Regulatory sequences may be found within, 5' and/or 3' to the coding region of the gene.
  • Inducible or regulatable promoters generally exhibit low activity in the absence of the inducer, and are up-regulated in the presence of the inducer.
  • the inducible promoter can be induced by a molecule (e.g. a small molecule or protein) heterologous to the cell in which the expression cassette is to be used.
  • inducible promoters are well-known to those of ordinary skill in the art including but not limited to the tetracycline responsive system and the lac operator-repressor system (see WO 03/022052 Al; and US 2002/0162126 Al), the ecdysone regulated system, or promoters regulated by glucocorticoids, progestins, estrogen, RU-486, steroids, thyroid hormones, cyclic AMP, cytokines, the calciferol family of regulators, or the metallothionein promoter (regulated by inorganic metals).
  • genes encoding SV40 TAg and/or reprogramming genes are operably linked to a tetracycline-inducible promoter.
  • genes encoding reprogramming proteins are linked by an internal ribosome entry site (IRES) and are operably linked to a tetracycline-inducible promoter.
  • genes encoding reprogramming proteins are linked by foot-and- mouth disease virus 2A element. Multicistronic and inducible expression systems are known in the art. See, for example, Chappell, S.A. et al. (2004) Proc Natl Acad Sci U S A.
  • genes encoding reprogramming proteins are linked by self- processing cleavage sites linking the two proteins.
  • the linking of proteins in the form of polyproteins in a single open reading frame is a strategy adopted in the replication of many viruses including picornaviridae.
  • virus-encoded proteinases mediate rapid intramolecular (cis) cleavage of a polyprotein to yield discrete mature protein products.
  • Foot and Mouth Disease viruses are a group within the picornaviridae which express a single, long open reading frame encoding a polyprotein of approximately 225 kD.
  • the full length translation product undergoes rapid intramolecular (cis) cleavage at the C-terminus of a self-processing cleavage site, for example, a 2A site or region, located between the capsid protein precursor (P1-2A) and replicative domains of the polyprotein 2BC and P3, with the cleavage mediated by proteinase-like activity of the 2A region itself (Ryan et al., J. Gen. Virol. 72:2727-2732, 1991); Vakharia et al, J. Virol. 61:3199-3207, 1987). Similar domains have also been characterized from aphthoviridea and cardioviridae of the picornavirus family (Donnelly et al, J. Gen. Virol. 78:13-21, 1997).
  • marker refers to a polynucleotide sequence whose expression product, reporter, or marker, (whether
  • Detection can be by any means, including but not limited to visible to the naked eye, spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • Reporter molecules of the invention are known in the art. Recombinant DNA reporter gene systems were developed to enable quantitative, rapid and inexpensive measurement of the activity of the study of transcriptional promoters and enhancers
  • TREs transcriptional regulatory elements
  • Recombinant DNAs encoding enzyme are often used as reporter genes due to the sensitivity of enzyme assays.
  • enzymes used as reporter genes include chloramphenicol acetyltransferase (CAT; Gorman CM et al, 1982 Mol. Cell. Biol. 2:1044), beta-galactosidase ( ⁇ -gal), beta-lactamase (BLA) Zlorkanik G, et al., 1998 Science 279:84-88), secreted alkaline phosphatase (SEAP; Berger J et al, 1988 Gene 66:1-10), and beta-glucuronidase (GUS) Jefferson RA, et al, 1987 EMBO J. 6:3901-3907).
  • CAT chloramphenicol acetyltransferase
  • ⁇ -gal beta-galactosidase
  • BLA beta-lactamase
  • SEAP secreted alkaline phosphatase
  • GUS beta-glucuroni
  • LOC luciferases
  • fluorescent proteins have found wide use as reporters for gene expression.
  • the most commonly used fluorescent protein is the green fluorescent protein (GFP) from the jellyfish, Aequorea victoria (Chalfie M, et al, 1994 Science 263:802-805).
  • GFP green fluorescent protein
  • the gene for GFP has been mutated for improved stability, spectroscopic properties, and expression in eukaryotes as well as different fluorescent colors.
  • fluorescent proteins include yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), orange fluorescent protein (OFP) and red fluorescent protein (RFP).
  • the preferred detection reagent for detection of the marker will depend on the identity of the marker.
  • the preferred detection reagent is a substrate, whether natural or synthetic, that is detectable after processing by the enzyme. Any type of substrate in which the processed product can be assayed should be suitable, although chromogenic and fluorogenic ⁇ e.g., substrates which become colored or fluorescent after enzyme processing) are preferred.
  • enzyme-substrate combinations include beta-galactosidase and O-nitrophenol-b-D-pyranogalactoside (ONPG), beta-galactosidase and fluoroscein din-b-galactopyranoside (FDG) beta-galactosidase and galacton, firefly luciferase and D-luciferin, Renilla luciferase and coelenterazine, Gaussia luciferase and coelenterazine and alkaline phophotase and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP).
  • Another reporter molecule and detection reagent pair is ⁇ -lactamase and CCF2.
  • CCF2 fluoresces green in its native state but cleavage of the ⁇ -lactam ring of CCF2; for example by ⁇ - lactamase, results in blue fluorescence.
  • the reporter molecule is a fluorescent reporter, for example; GFP, YFP,
  • reporter expression may be determined by any method known in the art to detect and/or measure fluorescence.
  • cells expressing GFP may be detected by fluorescence microscopy or by fluorescence activated cell sorting analysis. In other cases, fluorescence may be measured with a fluorometer.
  • Episomal vectors may be introduced into a somatic cell using standard techniques.
  • transfection or “transformation” refer to the insertion of an exogenous polynucleotide into a host cell.
  • transfection techniques include, but are not limited to, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation and microinjection.
  • the method of introducing polynucleotides into a differentiated cell is optimized for a specific cell type. For example, if the differentiated cell of the invention is a dermal fibroblast, an optimal introduction technique may be determined among techniques known in the art. Suitable methods for transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, and other laboratory textbooks.
  • a mixture of replicating episomal plasmids that encode reprogramming genes may be used to generate iPSC from differentiated cells without integration of the vectors in the cellular genome.
  • Episomal vectors are eukaryotic expression vectors which are maintained and replicate extrachromasomally whenever the necessary trans-acting factors are provided. Such vectors are advantageous under conditions where one does not desire integration of introduced polynucleotides into the host cell chromosomes.
  • Replicating episomal vectors are often based on DNA virus in which the viral origin of replication is provided in cis and the necessary viral and cellular factors to stimulate replication are provided in trans.
  • episomal vectors include, but are not limited to vectors based on sequences from DNA viruses such as SV40, Polyoma virus (PyV), Epstein-Barr virus (EBV), BK virus (BKV), and bovine papilloma virus (BPV) (Gassmann, M. et al. 1995 Proc. Natl. Acad. Sci. USA 92:1292-1296; Camenisch, G. et al. 1996 Nucl. Acids Res. 24:3707-3713; Craenenbroeck, K. et al. 2000 Eur. J. Biochem.
  • DNA viruses such as SV40, Polyoma virus (PyV), Epstein-Barr virus (EBV), BK virus (BKV), and bovine papilloma virus (BPV)
  • SV40 TAg is a hexamer protein derived from the polyoma virus SV40 which is capable for transforming a variety of cell types.
  • TAg is a product of an early gene transcribed during viral infection and is involved in genome replication and regulation of host cell cycle. After entering the cell, viral genes are transcribed by host cell RNA polymerase II to produce early mRNAs, including mRNA encoding TAg.
  • SV40 replication is initiated by binding of TAg to the SV40 ori.
  • the function of TAg is controlled by phosphorylation, which attenuates its binding to the SV40 ori.
  • Protein-protein interactions between TAg and DNA polymerase a directly stimulate replication of the virus genome.
  • SV40-based episomal systems permit transient expression of reprogramming genes that induce iPSC production without permanent introduction of these genes into the somatic cell genome.
  • Each plasmid including the plasmid encoding the SV40 TAg itself, contains the SV40-origin. In the presence of functional SV40 TAg, these plasmids are maintained as episomes within populations of mammalian cells.
  • SV40 TAg function is regulated by use of a temperature sensitive mutant (tsA357R-K) of TAg (Reynisdottir, 1992).
  • tsA357R-K is also known as tsA30, these names may be used interchangeably.
  • a V5 epitope-tagged, tsA357R-K mutant of SV40 large TAg is generated by PCR mutagenesis of tagged wild type TAg, and subcloned into a suitable vector comprising an SV40 origin of replication, such as pcDNA6.2.
