Programming and reprogramming of cells

Abstract

To provide dedifferentiation of differentiated somatic cells; methods of generating secondary iPS cells and the secondary iPS cells produced by the methods; chimeric animals, e.g., mice, produced from the secondary iPS cells; and methods of screening for reprogramming agents utilizing the secondary iPS cells and chimeric animals.SOLUTION: The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully / terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to chimeric animals comprising reprogrammed somatic cells of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Patent No. 61 / 061,525, filed June 13, 2008, and U.S. Patent No. 61 / 077,068, filed June 30, 2008, the entire teachings of which are incorporated herein by reference.

[0002] government support This invention was supported in whole or in part by grants 5-RO1-HD045022, 5-R37-CA084198, and 5-RO1-CA087869 from the National Institutes of Health. The United States Government has certain rights in this invention. [Background technology]

[0003] Embryonic development and cell differentiation are considered to be unidirectional pathways, because cells undergo progressive loss of developmental potency during cell fate specification. Two categories of pluripotent stem cells are known to date: embryonic stem cells and embryonic germ cells. Embryonic stem cells are pluripotent stem cells derived directly from an embryo. Embryonic germ cells are pluripotent stem cells derived directly from the fetal tissue of aborted fetuses. For the purpose of simplicity, embryonic stem cells and embryonic germ cells are collectively referred to herein as "ES" cells.

[0004] The generation of living animals by nuclear transfer (NT) has demonstrated that the epigenetic state of somatic cells, including that of terminally differentiated cells, is unstable and can be reset to an embryonic state capable of directing the development of new organisms. Nuclear cloning technology is of potential interest for transplantation medicine, but any medical application is hindered by the inefficiency of the cloning process, lack of knowledge of the underlying mechanisms, and ethical concerns. A major breakthrough in resolving these issues was the in vitro induction of reprogrammed somatic cells (referred to as "induced pluripotent stem cells" or "iPS" cells) by ectopic expression of four transcription factors Oct4, Sox2, c-myc, and Klf4 (hereafter referred to as "reprogramming factors" or "factors") by Yamanaka (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takahashi and Yamanaka, Cell 126:663-676 (2006) Summary of the Invention [Problem to be solved by the invention]

[0006] Further progress in the area of ​​reprogramming will be facilitated by establishing robust methods for reprogramming human somatic cells, as well as by defining effective protocols for manipulating human ES and iPS cells. [Means for solving the problem]