  • episomal vectors expressing reprogramming genes have an SV40 origin of replication. Such vectors may be maintained and replicated in the presence of SV40 TAg.
  • the invention provides methods to generate iPSC from populations of differentiated cells by the introduction of a mixture of plasmids, each containing an SV40-origin of replication, that encode reprogramming genes and a
  • tsTAg temperature sensitive mutant of SV40 large T antigen (tsTAg) to generate iPSC from differentiated cells.
  • tsTAg SV40 large T antigen
  • expression of SV40 TAg is under the control of an inducible promoter.
  • Inducible promoters may be any inducible promoter known in the art. Such inducible systems include, for example, tetracycline inducible systems,
  • a polynucleotide encoding an SV40 large TAg may be under the control of a tetracycline inducible promoter (May, T. et al., 2005 Biochem. Biophys. Res. Commun. 327:734-741).
  • SV40-based episomal vectors are used to deliver reprogramming genes to generate iPSC from populations of differentiated cells.
  • SV40 TAg may be expressed under the control of an inducible promoter. Plasmids encoding reprogramming genes, as well as plasmids encoding TAg, are encoded on plasmids containing a SV40 ori. In the presence of the activator of the inducible promoter, TAg is expressed and the SV40 ori containing plasmids are maintained as episomes within populations of mammalian cells. By removing the activator of the inducible promoter, expression of TAg is diminished and therefore loses its ability to maintain and replicate the episomes. Consequently, the episomes are progressively lost from the population as the cells divide.
  • SV40 TAg As an alternative to using SV40 TAg to stimulate replication and maintenance of SV40-based episomal plasmids, a system where TAg is replaced by the scaffold/matrix attached region (S/MAR) from the 5' region of the human interferon gene may be used (Piechaczek, C. et al 1999 Nucl. Acids Res. 27:426-428). Here, expression of S/MAR is under the control of an inducible promoter. Plasmids encoding reprogramming genes as well as encoding S/MAR itself, contain the SV40-origin.
  • S/MAR scaffold/matrix attached region
  • SV40- based episomal vectors are used to deliver reprogramming genes to generate iPSC from populations of differentiated cells and S/MAR is used to maintain and replicate the SV40- based episomes.
  • S/MAR is expressed under the control of an inducible promoter.
  • plasmids encoding reprogramming genes, as well as plasmids encoding S/MAR are introduced to differentiated cells.
  • S/MAR is expressed and the SV40 ori containing plasmids are maintained as episomes within populations of mammalian cells. By removing the activator of the inducible promoter, expression of S/MAR is diminished and therefore loses its ability to maintain and replicate the episomes.
  • Polyoma virus DNA replicates as unintegrated minichromosomes in mouse cells.
  • the replication of polyoma DNA requires only the viral origin of replication, the transacting protein large T, and cellular proteins needed for DNA synthesis (Gassmann, M. et al. 1995 Proc. Natl. Acad. Sci. USA 92:1292-1296; Camenisch, G. et al. 1996 Nucl. Acids Res. 24:3707-3713).
  • Polyoma-based vectors have been shown to replicate in ES cells (Niwa, H. et al. 2002 Mol. Cell. Biol. 22:1526-1536; Aubert, J. et al. 2002 Nat. Biotech. 20:1240-1245).
  • episomal vectors based on murine polyoma virus may be used to deliver reprogramming genes to generate iPSC from populations of differentiated cells.
  • Reprogramming gene episomes and ts large T episomes are introduced into differentiated cells and incubated at the permissive temperature of ⁇ 32°C to allow replication of episomes and expression of reprogramming genes.
  • the temperature is shifted to the nonpermissive temperature of ⁇ 39°C.
  • the resulting iPS cells may be maintained at -37 °C.
  • a modified early region of polyoma virus that encodes a PyV large T antigen may be expressed under the control of an inducible promoter.
  • Plasmids encoding reprogramming genes, as well as plasmids encoding large T are constructed to also contain a Py-origin.
  • PyV- based plasmids encoding reprogramming genes and Py large T are introduced to
  • differentiated cells In the presence of the activator of the inducible promoter, large T is expressed and the Py-ori containing plasmids are maintained as episomes within populations of mammalian cells. Reprogramming genes are expressed to induce dedifferentiation of the differentiated cells. By removing the activator of the inducible promoter, expression of large T is diminished and therefore loses its ability to maintain and replicate the episomes.
  • the Py large-T is derived from plasmid PGKhphA LT20 (Gassmann, M. et al. 1995 Proc. Natl. Acad. Sci. USA
  • BKV is a polyoma virus that infects human cells and can transform hamster, mouse, rat, rabbit and monkey cells in tissue culture. Replication of BKV DNA requires only the viral origin of replication and the trans-acting large T antigen with its DNA-binding and helicase activites intact (Craenenbroeck, K. et al. 2000 Eur. J. Biochem. 267:5665-5678).
  • episomal vectors based on BKV may be used to deliver reprogramming genes to generate iPSC from populations of differentiated cells. For example, a BKV large T antigen may be expressed under the control of an inducible promoter.
  • Reprogramming genes are encoded on episomal plasmids containing a BK-ori.
  • large T is expressed and the BK-ori containing plasmids are maintained as episomes within populations of mammalian cells.
  • Reprogramming genes are expressed to induce generation of iPSC from the differentiated cells.
  • the activator of the inducible promoter is removed from the population of cells and expression of large T is diminished. As such, large T loses its ability to maintain and replicate the episomes and consequently, the episomes are progressively lost from the population as the cells divide.
  • Bovine papilloma virus can transform a variety of rodent cells in culture. Viral plasmid DNA replication is divided into two stages: establishment and maintenance. BPV replication requires two viral encoded proteins, El and E2 in trans and the BPV ori in cis (Ustav, M. and Stenlund, A. 1991 EMBO J. 10:449-457). BPV replication has been demonstrated in human cells when El and E2 are expressed from a heterologous promoter. A minimal ori (MO) has been identified (Ustav, E. et al. 1993 Proc. Natl. Acad. Sci. USA 90:898-902).
  • MO minimal ori
  • the invention provides methods to generate iPSC from populations of differentiated cells by delivering
  • reprogramming genes encoded on BPV-based vectors are encoded on episomal plasmids containing a BPV MO and a BPV MME.
  • plasmids containing a BPV MO and a BPV MME are constructed to express BPV El and BPV E2 under the control of an inducible promoter.
  • BPV El and BPV E2 are under the control of the same inducible promoter.
  • BPV El and BPV E2 are under the control of different inducible promoters.
  • only BPV El is under the control of an inducible promoter.
  • BPV E2 is under the control of an inducible promoter.
  • BPV El and BPV E2 are encoded on the same plasmid under the control of one inducible promoter.
  • polynucleotides encoding BPV El and BPV E2 are linked by an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • polynucleotides encoding BPV El and BPV E2 are linked to express a fusion protein of El and E2 (see, for example, U.S. Pat. No. 5,674,703).
  • BPV-based plasmids encoding reprogramming genes and BPV El and BPV E2 are introduced to differentiated cells.
  • the activator(s) of the inducible promoters is added to the cells to activate expression of BPV El and/or BPV E2.
  • BPV MO and BPV MME-containing plasmids are replicated and maintained in the cells thereby allowing expression of reprogramming genes.
  • the activator(s) of the inducible promoter(s) is removed from the culture and episomes are progressively lost from the population of iPSC as the cells divide.
  • human papilloma virus ori, MME, El and E2 elements are used.
  • EBV-based vectors based on EBV have been used successfully in biotechnology and have been shown to replicate in a variety of human cell lines as well as monkey and dog cell lines. A recent study has shown that EBV-based vectors are maintained and replicate in hES cells (Ren, C. et al. 2006 Stem Cells 24:1338-1347).
  • An example of a commercially available episomal vector based on EBV is pCEP4 (Invitrogen). The only viral elements necessary for stable episomal maintenance in the cell are the oriP and EBNAl sequences (Craenenbroeck, K. et al. 2000 Eur. J. Biochem. 267:5665-5678).
  • OriP is composed of two noncontiguous regions, a family of repeats and the dyad symmetry element and is required in cis for plasmid replication.
  • EBNAl binds directly to oriP and is required in trans for plasmid replication.
  • the invention provides methods to generate iPSC from differentiated cells by delivering reprogramming genes encoded on EBV-based vectors.
  • reprogramming genes are encoded on episomal plasmids containing oriP.
  • a plasmid expressing EBNAl under the control of an inducible promoter is encoded on an episomal plasmid containing oriP.
  • EBV- based plasmids encoding reprogramming genes and EBNAl are introduced to differentiated cells.
  • the activator of the inducible promoter is added to the cells to activate expression of EBNAl and the oriP-containing plasmids are replicated and maintained in the cells thereby allowing expression of reprogramming genes.
  • the activator of the inducible promoter is removed from the culture and oriP episomes are progressively lost from the population of iPSC as the cells divide.
  • the vectors used to generate iPSC from differentiated cells by delivering reprogramming genes encoded on episomal vectors are not EBV-based vectors.
  • Reprogramming genes encode factors that play a role in the induction of totipotency or pluripotency to somatic cells. In some cases, reprogramming factors play a role in the maintenance of embryonic stem (ES) cell identity. In some aspects of the invention, reprogramming factors include, but are not limited to OCT3/4 (POU5FL), SOX2, KLF4 and MYC. In some aspects of the invention; genes encoding one or more of OCT3/4, SOX2, KLF4 and MYC are introduced into populations of differentiated cells to induce reprogramming of the differentiated cells into pluripotent cells.
  • additional genes encoding potential reprogramming factors are introduced to differentiated cells to induce reprogramming of the differentiated cells into pluripotent cells.
  • additional potential reprogramming factors include, but are not limited to, NANOG, FOXD3, UTF1, ZNF206, MYB12, LIN28, ESG1, OTX2.
  • Polynucleotides encoding reprogramming factors may be from any source and subcloned into a suitable episomal vector; for example, a vector comprising an SV40 origin of replication.
  • reprogramming genes are cloned into pCMV-Script.
  • reprogramming genes may also be cloned under the control of an inducible regulatory element.
  • one or more reprogramming genes may be under the control of a tetracycline-inducible promoter, thereby permitting modulation of expression levels at initial and later states of reprogramming.
  • two or more reprogramming genes are on the same vector.
  • two or more reprogramming genes are on the same vector but under the control of different regulatory elements.
  • two or more reprogramming genes are on the same vector and under the control of the same regulatory element.
  • two reprogramming genes may be under the control of the same regulatory element and separated by an internal ribosome entry site (IRES).
  • a vector encoding SV40 TAg also encodes one or more reprogramming genes. V. Differentiated cells
  • the differentiated cells of the invention used to generate iPSC may be from any source and may be obtained by one of skill in the art.
  • Differentiated cells of the invention are non-embryonic cells obtained from a fetal, newborn, juvenile or adult mammal, including humans.
  • Examples of differentiated cells that can be used with the methods of the invention include, but are not limited to, fibroblast cells, hepatic cells, bone marrow cells, epithelial cells, hematopoietic cells, intestinal cells, mesenchymal cells, myeloid precursor cells and spleen cells.
  • the differentiated cells may be cells that can themselves proliferate and further differentiate into other types of cells, including blood stem cells, muscle/bone stem cells, brain stem cells and liver stem cells.
  • the differentiated cells are fibroblasts.
  • the differentiated cells are dermal fibroblasts.
  • the invention provides methods of iPSC induction to generate multiple iPSC lines using donor cells from individuals of diverse genetic backgrounds.
  • the invention provides methods of iPSC induction to generate multiple iPSC lines using donor cells from individuals of diverse ethnic backgrounds, with subsequent detailed genotypic analysis of several drug metabolism genes in each line.
  • dermal fibroblasts from donors of Caucasian, Asian and Latino backgrounds may be used to create a panel of iPSC derived hepatocytes. These ethnic backgrounds have been shown to represent three major classes of drug metabolism alleles (Ingelman-Sundberg, M. et al. 2007 Pharmacol Ther. 116:496-526).
  • the invention provides methods of induction of iPSC that are modified from that described in Takahashi, K and Yamanaka, S. (2006) Cell 126:663-676.
  • Plasmids are introduced into populations of differentiated cells by methods known in the art.
  • plasmids may be introduced into populations of differentiated cells using the Amaxa ® nucleofectin system.
  • the differentiated cells are normal human dermal fibroblast cells. Optimization of cell culture at permissive and non-permissive temperatures allows maintenance and elimination, respectively, of the episomes, without compromising cell viability.
  • plasmid mixtures encoding tsA357R-K TAg and destabilized GFP are used to optimize the kinetics of establishment or loss of episomal constructs.
  • real-time PCR primer pairs designed to detect the SV40-origin sequence is used to detect episomal DNA.
  • the kinetics of TAg expression may be monitored by staining for the V5 epitope tag and GFP fluorescence reflects the establishment and loss of episomally encoded proteins.
  • each dose/ratio is scored by counting the number of iPSC colonies; for example, based upon colony morphological characteristics (Meissner, A. et al. 2007 Nat. Biotechnol. 25:1177-1181). In some cases, representative colonies are selected to validate the iPSC induction process using real-time PCR primer pairs that distinguish transcripts of the plasmid-encoded reprogramming genes from those of their endogenous counterparts, as well as pairs detecting hTERT expression.
  • the endpoint of the dose optimization is the determination of the dose and ratio of the TAg and reprogramming gene constructs that yields reliable and efficient iPSC colony induction.
  • the relative ratio of reprogramming genes may be adjusted to increase reprogramming efficiency.
  • reprogramming genes may be introduced to cells on separate vectors.
  • the ratio of the quantities of vectors may be altered to optimize the efficiency of reprogramming.
  • vectors encoding OCT3/4, SOX3, KLF and c-MYC may be introduced to differentiated cells at a 1:1:1 :1 ratio or the vectors may be introduced wherein one or more vectors encoding reprogramming genes represent a larger proportion of the pool of vectors.
  • One of skill in the art may optimize the ratio of vectors which results in the greatest efficiency of induction of iPSC.
  • the efficiency of induction of iPSC may be determined by characterization of the iPSC as described below.
  • the relative ratio of reprogramming gene products may be adjusted by operably linking the reprogramming genes to different regulatory elements. For example, one or more reprogramming gene products that may be needed in greater abundance may be expressed under the control of a strong promoter element whereas one or more reprogramming gene products that may be needed in lower abundance may be under the control of a weak promoter.
  • a strong promoter element whereas one or more reprogramming gene products that may be needed in lower abundance may be under the control of a weak promoter.
  • expression levels of specific promoters in specific cell types can be determined; for example, by placing a reporter gene under the control of the promoter.
  • expression levels of reprogramming genes may be regulated by the use of episomal vectors with different copy numbers.
  • a reprogramming gene needed in abundance may be encoded on a high copy number plasmid such as SV40-based plasmids and a reprogramming gene needed in low abundance is encoded on a low copy number plasmid such as EBV-based plasmids.
  • reprogramming genes are expressed under the control of inducible promoters.
  • control of expression may be controlled by the amount and the timing of introduction to the inducer of the inducible promoter.
  • the reprogramming genes are expressed differentially relative to timing.
  • the introduction of vectors encoding reprogramming genes may be spaced out over time; for example, depending on if the reprogramming gene is required early or late in the induction process.
  • temporal control of reprogramming gene expression may be the controlled through the use of inducible promoters.
  • temporal control of gene expression may be obtained by introducing inducers at different points in time.
  • the invention provides methods to generate iPSC from a population of mature differentiated cells.
  • An illustrative but non-limiting example is given by the induction of iPSC by reprogramming normal human dermal fibroblasts (NHDF).
  • NHDF normal human dermal fibroblasts
  • FBS Fetal bovine serum
  • episomal plasmids containing an SV40 origin of replication and encoding tsTAg, OCT3/4, SOX2, KLF4 and c-MYC are introduced to the fibroblasts by
  • Fibroblasts are incubated at the permissive temperature of 32°C.
  • fibroblasts are harvested by trypsinization and replated on a STO feeder layer. Cells are continued to be maintained at 32°C.
  • the media is replaced with primate ES cell medium supplemented with 4 ng/ml bFGF. The medium is changed approximately every two days.
  • ES-like colonies are picked. Colonies are mechanically dissociated and transferred to STO feeder cells. The temperature is then shifted to the nonpermissive temperature of 39°C.
  • Cells are passaged by treatment with collagenase and at various times after the shift to 39°C, cells are assayed for the presence of the tsTAg and reprogramming gene vectors. When it is determined that cells no longer harbor vectors encoding tsTAg, the temperature is shifted to and maintained at 37 °C.
  • Induced pluripotent stem cells may be cultured in any medium used to support growth of pluripotent cells.
  • Typical culture medium includes, but is not limited to, a defined medium, such as TeSRTM (StemCell Technologies, Inc.; Vancouver, Canada), mTeSRTM (StemCell Technologies, Inc.) and StemLineTM serum-free medium (Sigma; St. Louis, Mo.), as well as conditioned medium, such as mouse embryonic fibroblast (MEF)-conditioned medium.