[0007] The present invention relates generally to dedifferentiation of differentiated somatic cells, methods of generating second iPS cells and the second iPS cells produced by those methods, chimeric animals, e.g., mice, produced from the second iPS cells, and methods of screening reprogramming factors utilizing the second iPS cells and the chimeric animals. The present invention provides, for example, the following items. (Item 1) A nucleic acid construct comprising at least two coding regions, each linked to another by a nucleic acid encoding a self-cleaving peptide so as to form a single open reading frame, the coding regions encoding first and second reprogramming factors capable of reprogramming a mammalian somatic cell to pluripotency, either alone or in combination with one or more additional reprogramming factors. (Item 2) The nucleic acid construct of item 1, further comprising a third coding region encoding a third reprogramming factor, wherein the three coding regions are linked to each other by a nucleic acid encoding a self-cleaving peptide so as to form a single open reading frame. (Item 3) The nucleic acid construct of item 1, further comprising a third and fourth gene encoding a third and fourth reprogramming factor, wherein the four coding regions are linked to each other by a nucleic acid encoding a self-cleaving peptide to form a single open reading frame. (Item 4) The nucleic acid construct described in Item 1, wherein the self-cleaving peptide is a viral 2A peptide. (Item 5) The nucleic acid construct described in Item 1, wherein the self-cleaving peptide is an aphthovirus 2A peptide. (Item 6) The nucleic acid construct described in Item 1, wherein the reprogramming factor is selected from Oct4, Nanog, Sox2, Klf4, Lin28, and c-Myc. (Item 7) The nucleic acid construct described in Item 1, wherein the coding region encodes a first and a second reprogramming factor that require at least one additional reprogramming factor to reprogram a mammalian somatic cell to pluripotency. (Item 8) A nucleic acid construct according to item 1, which does not encode Oct4. (Item 9) The nucleic acid construct of item 1, which does not encode Klf4. (Item 10) The nucleic acid construct of item 1, which does not encode Sox2. (Item 11) A nucleic acid construct according to item 1, which does not encode c-Myc. (Item 12) A nucleic acid construct according to item 1, which does not encode Lin28. (Item 13) The nucleic acid construct of item 1, which does not encode Nanog. (Item 14) The nucleic acid construct of item 1, wherein the coding region encodes a set of reprogramming factors selected from the group consisting of (i) a set consisting of Oct4 and Sox2; (ii) a set consisting of Oct4 and Klf4; (iii) a set consisting of Oct4 and Nanog; (iv) a set consisting of Oct4, Klf4, and Sox2; (v) a set consisting of Oct4, Nanog, and Lin28; (vi) a set consisting of Oct4, Nanog, and Sox2; and (vii) a set consisting of any of (i) to (vi) and further including c-Myc. (Item 15) An expression cassette comprising the nucleic acid construct described in Item 1 operably linked to a promoter, wherein the promoter drives transcription of a polycistronic message encoding the reprogramming factors, and each reprogramming factor is linked to at least one other reprogramming factor by a self-cleaving peptide. (Item 16) The expression cassette described in Item 15, further comprising one or more sites that mediate integration into the genome of a mammalian cell. (Item 17) An expression vector comprising the expression cassette described in Item 15. (Item 18) The expression vector described in Item 17, which is retroviral. (Item 19) The expression vector described in Item 18, wherein the promoter is inducible. (Item 20) A mammalian cell comprising the expression cassette described in Item 15. (Item 21) The mammalian cell described in Item 20, which is a somatic cell. (Item 22) The mammalian cell described in Item 20, which is a terminally differentiated somatic cell. (Item 23) The mammalian cell described in Item 20, which is an iPS cell. (Item 24) A chimeric mouse generated at least in part from the mammalian iPS cell according to Item 23. (Item 25) The chimeric mouse described in Item 24, produced by injecting the mammalian iPS cell into a mouse blastocyt and growing the blastocyt in vivo into a mouse. (Item 26) A cell obtained from the mouse described in Item 24, which is derived from the iPS cell. (Item 27) The mammalian cell described in Item 20, which is a human cell. (Item 28) The mammalian cell described in Item 20, wherein the expression cassette is integrated into a genomic locus and disruption of the genomic locus has minimal or no effect on the cell. (Item 29) The mammalian cell described in Item 20, expressing two reprogramming factors from the expression cassette. (Item 30) The mammalian cell described in Item 20, expressing three reprogramming factors from the expression cassette. (Item 31) The mammalian cell described in Item 20, expressing three reprogramming factors from the expression cassette, wherein the three reprogramming factors are not sufficient to reprogram mammalian fibroblasts with an efficiency of at least 0.05%. (Item 32) The mammalian cell described in Item 20, expressing three reprogramming factors from the expression cassette, wherein the three reprogramming factors are sufficient to reprogram mammalian fibroblasts with an efficiency of at least 0.05%. (Item 33) The mammalian cell described in Item 20, further comprising a reporter gene integrated into the locus, wherein activation of the reporter gene serves as a marker of reprogramming to pluripotency. (Item 34) The mammalian cell of item 31, wherein the locus is selected from Nanog and Oct4. (Item 35) A composition comprising a plurality of essentially genetically identical cells according to Item 20. (Item 36) A composition for identifying a reprogramming factor, comprising one or more mammalian cells described in Item 20 and a test agent. (Item 37) The composition described in Item 34, further comprising an agent that induces expression of a reprogramming factor from the expression cassette. 