  • Defined medium refers to a biochemically defined formulation comprised solely of biochemically-defined constituents.
  • a defined medium may also include solely constituents having known chemical compositions.
  • a defined medium may further include constituents derived from known sources.
  • Conditioned medium refers to a growth medium that is further supplemented with soluble factors from cells cultured in the medium.
  • cells may be maintained on MEFs in culture medium.
  • cells are maintained on MEFs in DSR high glucose DMEM with KSR, glutamine, non-essential amino acids and -mercaptoethanol (Humphrey, R.K., et al, (2004) Stem Cells 22(4): 522- 530).
  • the invention provides methods to phenotypically, genetically, and functionally characterize the iPSC lines induced prepared by the methods described above. Validation of these cell lines are based upon multiple, established criteria (Takahashi, K. and Yamanaka, S. 2006 Cell 126:663-676; Park, I.H. et al, 2008 Nature 452:141-146; Yu, J. et al, 2007 Science 318:1917-1920; Wernig, M. et al 2007 Nature 448:318-324).
  • iPSC are examined for colonies with distinct, sharp boundaries and cells with large nuclei and minimal cytoplasm.
  • iPSC may be tested for markers of undifferentiated stem cells, including alkaline phosphatase activity and expression of antigens such as SSEA- 4, TRA-1-60, and TRA-1-81. Additionally, reactivation of telomerase activity may be confirmed using commercially available telomerase detection kits (Chemicon). Successfully induced iPSC express markers of undifferentiated stem cells and exhibit telomerase activity, whereas uninduced dermal fibroblasts do not.
  • iPSC may be validated by a number of criteria include karyotyping, FISH analysis, and DNA fingerprinting. These genetic assays not only verify that iPSC lines have been created with normal chromosomal and genetic composition, but these analyses also ensure that there has been no cross-contamination between the iPSC lines and other cell lines. Short-tandem repeat analysis may be performed to confirm that the iPSC lines are derived from the starting fibroblasts, while methylation analysis for the OCT4 and NANOG genomic loci may be used to confirm successful reprogramming of the fibroblasts to iPSC (Park, I.H. et al, 2008 Nature 452:141-146).
  • Gene expression may be characterized in the iPSC lines by RT-PCR to verify expression of a panel of known markers of undifferentiated, pluripotent human cells that, by comparison, would not ordinarily be expressed in uninduced dermal fibroblasts.
  • the iPSC lines may be tested for their potential to differentiate into derivatives of all three primary germ layers: ectoderm, mesoderm, and endoderm.
  • embryoid body (EB) outgrowths of the iPSC may be immunostained for markers of each of the three lineages; for example, alpha-fetoprotein (AFP) for endoderm (liver); myosin for mesoderm (muscle); and ⁇ -tubulin-III for ectoderm (neurons).
  • RT-PCR for a panel of differentiation markers of each lineage may be performed on RNA isolated from the EB outgrowths.
  • Directed differentiation may be performed using established protocols to differentiate the iPSC lines specifically into hepatocytes, cardiomyocytes, and neurons.
  • Pluripotency may also be assessed by teratoma testing in SCID mice. Resultant tumors are analyzed histologically for tissue derivatives of all three lineages.
  • the absence of reprogramming gene constructs in the final iPSC lines may be tested by standard assays known in the art. For example, real-time PCR may be performed to determine if residual exogenous reprogramming transcripts remain.
  • FISH probes prepared from the backbones of the episomal plasmids or from the cDNA encoding the TAg may be used to demonstrate the lack of randomly integrated episomal plasmid DNA.
  • An iPSC population of the present invention is capable of developing into cells of mesodermal lineage, of ectodermal lineage or of endodermal lineage.
  • mesodermal cells include cells of connective tissue, bone, cartilage, muscle, blood and blood vessels, lymphatic and lymphoid organ, notochord, pleura, pericardium, peritoneum, kidney and gonad.
  • Ectodermal cells include epidermal tissue cells, such as those of nail, hair, glands of the skin, the nervous system, the external sense organs (e.g., eyes and ears), and mucous membranes (such as those of the mouth and anus).
  • Endodermal cells include cells of the epithelium such as those to the pharynx, respiratory tract (except the nose), digestive tract, bladder and urethra cells.
  • cells within an iPSC population of the present invention include at least one of the following cellular lineages: hematopoietic cell lineage, endothelial cell lineage, epithelial cell lineage, muscle cell lineage, hepatic cell lineage, endocrine cell lineage, and neural cell lineage.
  • Another aspect of the present invention is a method to produce a cell type, such as a mesodermal cell, an ectodermal cell and/or an endodermal cell from iPSC that includes the steps of: (a) selecting a desired cell type to produce; and (b) culturing an iPSC population of the present invention under conditions suitable to contain the desired cell type.
  • Suitable culture conditions for obtaining a desired cell type include culturing an iPSC population in a medium including one or more growth factors that is able to stimulate the iPSC population to differentiate to the desired cell type(s).
  • an iPSC population may be cultured in medium including a growth factor capable of promoting differentiation of the cell population into an endoderm cell type.
  • an endoderm cell type is a hepatocyte cell or hepatocyte-like cell.
  • one or more differentiation genes are introduced into the iPSC.
  • differentiation genes under the control of an inducible promoter are introduced into the iPSC.
  • differentiation genes under the control of an inducible promoter may be introduced into differentiated cells before reprogramming to iPSC. Once iPSC are induced, the differentiation gene may be activated to differentiate the iPSC to the desired cell lineage.
  • Cell populations enriched for endoderm may be obtained by culturing iPSC in the absence of serum and in the presence of the growth factor activin, for about two to about ten days, and isolating cells that express brachyury.
  • the amount of activin is sufficient to induce differentiation of iPSC to endoderm.
  • Such differentiation may be measured by assaying for the expression of genes associated with endoderm development, including for example HNF3 ?, mixl-1, soxl7, hex-1 or pdx-1.
  • the concentration of activin is at least about 30 ng/ml. In another aspect of the invention, the concentration of activin is about 100 ng/ml.
  • Cell populations enriched for mesoderm may be obtained by culturing iPSC in the absence of serum and the presence of activin for about two to about ten days, and isolating cells that express brachyury.
  • the amount of activin is sufficient to induce differentiation of iPSC to mesoderm, but insufficient to induce differentiation to endoderm.
  • Differentiation to mesoderm may be measured by assaying for the expression of genes associated with mesoderm development, including for example GATA-1, and the absence of expression of genes associated with endoderm development.
  • the concentration of activin is less than 30 ng/ml. In another aspect of the invention, the concentration of activin is about 3 ng/ml.
  • iPS cells are maintained on MEF feeder cells. Cells are then passaged onto plates without MEF feeder cells for about one day. On day 0, iPSCs are induced to form embryoid bodies (EBs). On about day 2, EBs are incubated in the presence of activin A to form endoderm. In some cases, on about day 9, cells are harvested for analysis. Cells can be analyzed for endoderm cell characteristics by a number of methods known in the art including, but not limited to RT-PCR, immunohistochemistry and enzyme assays. For example, expression of genes associated with endoderm
  • iPSC iPSC-derived endoderms
  • Undifferentiated iPSCs are maintained on MEF feeder cells.
  • cells are plated on gelatinized culture dishes in the absence of MEF feeder cells.
  • cells are passaged in a pre-differentiation step.
  • EBs are induced by culture in SFD complete medium (75% IMDM and 25% Ham's F12 medium (Gibco) supplemented with 0.5 % N2 and 1% B27 (with RA) supplements (Gibco), 1% penicillin/streptomycin, 0.05% bovine serum albumin, 2 mM glutamine, 0.5 mM ascorbic acid and 4.5 x 10 "4 M MTG).
  • SFD complete medium 75% IMDM and 25% Ham's F12 medium (Gibco) supplemented with 0.5 % N2 and 1% B27 (with RA) supplements (Gibco), 1% penicillin/streptomycin, 0.05% bovine serum albumin, 2 mM glutamine, 0.5 mM ascorbic acid and 4.5 x 10 "4 M MTG).
  • SFD complete medium 75% IMDM and 25% Ham's F12 medium (Gibco) supplemented with 0.5 % N2 and 1% B27 (with RA) supplements (
  • iPSC have many different potential applications, including treatment of diabetes, Parkinson's disease, or spinal cord injuries, or for elucidating developmental pathways; the creation of iPSC with defined genotypes could facilitate drug discovery and development via novel in vitro assays.
  • iPSC may provide a consistent, renewable source of non-transformed, non- embryo-derived cells, which may be differentiated into hepatocyte cultures for hepatotoxicity and drug metabolism studies.
  • Cell-based assays using iPSC lines allow earlier identification of drug-induced hepatotoxicity, decrease the number of animal studies necessary for drug development, permit in vitro assessment of the variation in drug metabolism due to pharmacogenetic variation, and ultimately result in a more efficient process for drug development.
  • Hepatocyte or hepatocyte-like cultures may be derived from iPSC prepared as described above. Any differentiation protocol may be used to differentiate iPSC to endoderm and ultimately to hepatocyte or hepatocyte-like cultures. For example, differentiation protocols used to differentiate embryonic stem cells to hepatocyte cultures, such as those described in U.S. Patent Application Publication 2006/0003446 may be used differentiate iPSC to hepatocyte-like cultures. In these protocols, hES cultures progress through a CXCR4 + /cKIT + (e.g., definitive endoderm) intermediate, ultimately resulting in the formation of cells that express both albumin (ALB) and AFP, characteristic markers of hepatocytes.
  • CXCR4 + /cKIT + e.g., definitive endoderm
  • iPSC-derived hepatocytes may be validated by several established methods (Gouon-Evans, V. et al. 2006 Nat. Biotech. 24:1402-1411; Baharvand, H. et al. 2006 Int. J. Dev. Biol.
  • definitive endoderm formation may be monitored by flow cytometry.
  • RT-PCR may be performed to detect expression of a panel of known hepatocyte markers (i.e., ALB, AFP, AAT, TO, TTR, and HNF4a) and immunocytochemistry may be used to detect ALB, AFP, HNF4a, and AAT proteins in iPSC-derived hepatocytes.
  • Cells may be analyzed for indocyanine green uptake, an organic anion exclusively taken up and eliminated by hepatocytes (Yamada, T. et al., 2002 Stem Cells 20:146-154).
  • Glycogen storage may be assessed by periodic acid-Schiff staining.
  • ALB and AFP secretion may be measured by means known in the art.
  • iPSC-derived hepatocytes are analyzed to determine the expression of specific metabolizing enzymes responsible for metabolism of the majority of clinically prescribed drugs.
  • Expression profiling may be performed on populations of iPSC-derived hepatocytes using, for example, CodeLink human whole genome microarrays, and may be compared to profiles of commercially available RNA from mature human hepatocytes, human fetal liver, and human adult liver.
  • Expression analysis may include 128 select phase I and phase II drug metabolizing enzymes in the iPSC-derived cultures, including the CYP1, 2 and 3 family of isozymes, and members of the FMO, ADH1, NAT, GST, SULT, and UGT families. Additionally, expression levels of several nuclear receptors linked to CYP induction may be determined (Lin, J. 2006 Pharmaceut. Res.
  • CYP3A4 is responsible for metabolizing about 60% of currently used therapeutics, accounts for about 40% of the total P450 content in adult human livers and induction and inhibition of this P450 isozyme is responsible for clinically relevant drug-drug interactions (Lin, J. 2006 Pharmaceut. Res. 25:1089-1116; Dresser, G. et ah, 2000 Clin. Pharmacokinet. 38:41-57). Characterization of CYP3A4 activity may be performed incubating whole cells with fluorogenic probe substrates in plate-based assays (Donato et ah, 2004 Drug Metaboh Dispos. 32:699-706).
  • CYP3A4 activity may be confirmed by metabolite detection using HPLC-based assays with testosterone-6b as a probe substrate (Rendic, S. and DiCarlo, F. 1997 Drug Metab. Rev. 29:413-580).
  • CYP3A4 induction assays may be performed using 3 day preincubation with rifampin prior to exposure of cells to the probe substrates (Madan, A. et ah 2003 Drug Metaboh Dispos. 31:421-431), while co- incubation of ketoconazole and probe substrate may be performed to confirm inhibition of CYP3A4 activity.
  • the activities of various other CYP enzymes may be characterized by incubating the cultures with fluorogenic or standard CYP isozyme- selective probe substrates.
  • the iPSC induction methods of the present invention may be used to readily create donor- specific iPSC lines.
  • an extensive panel of iPSC with relevant defined and variant genotypes useful in drug development, metabolism, and toxicity screens may be created.
  • the pharmacogenetic diversity found in human populations may be utilized to create such a panel.
  • dermal fibroblasts from donors of Caucasian, Asian and Latino backgrounds may be used to create a panel of iPSC derived hepatocytes. These ethnic backgrounds have been shown to represent three major classes of drug metabolism alleles (Ingelman-Sundberg, M. et ah 2007 Pharmacol. Ther.. 116:496-526).
  • genotyping of the donor fibroblasts may be limited to twenty-two alleles selected for their demonstrated pharmaceutical relevance and relatively high penetrance in at least one of the these populations (Roden, D. and George, A. 2001 Nat. Rev. Drug Discov. 1:37-44; Phillips, K. et al. 2001 JAMA 286:2270-2279; Ingelman-Sundberg, M. 2001 J. Intern. Med. 250:186-200; Ingelman-Sundberg, M. 2007 Pharmacol. Ther. 116:496-526).
  • Fibroblasts may be genotyped by established PCR-based assays (Doll, M. and Hein, D. 2001 Anal. Biochem. 288:106-108; Hersberger, M. et al, 2001 Clin. Chem. 47:772- 774; Ji, L., et al, 2002 Clin. Chem. 48:983-988; Zainuddin, Z. et al, 2003 Clin. Chem. Acta 336:97-102; Ledesma, M.
  • the derived cultures may be characterized for expression of phase I and phase II enzymes and for function of selected CYPs.
  • genes involved in drug metabolism in the rat include, but are not limited to, drug transporters such as metallothioneins, Mt3; P-glycoprotein family genes including Abcbl, Abcbla, Abcb4, Abccl, and Gpi; phase I metabolizing enzymes including P450 family genes Cypl7al, Cypl9al, Cyplal, Cypla2, Cyplbl, Cyp27bl, Cyp2bl5, Cyp2b6, Cyp2C13, Cyp2C6, Cyp2C7, Cyp2el, Cyp3a3, Cyp4bl; phase II metabolizing enzymes including carboxylesterases such as Cesl, and Ces2; decarboxylases including Gadl and Gad2; dehydrogenases including Adhl, Adh4, Alad, Aldhlal, Hsdl7bl, Hsdl7b2 and Hsdl7b3, glutathione peroxidases including Gpx
  • cytochrome P450 genes include, but are not limited to, cytochrome P450 genes: CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP1A1, CYP1A2, CYP1B1, CYP21A2, CYP24A1, CYP26A1, CYP26B1, CYP26C1, CYP27A1, CYP27B1, CYP2A13, CYP2A6, CYP2A7, CYP2B6, CYP2C18, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP2W1, CYP3A4, CYP3A43, CYP3A5, CYP3A7, CYP4A11, CYP4A22, CYP4B1, CYP4F11, CYP4F12, CYP3
  • the present invention further provides methods of identifying agents that affect the proliferation, differentiation or survival of the iPSC populations of the invention.
  • the methods comprise culturing iPSCs produced by the methods of the invention in the absence and presence of an agent to be tested, and determining whether the agent has an effect on proliferation, differentiation or survival of the iPSC population.
  • the agent to be tested may be natural or synthetic, one compound or a mixture, a small molecule or polymer including polypeptides, polysaccharides, polynucleotides and the like, an antibody or fragment thereof, a compound from a library of natural or synthetic compounds, a compound obtained from rational drug design, or any agent the effect of which on the cell population may be assessed using assays known in the art, for example standard proliferation and differentiation assays as described in U.S. Pat. No. 6,110,739. Such agents are useful for the control of cell growth and differentiation in vivo and in vitro.
  • the present invention further provides a method of identifying genes involved in cell differentiation and development of specific lineages and tissues.
  • the method comprises isolating populations of iPSC of the invention after different amounts of time in culture, comparing gene expression profiles in the different populations, and identifying genes that are uniquely expressed in a population.
  • microarray analysis and subtractive hybridization are used to compare gene expression profiles.
  • Hepatocytes or hepatocyte-like cells differentiated from iPSC of the invention may be used to screen for factors that affect the characteristics of differentiated cells of the hepatocyte lineage derived from iPSC.
  • factors include but are not limited to solvents, small molecule drugs, peptides, and polynucleotides.