38. A method for identifying a reprogramming factor, comprising: (i) maintaining the composition of item 34 for a period of time under conditions in which the reprogramming factors are expressed from the expression cassette and cell proliferation occurs; and (ii) assessing the extent to which the cells become reprogrammed, wherein the test agent is identified as a reprogramming factor or enhancer of reprogramming when reprogramming occurs at a frequency significantly greater than occurs when the composition lacks the test agent. The method includes: (Item 39) The method according to Item 36, wherein the composition is maintained for at least X days. 40. The method of claim 36, wherein the test agent is present for at least X days. 41. The method of claim 36, wherein the test agent is identified as a reprogramming factor when the cells do not undergo reprogramming at a detectable frequency when maintained in the absence of the test agent for a period of time, but do undergo reprogramming at a detectable frequency when maintained in the presence of the test agent for at least a portion of the period. (Item 42) The method described in Item 36, wherein the test agent is identified as an enhancer of a reprogramming factor when the cells become reprogrammed at a detectable frequency when maintained in the absence of the test agent for a certain period of time and at a significantly higher frequency when maintained in the presence of the test agent for at least a portion of the period. (Item 43) The method of item 36, wherein the cell expresses a set of reprogramming factors from the expression cassette, wherein the set consists of any of (i) to (vi), further comprising: (i) Oct4 and Sox2; (ii) Oct4 and Klf4; (iii) Oct4 and Nanog; (iv) Oct4, Klf4, and Sox2; (v) Oct4, Nanog, and Lin28; (vi) Oct4, Nanog, and Sox2; and (vii) c-Myc. 44. A method for reprogramming a differentiated somatic cell to a pluripotent state, comprising: (a) contacting a differentiated somatic cell with at least one reprogramming factor that contributes to reprogramming said cell to a pluripotent state; (b) maintaining the cells under conditions suitable for proliferation of the cells and for activity of the at least one reprogramming factor for a period of time sufficient to initiate reprogramming of the cells; and (c) functionally inactivating said at least one reprogramming factor. The method includes: 45. A method for reprogramming a differentiated somatic cell to a pluripotent state, comprising: (a) providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming the differentiated somatic cell to a pluripotent state; (b) maintaining the cells under conditions suitable for proliferation of the cells and for activation of the at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; and (c) functionally inactivating said at least one exogenously introduced factor. The method includes: 46. ​​A method for selecting differentiated somatic cells reprogrammed to a pluripotent state, comprising: (a) providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming the differentiated somatic cell to a pluripotent state; (b) maintaining said cells under conditions suitable for proliferation of said cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; and (d) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not. The method includes: (Item 47) An isolated pluripotent cell derived from a reprogrammed differentiated somatic cell. (Item 48) A purified somatic cell population comprising at least 70% pluripotent cells derived from reprogrammed differentiated somatic cells. (Item 49) An isolated pluripotent cell, comprising the steps of: (a) providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming the differentiated somatic cell to a pluripotent state; (b) maintaining said cells under conditions suitable for proliferation of said cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; and (d) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not; A cell produced by a method comprising the steps of: (Item 50) A purified somatic cell population comprising at least 70% pluripotent cells derived from reprogrammed differentiated somatic cells, comprising the steps of: (a) providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming the differentiated somatic cell to a pluripotent state; (b) maintaining said cells under conditions suitable for proliferation of said cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; and (d) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not; 2. A purified somatic cell population produced by a method comprising: (Item 51) A method for producing pluripotent cells from somatic cells, comprising the steps of: (a) providing one or more somatic cells, each of which comprises at least one exogenously introduced factor that contributes to reprogramming the somatic cells to a pluripotent state, wherein the exogenously introduced factor is introduced using an inducible vector that has not been subject to methylation-induced silencing; (b) maintaining said one or more cells under conditions suitable for proliferation of said one or more cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; (d) selecting one or more cells that exhibit markers of pluripotency; (e) generating a chimeric embryo using one or more cells that exhibit markers of pluripotency; (f) obtaining one or more somatic cells from the chimeric embryo; (g) maintaining said one or more somatic cells