  • differentiated along the hepatocyte differentiation pathway may be used to screen factors that promote maturation of cells along the hepatocyte lineage, or promote proliferation and maintenance of such cells in long-term culture.
  • the invention provides methods to screen pharmaceutical compounds in drug research.
  • Hepatocytes or hepatocyte-like cells derived from iPSC of the invention may be used for standard drug screening and toxicity assays. See, for example, "in vitro Methods in Pharmaceutical Research", Academic Press, 1997.
  • the methods of the invention generally involve combining hepatocytes or hepatocyte-like cells derived from iPSC of the invention with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound compared with untreated cells or cells treated with an inert compound, and then correlating the effect of the compound with the observed change.
  • the screening may be done either because the compound is designed to have a pharmacological effect on hepatocytes, or because a compound designed to have effects elsewhere may have unintended hepatic side effects.
  • Two or more drugs may be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects.
  • the invention provides methods to screen pharmaceutical compounds in drug research using hepatocyte or hepatocyte-like cells derived from iPSC from different genetic backgrounds as discussed above.
  • Panels of iPSC with relevant defined and variant genotypes useful in drug development, metabolism, and toxicity screens may be used in drug screening studies.
  • the pharmacogenetic diversity found in human populations may be utilized to create such a panel.
  • genotyping of the donor fibroblasts may be limited to twenty-two alleles selected for their demonstrated pharmaceutical relevance and relatively high penetrance in at least one of the these populations.
  • compounds are screened initially for potential hepatotoxicity (Castell et al., pp 375-410 in "In vitro Methods in Pharmaceutical Research," Academic Press, 1997). Cytotoxicity may be determined in the first instance by the effect on cell viability, survival, morphology, and leakage of enzymes into the culture medium. More detailed analysis is conducted to determine whether compounds affect cell function (such as gluconeogenesis, ureogenesis, and plasma protein synthesis) without causing toxicity.
  • An example of a hepatotoxicity marker is lactate dehydrogenase (LDH).
  • the LDH hepatic isoenzyme (type V) is stable in culture conditions thereby allowing reproducible measurements in culture supernatants after 12-24 h incubation. Leakage of enzymes such as mitochondrial glutamate oxaloacetate transaminase and glutamate pyruvate transaminase may also be used. Gomez-Lechon et al. (Anal. Biochem. 236:296, 1996) describe a microassay for measuring glycogen, which may be applied to measure the effect of pharmaceutical compounds on hepatocyte gluconeogenesis.
  • hepatotoxicity assessments include determination of the synthesis and secretion of albumin, cholesterol, and lipoproteins; transport of conjugated bile acids and bilirubin; ureagenesis; cytochrome P450 levels and activities; glutathione levels; release of a-glutathione s-transferase; ATP, ADP, and AMP metabolism; intracellular K + and Ca 2+ concentrations; the release of nuclear matrix proteins or oligonucleosomes; and induction of apoptosis (indicated by cell rounding, condensation of chromatin, and nuclear fragmentation). DNA synthesis can be measured as [ H] -thymidine or BrdU incorporation.
  • Effects of a drug on DNA synthesis or structure can be determined by measuring DNA synthesis or repair.
  • [ H] -thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect.
  • Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. See; for example, A. Vickers (pp 375-410 in "In vitro Methods in
  • the invention provides methods for assessing the toxicity of chemical compositions using hepatocytes, or hepatocyte-like cells, derived from iPSC of the invention.
  • the invention is directed to methods of creating a molecular profile of a chemical composition are generated by comprising the steps of a) contacting a population of hepatocytes or hepatocyte-like cells derived from iPSC with the chemical composition; and b) recording alterations in gene expression or protein expression in the iPSC-derived hepatocytes in response to the chemical composition to create a molecular profile of the chemical composition.
  • molecular profiles of chemical compositions may be generated by contacting populations of iPSC-derived hepatocytes, with known genetic backgrounds relative to drug metabolism genes.
  • the invention further encompasses methods of compiling libraries of molecular profiles of chemical compositions having predetermined toxicities.
  • the present invention provides methods for typing toxicity of a test chemical composition by comparing its molecular profile in iPSC-derived hepatocytes with known genetic backgrounds relative to drug metabolism genes with that of an identified chemical composition with predetermined toxicity.
  • the invention provides methods to screen pharmaceutical compounds in drug metabolism studies.
  • Hepatocytes or hepatocyte-like cells derived from iPSC of the invention may be used for standard metabolism assays.
  • the methods of the invention generally involve combining hepatocytes or hepatocyte-like cells derived from iPSC of the invention with the candidate compound, determining any change in the candidate compound. Changes in candidate compounds may be determined by analytic techniques known in the art. Examples include, but are not limited to, spectroscopy, HPLC, gas chromatography, mass spectrometry, GC-mass spectrometry, crystallography, and NMR. Two or more drugs may be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects.
  • the invention also provides methods to evaluate pharmaceutical compounds in drug metabolism studies using hepatocyte or hepatocyte-like cells derived from iPSC from different genetic backgrounds as discussed above.
  • Panels of iPSC with relevant defined and variant genotypes useful in drug metabolism screens may be used.
  • the pharmacogenetic diversity found in human populations may be utilized to create such a panel.
  • genotyping of the donor fibroblasts may be limited to twenty-two alleles selected for their demonstrated pharmaceutical relevance and relatively high penetrance in at least one of the these populations.
  • variations in drug metabolism in individuals of diverse genetic backgrounds may be determined as part of drug discovery processes.
  • the invention provides therapeutic compositions comprising populations of iPSC capable of serving as progenitors of various cell lineages in vivo.
  • the therapeutic composition may be useful to repopulate one or more lineages in an animal.
  • the therapeutic composition may comprise a cell population that can be administered to an animal to restore a lineage of cells.
  • Autologous or allogenic populations of iPSCs or iPSC- derived cells may be used in cell replacement therapies.
  • a therapeutic composition of the present invention may be useful for the treatment of disease, such as anemia, leukemia, diabetes, Parkinson's disease, breast cancer and other solid tumors, and AIDS.
  • the present invention also provides a method for generating mammalian cells in vitro.
  • the method comprises culturing an iPSC-derived cell population enriched in mesendoderm and/or mesoderm cells under conditions effective for the differentiation of mesoderm into cardiac muscle, vascular smooth muscle, endothelium or hematopoietic cells and the like. Conditions effective for differentiation into the various cell types in vitro are known in the art.
  • the method comprises culturing an iPSC-derived cell population enriched in endoderm cells under conditions effective for the differentiation of endoderm into liver cells or pancreatic cells and the like. Effective conditions for such differentiation are known in the art.
  • Such cells are useful, for example, for cell replacement therapy for the treatment of disorders that result from destruction or dysfunction of a limited number of cell types; for example, diabetes mellitus, liver failure, heart failure, cardiovascular and other vascular disease, Duchenne's muscular dystrophy, osteogenesis imperfecta, and disorders treatable by bone marrow transplant, for example leukemias and anemias. See, Odorico et ah, (2001) Stem Cells 19:193-204.
  • the iPSC populations of the present invention are useful for generating differentiated cells and tissues for cell replacement therapies.
  • the suitability of the cell populations of the present invention for cell replacement therapy may be assessed by transplanting the iPSC-derived cells into animal models of disorders that are associated with the destruction or dysfunction of a limited number of cell types. For example, the
  • FAH deficient mouse disclosed for example by Grompe et al. (1993) Genes & Dev. 7:2298, provides a model for liver failure.
  • FAH deficient mice suffer from progressive liver failure and renal tubular damage unless treated with NTBC (2-(2-nitro-4- trifluoromethyl benzoyl)- 1,3-cyclohexedione) or transplanted with normal hepatocytes. These mice thus provide an ideal model for testing the potential of cells with characteristics of immature hepatocytes generated from iPSCs.
  • Methods for transplantation of hepatocytes into FAH deficient mice removed from NTBC are known in the art and disclosed for example by Oversturf et al. (1996) Nature Genet. 12:266-273.
  • Normal liver function is indicated by survival of the mice, and may also be assessed by measuring serum aspartate transaminase levels, plasma bilirubin levels, and by determining normal structure of the regenerated liver.
  • Another aspect of the present invention is the use of a cell population of the present invention for the treatment of genetic diseases.
  • Genetic diseases associated with various lineages may be treated by genetic modification of autologous or allogenic populations of iPSCs or iPSC-derived cells of the present invention.
  • diseases such as beta-thalassemia, sickle cell anemia, adenosine deaminase deficiency, hemophilia, and other genetic diseases related to a deficiency or malfunction of a cell or gene product can be corrected by introduction of a wild type gene into the iPSC or iPSC-derived cell population.
  • Methods for transformation and expression of genes in an iPSC population of the present invention are standard to those in the art (see, for example, Sambrook et ah, ibid.).
  • iPSCs or iPSC-derived cells may be used in cell therapies.
  • differentiated cells from an individual may be cultured and reprogrammed to iPSC by the methods described above.
  • the iPSC may subsequently be differentiated to the desired cell lineage and then implanted back into the individual in order to provide a patient specific therapy.
  • allogeneic iPSCs or iPSC-derived cell lines are established for cell therapies.
  • compositions of the present invention may be administered to any animal; for example, mammals such as humans.
  • therapeutic compositions may be formulated in an excipient that the animal to be treated can tolerate and that maintains the integrity of the iPSC population or the iPSC-derived cell population.
  • excipients examples include aqueous physiologically balanced salt solutions.
  • Excipients may also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • SV40 large T antigen (TAg) maintains expression of co-transfected plasmids that contain the SV40 origin of replication (SV40 ori)
  • pTAg V5-epitope tagged TAg
  • pTAg was co-nucleoporated into NHDF along with pTimer-1, a plasmid which contains an SV40 ori and encodes a GFP variant that fluoresces green when newly transfected, yet will shift to red wavelengths as the GFP protein ages.
  • Nucleoporated cells were plated and examined at 24 hour intervals after nucleofection for up to 168 hours. As shown in Figure 2, cells transfected with both plasmids demonstrate green and red fluorescence at 48 and 168 hours after transfection, whereas cells lacking TAg lose all green and some red fluorescence by 168 hours post-nucleofection.
  • the design for induction of iPSC from NHDF involves culturing NHDF at a permissive temperature of 32°C to allow expression of the temperature sensitive TAg mutant, tsA357R-K TAg. It is essential that NHDF are capable of being cultured at this lower temperature.
  • 1.3 x 10 5 NHDF cells were plated per well of a 6-well dish on day 0. One plate was cultured at 32°C, and the other was cultured at 37°C. Every two days, two wells from each plate were harvested, trypsinized, and counted. As shown in Figure 3, NHDF cells did grow well at 32°C, albeit at a slower rate than at 37°C. The cells looked healthy morphologically (data not shown).
  • reprogramming proteins under the control of the CMV promoter and contained an SV40 ori to permit episomal replication of the vector.
  • An episomal plasmid containing an SV40 ori and expressing ts TAg with an additional two mutations in the transactivating domain of TAg was constructed using pcDNA6.2 GW/D as a backbone.
  • NHDF were plated at 5 x 10 5 cells per well in two six well dishes.
  • the culture media was NHDF media (Lonza) supplemented with Fibroblast Cell Basal Medium, hFGF-B, insulin, FBS and GA-1000. The following day cells episomal plasmids were transfected into NHDF as described in Example 1.
  • the following mixtures of plasmids were used in duplicate: 1) 1 ⁇ g TAg plasmid; 1 ⁇ g mixture of SOX2, OCT3/4, KLF4 and c-MYC episomal plasmids; and 0.1 ⁇ g CMV-Timer (as described in Example 2); 2) 2 ⁇ g TAg plasmid; 1 ⁇ g mixture of SOX2, OCT3/4, KLF4 and c-MYC episomal plasmids; and 0.1 ⁇ g CMV-Timer; 3) 0.1 ⁇ g TAg plasmid; 1 ⁇ g mixture of SOX2, OCT3/4, KLF4 and c-MYC episomal plasmids; and 0.1 ⁇ g CMV-Timer; and 4) 4 ⁇ g pcDNA6.2 CAT control.
  • Differentiation media was SFD RA, 50 ⁇ g/ml ascorbic acid, 1 mM glutamine, 3 ⁇ /ml MTG and 1 ng/ml BMP4.
  • SFD RA media was 75% IMDM, 25% F12 supplemented with 0.5 X N2 Supplement (Gibco), 0.5 X B27 with retinoic acid, 0.05% BSA, glutamine and 37.5 u/ml penicilliin-37.5 ⁇ g/ml streptomycin.
  • EBs were harvested and left to settle.
  • the supernatant was aspirated and replaced with media containing Activin A (100 ng/ml), BMP4 (0.5 ng/ml) and bFGF (0.25 ng/ml). 2 ml/well were replated into new low cluster 6- well plates and incubation was continued at low oxygen (5% 0 2 ). On day 4, the harvest protocol of day 1 was repeated using the same media. On day 5 or day 6, samples were removed and analyzed by flow cytometry using labeled antibodies specific for expression of CXCR4 as measured by APC, cKIT as measured by PE and CD31 as measured by FITC. Control cells were not treated with antibodies.
  • EBs were harvested from 12 well plates into a 50 ml tube. Wells were washed with 1 ml IMDM. The tubes were allowed to sit for 15 min ant then the media was gently aspirated to leave the EB pellet. Two ml of trypsin- EDTA with DNAse at 30 ⁇ /ml were added to the tubes and the tubes were incubated in a 37°C water bath for 4 min. Samples were vortexed and pipetted using a 1000 ⁇ pipette tip. The wash media was added to the tube and then the tube was centrifuged for 5 min at 1000 rpm. Cells were then counted and plated at 5 x 10 5 cells/well in P12 plus gelatin or matrigel.
  • T4 medium was SFD RA supplemented with 50 ⁇ g/ml ascorbic acid, 1 mM glutamine, 3 ⁇ /ml MTG, 50 ng/ml activin A, 1 ng/ml BMP4, 5 ng/ml bFGF and 10 ng/ml VEGF.
  • the media was changed media on day 7 or day 8 to SFD RA supplemented with 50 ⁇ g/ml ascorbic acid, 1 niM glutamine, 3 ⁇ /ml MTG, 20 ng/ml hHGF, 50 ng/ml hBMP4, 10 ng/ml hbFGF, 10 ng/ml hVEGF, 10 ng/ml hEGF, 20 ng/ml TGFa and 40 ng/ml Dex.
  • Cells were transferred from the wells of six-well plates into 15 ml tubes containing 6 ml of IMDM (wash). Tubes were centrifuged for 5 min at 1000 rpm. Supernatants were removed from tubes and 6 ml of liver induction media was added to two tube cell pellets. 12 wells of a 12- well plate went into 3 wells of a low cluster 6- well plate. The media was pipetted up and down with a 5 ml pipette to one well and then 2 mis were transferred to the next well until 3 wells of a low cluster plate were filled. Cells were incubated in low oxygen. On day 22 cells were analyzed for AFP and ALB staining and cells were collected for RNA analysis.
  • fibroblasts 100 mm dish in DMEM with 10% Fetal bovine serum (FBS) at 37°C one day prior to transfection.
  • FBS Fetal bovine serum
  • episomal plasmids containing an SV40 origin of replication and encoding tsTAg, OCT3/4, SOX2, KLF4 and c-MYC are introduced to the fibroblasts by electroporation/nucleofection as described in Example 1.
  • Fibroblasts are incubated at the permissive temperature of 32°C.
  • fibroblasts are harvested by trypsinization and replated at 5 x 10 4 cells per 100 mm dish on a MEF feeder layer. Cells are maintained at 32°C.
  • the media On day 7 following transduction, the media is replaced with DSR medium supplemented with 4 ng/ml bFGF. The medium is changed approximately every two days. On about day 30-40 following transduction, ES-like colonies are picked and transferred into 0.2 ml ES cell medium. Colonies are mechanically dissociated and transferred to STO feeder cells in 24- well plates. The temperature is shifted to the nonpermissive temperature of 39°C. Cells are passaged by treatment with collagenase and at various times after the shift to 39°C, cells are assayed for the presence of the tsTAg and reprogramming gene vectors. When it is determined that cells no longer harbor vectors encoding tsTAg and reprogramming genes, the temperature is shifted to and maintained at 37°C.
  • Example 7 Induction of hepatocytes from iPSC
  • the differentiation protocol used to differentiate iPSC to hepatocyte-like cultures is similar to that used for differentiating hESC to hepatocyte-like cells.
  • iPSC are differentiated for 2 days in Stem Pro 34 medium without serum.
  • the developing EBs are harvested and recultured in IMDM supplemented with serum replacement (serum free) and Activin A at a concentration of 100 ng/ml.
  • a sample of EBs are harvested at different days and assayed for expression of genes indicative of ectoderm, mesoderm and endoderm development.
  • the formation of definitive endoderm as assessed by the markers c-KIT and CXCR4 is determined.
  • Extended culture of the CXCR4 + /cKIT + cells in media formulated to promote hepatoblast and hepatocyte differentiation leads to cultures expressing ALB transcripts at day 32.
  • Pancreatic endocrine progenitor cells can be derived from iPSC by differentiation of iPSC into endoderm by treatment with activin, as described above, followed by expression of Pdxl and Ngn3 in the endoderm cells.
  • polynucleotides expressing Pdxl and Ngn3 are stably introduced to a population of iPSC prior to
  • polynucleotides expressing Pdxl and Ngn3 are introduced to a population of endoderm derived from iPSC.
  • the polynucleotides expressing Pdxl and Ngn3 may be under the control of an inducible promoter.
  • a population of undifferentiated iPSCs maintained on MEF feeder cells is used.
  • cells are plated on gelatinized culture dishes in the absence of MEF feeder cells.
  • On about day -2 cells are passaged in a pre-differentiation step.
  • EBs are induced by culture in SFD complete medium.
  • EBs are dissociated and replated in the presence of activin A (30 - 100 ng/ml).
  • EBs are reaggregated and Pdxl, Ngn3 and MafA expression is induced; for example, by addition of Dox to the media.
  • cells are expanded on low attachment plates. Induction of expression of Pdxl and Ngn3 is continued.
  • 11 and 13 cells are fed and induction of expression of Pdxl and Ngn3 is continued.
  • cells are harvested and analyzed. Cells are analyzed for pancreatic endocrine progenitor cell characteristics by a number of methods known in the art including, but not limited to RT-PCR,
  • a polynucleotide encoding a reporter gene such as beta-lactamase or GFP under the control of insulin- 1 regulatory elements is also stably introduced into to the iPS cells.
  • cells can be assayed for development of pancreatic endocrine progenitor characteristics by BLA assay or FACS.
  • pancreatic endocrine progenitor cell from iPS cells in which Pdxl and Ngn3 are stably introduced is provided as follows.
  • Undifferentiated iPSCs are maintained on MEF feeder cells.
  • cells are plated on gelatinized culture dishes in the absence of MEF feeder cells.
  • cells are passaged in a pre-differentiation step.
  • iPS cells are plated as a monolayer in SFD complete medium.
  • cells are dissociated and replated in the presence of activin A (30 - 100 ng/ml).
  • cells are dissociated and Pdxl and Ngn3 expression is induced; for example, by addition of Dox to the media.
  • cells are expanded. Induction of expression of Pdxl and Ngn3 is continued.
  • pancreatic endocrine progenitor cell characteristics are analyzed for pancreatic endocrine progenitor cell characteristics by a number of methods known in the art including, but not limited to RT-PCR, immunohistochemistry and enzyme assays.
  • a polynucleotide encoding a reporter gene, such as beta-lactamase or GFP, under the control of insulin-1 regulatory elements is also stably introduced into to the iPS cells.
  • cells are assayed for development of pancreatic endocrine progenitor characteristics by BLA assay or FACS. The resulting pancreatic endocrine progenitor cells are maintained as a monolayer.
  • Primitive beta-islet cells can be derived from iPSC by differentiation of iPSC into endoderm by treatment with activin, as described above, followed by expression of Pdxl, Ngn3 and MafA in the endoderm cells.
  • polynucleotides expressing Pdxl, Ngn3 and MafA are stably introduced to a population of iPSC prior to differentiation.
  • polynucleotides expressing Pdxl, Ngn3 and MafA are introduced to a population of endoderm derived from iPSC.
  • the polynucleotides expressing Pdxl, Ngn3 and MafA may be under the control of an inducible promoter.
  • a population of undifferentiated iPSCs maintained on MEF feeder cells is used.
  • cells are plated on gelatinized culture dishes in the absence of MEF feeder cells.
  • cells are passaged in a pre-differentiation step.
  • EBs are induced by culture in SFD complete medium.
  • EBs are dissociated and replated in the presence of activin A (30 - 100 ng/ml).
  • EBs are reaggregated and Pdxl, Ngn3 and MafA expression is induced; for example, by addition of Dox to the media.
  • cells are expanded on low attachment plates.
  • Induction of expression of Pdxl, Ngn3 and MafA is continued. On about days 9, 11 and 13 cells are fed and induction of expression of Pdxl, Ngn3 and MafA is continued. On about day 16, cells are harvested and analyzed. Cells are analyzed for beta-islet cell characteristics by a number of methods known in the art including, but not limited to RT-PCR, immunohistochemistry and enzyme assays. In some cases, a polynucleotide encoding a reporter gene such as beta-lactamase or GFP under the control of insulin- 1 regulatory elements is also stably introduced into to the iPS cells. In these cases, cells can be assayed for development of primitive beta-islet cell characteristics by BLA assay or FACS.
  • Example II Induction of primitive beta-islet cells from iPSC
  • polynucleotides expressing Pdxl, Ngn3 and MafA are stably introduced to a population of iPSC prior to differentiation.
  • polynucleotides expressing Pdxl, Ngn3 and MafA are introduced to a population of endoderm derived from iPSC.
  • the polynucleotides expressing Pdxl, Ngn3 and MafA may be under the control of an inducible promoter. Undifferentiated iPSCs are maintained on MEF feeder cells.
  • cells are plated on gelatinized culture dishes in the absence of MEF feeder cells.
  • cells are passaged in a pre-differentiation step.
  • iPS cells are plated as a monolayer in SFD complete medium.
  • cells are dissociated and replated in the presence of activin A (30 - 100 ng/ml).
  • cells are dissociated and Pdxl, Ngn3 and MafA expression is induced; for example, by addition of Dox to the media.
  • cells are expanded. Induction of expression of Pdxl, Ngn3 and MafA is continued.
  • cells are fed and induction of expression of Pdxl, Ngn3 and MafA is continued. In some cases, cells are harvested and analyzed on about day 16. Cells are analyzed for primitive beta-islet cell characteristics by a number of methods known in the art including, but not limited to RT-PCR, immunohistochemistry and enzyme assays. In some cases, a polynucleotide encoding a reporter gene, such as beta-lactamase or GFP, under the control of insulin- 1 regulatory elements is also stably introduced into to the iPS cells. In these cases, cells are assayed for development of primitive beta-islet cell characteristics by BLA assay or FACS. The resulting primitive beta-islet cells are maintained as a monolayer.
  • a reporter gene such as beta-lactamase or GFP

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Abstract

La présente invention concerne des cellules pluripotentes induites générées à partir de cellules différenciées par l'expression transitoire épisomique de gènes de reprogrammation. En utilisant des cellules différenciées dont les contextes génétiques sont connus et diversifiés, il est possible de générer une collection d'iPSC génétiquement diversifiées. Les iPSC sont utiles pour la recherche de nouveaux médicaments et des thérapies de remplacement cellulaire.
PCT/US2011/029972 2010-03-25 2011-03-25 Induction de cellules ips en utilisant des vecteurs épisomiques transitoires WO2011119942A1 (fr)

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CN103923920A (zh) * 2014-04-04 2014-07-16 清华大学 一种增强人类细胞中非整合性基因表达的方法
KR20150014512A (ko) * 2012-05-23 2015-02-06 고쿠리츠 다이가쿠 호진 교토 다이가쿠 효율적 인공 다능성 간세포 수립 방법
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US20180161455A1 (en) * 2015-06-10 2018-06-14 American Gene Technologies International, Inc. Non-integrating viral delivery system and methods of use therof
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US11007209B2 (en) 2008-10-17 2021-05-18 American Gene Technologies International Inc. Safe lentiviral vectors for targeted delivery of multiple therapeutic molecules
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US11274279B2 (en) 2020-03-11 2022-03-15 Bit Bio Limited Method of generating hepatic cells
US11519006B2 (en) 2016-01-15 2022-12-06 American Gene Technologies International Inc. Methods and compositions for the activation of gamma-delta T-cells
US11583562B2 (en) 2016-07-21 2023-02-21 American Gene Technologies International Inc. Viral vectors for treating Parkinson's disease
US11820999B2 (en) 2017-04-03 2023-11-21 American Gene Technologies International Inc. Compositions and methods for treating phenylketonuria
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US10519425B2 (en) 2012-05-23 2019-12-31 Kyoto University Highly efficient method for establishing induced pluripotent stem cell
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JPWO2013176233A1 (ja) * 2012-05-23 2016-01-14 国立大学法人京都大学 効率的な人工多能性幹細胞の樹立方法
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CN102965393A (zh) * 2012-11-06 2013-03-13 上海交通大学 人诱导多能干细胞的制备方法及其用途
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