under conditions suitable for proliferation of said one or more somatic cells and for activity of said at least one exogenously introduced factor for a time sufficient to activate at least one endogenous pluripotency gene; and (h) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not. The method includes: (Item 52) An isolated pluripotent cell, comprising the steps of: (a) providing one or more somatic cells, each of which comprises at least one exogenously introduced factor that contributes to reprogramming the somatic cells to a pluripotent state, wherein the exogenously introduced factor is introduced using an inducible vector that has not been subject to methylation-induced silencing; (b) maintaining said one or more cells under conditions suitable for proliferation of said one or more cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; (d) selecting one or more cells that exhibit markers of pluripotency; (e) generating a chimeric embryo using one or more cells exhibiting markers of pluripotency; (f) obtaining one or more differentiated somatic cells from the chimeric embryo; (g) maintaining said one or more differentiated somatic cells under conditions suitable for proliferation of said one or more differentiated somatic cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; and (h) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not; A cell produced by a method comprising the steps of: 53. A purified somatic cell population comprising at least 70% pluripotent cells derived from reprogrammed differentiated somatic cells, comprising the steps of: (a) providing one or more somatic cells, each of which comprises at least one exogenously introduced factor that contributes to reprogramming the somatic cells to a pluripotent state, wherein the exogenously introduced factor is introduced using an inducible vector that has not been subject to methylation-induced silencing; (b) maintaining said one or more cells under conditions suitable for proliferation of said one or more cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; (d) selecting one or more cells that exhibit markers of pluripotency; (e) generating a chimeric embryo using one or more cells exhibiting markers of pluripotency; (f) obtaining one or more differentiated somatic cells from the chimeric embryo; (g) maintaining said one or more differentiated somatic cells under conditions suitable for proliferation of said one or more differentiated somatic cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; and (h) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not. A purified somatic cell population produced by a method comprising: 54. A method for reprogramming differentiated immune cells to a pluripotent state, comprising the steps of: (a) providing differentiated immune cells containing exogenously introduced Oct4, Sox2, Klf-4, and c-Myc, each under the control of an inducible vector, and further containing exogenously introduced C / EBPα; (b) maintaining the cells under conditions suitable for proliferation of the cells and for activity of Oct4, Sox2, Klf-4, c-Myc, and C / EBPα for a period of time sufficient to activate endogenous Nanog and / or Oct4; and (c) functionally inactivating exogenously introduced Oct4, Sox2, Klf-4, and c-Myc The method includes: 55. A purified immune cell population comprising at least 70% pluripotent cells derived from reprogrammed differentiated immune cells, comprising the steps of: (a) providing differentiated immune cells containing exogenously introduced Oct4, Sox2, Klf-4, and c-Myc, each under the control of an inducible vector, and further containing exogenously introduced C / EBPα; (b) maintaining the cells under conditions suitable for proliferation of the cells and for activity of Oct4, Sox2, Klf-4, c-Myc, and C / EBPα for a period of time sufficient to activate endogenous Nanog and / or Oct4; and (c) functionally inactivating exogenously introduced Oct4, Sox2, Klf-4, and c-Myc; 2. A purified immune cell population produced by a method comprising: 56. A method for identifying a reprogramming factor, comprising: (a) providing one or more somatic cells, each of which comprises at least one exogenously introduced factor that contributes to reprogramming the somatic cell to a pluripotent state, wherein each of the exogenously introduced factors is introduced using an inducible vector that is not subject to methylation-induced silencing and whose expression is controlled by a regulatory element that is induced by a separate inducer; (b) maintaining said one or more cells under conditions suitable for proliferation of said one or more cells and for activity of said at least one exogenously introduced factor for a period of time sufficient to reprogram said cells or to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; (d) selecting one or more cells that exhibit markers of pluripotency; (e) generating a chimeric embryo using one or more cells that exhibit markers of pluripotency; (f) obtaining one or more somatic cells from the chimeric embryo; (g) maintaining said one or more somatic cells under conditions suitable for proliferation of said one or more somatic cells and for activity of said at least one exogenously introduced factor, wherein activity of said at least one exogenously introduced factor is insufficient by itself to activate at least one endogenous pluripotency gene; (h) contacting the somatic cell of (g) with one or more candidate reprogramming factors; and (i) identifying cells contacted with the one or more candidate reprogramming factors that exhibit one or more markers of pluripotency, wherein a candidate reprogramming factor that induces the somatic cells of (g) to exhibit one or more markers of pluripotency is identified as a reprogramming factor. The method includes:

[0008] In one embodiment, the present invention relates to a method of reprogramming a differentiated somatic cell to a pluripotent state comprising: contacting a differentiated somatic cell with at least one reprogramming factor that contributes to reprogramming the cell to a pluripotent state; maintaining the cell under conditions suitable for proliferation of the cell and for activity of the at least one reprogramming factor for a sufficient time to initiate reprogramming of the cell; and functionally inactivating the at least one reprogramming factor.

[0009] In another embodiment, the present invention relates to a method of reprogramming a differentiated somatic cell to a pluripotent state comprising: providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming the differentiated somatic cell to a pluripotent state; maintaining the cell under conditions suitable for proliferation of the cell and for activity of the at least one exogenously introduced factor for a time sufficient to activate at least one endogenous pluripotency gene; and functionally inactivating the at least one exogenously introduced factor.

[0010] In a further embodiment, the present invention relates to a method for selecting differentiated somatic cells reprogrammed to a pluripotent state, comprising: providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming of the differentiated somatic cell to a pluripotent state; maintaining the cell under conditions suitable for proliferation of the cell and for activity of the at least one exogenously introduced factor for a sufficient time to activate at least one endogenous pluripotency gene; functionally inactivating the at least one exogenously introduced factor; and distinguishing or discriminating between cells that exhibit one or more markers of pluripotency and cells that do not. In one embodiment, distinguishing or discriminating between cells that exhibit one or more markers of pluripotency and cells that do not include selecting or enriching for cells that exhibit one or more markers of pluripotency and / or selecting against cells that do not exhibit one or more markers of pluripotency.

[0011] In some embodiments of the invention, the differentiated somatic cells are partially differentiated. In other embodiments of the invention, the differentiated somatic cells are fully differentiated.

[0012] In some embodiments of the invention, the differentiated somatic cells are hematopoietic lineage cells or mesenchymal stem cells; in some embodiments, the differentiated somatic cells are obtained from peripheral blood. In one embodiment of the invention, the differentiated somatic cells are immune system cells. In one embodiment, the differentiated somatic cells are macrophages. In one embodiment, the differentiated somatic cells are lymphoid cells. In other embodiments of the invention, the differentiated somatic cells are B cells, such as immature B cells (e.g., pro-B cells or pre-B cells) or mature B cells (e.g., non-naive). In yet other embodiments, the differentiated cells are neural progenitor cells, adrenal cells, keratinocytes, muscle cells, or intestinal epithelial cells.

[0013] In some embodiments of the present invention, at least one exogenously introduced factor is a polynucleotide. In other embodiments, at least one exogenously introduced factor is a polypeptide. In one embodiment, at least one exogenously introduced factor is selected from the group consisting of Oct4, Sox2, Klf-4, Nanog, Lin28, c-Myc, and combinations thereof. In certain embodiments of the present invention, the differentiated somatic cell comprises exogenously introduced Oct4, Sox2, and Klf-4, exogenously introduced Oct4, Sox2, Klf-4, and c-Myc.

[0014] In one embodiment of the present invention, the at least one exogenously introduced factor is selected from the group consisting of Oct4, Sox2, Klf-4, c-Myc, and combinations thereof, and the differentiated somatic cell further comprises at least one exogenously introduced factor (e.g., polynucleotide or polypeptide) capable of inducing dedifferentiation of the differentiated somatic cell. In one embodiment, the factor capable of inducing dedifferentiation of the differentiated somatic cell is selected from the group consisting of at least one polynucleotide that downregulates a B cell late stage specific marker, at least one polynucleotide that inhibits the expression of Pax5, at least one polypeptide that downregulates a B cell late stage specific marker, at least one polypeptide that inhibits the expression of Pax5, and combinations thereof. In one embodiment of the present invention, the factor capable of inducing dedifferentiation of the differentiated somatic cell is C / EBPα or a human homologue of C / EBPα.

[0015] In certain embodiments of the present invention, at least one exogenously introduced factor is introduced using vector, for example, inducible vector or conditional expression vector.In one embodiment, at least one exogenously introduced factor is introduced using vector that is not subject to methylation-mediated silencing.In yet another embodiment, at least one exogenously introduced factor is introduced using virus vector, such as retrovirus vector or lentivirus vector.

[0016] The present invention also provides methods for producing cloned animals. In these methods, somatic cells are isolated from animals with desired characteristics and reprogrammed using the methods of the present invention to produce one or more reprogrammed pluripotent somatic cells ("RPSCs"). The RPSCs are then inserted into a recipient embryo, and the resulting embryo is cultured to produce an embryo of appropriate size for implantation into a recipient female, which is then transferred to the recipient female to produce a pregnant female. The pregnant female is maintained under appropriate conditions to hold the embryo until a period that results in a chimeric animal offspring. The chimeric animal can be further mated with a wild-type animal as desired. The present invention further relates to the chimeric animals, such as chimeric mice, produced by the methods of the present invention.

[0017] The present invention further relates to isolated pluripotent cells produced by a method comprising the steps of: (a) providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to reprogramming the differentiated somatic cell to a pluripotent state; (b) maintaining the cell under conditions suitable for proliferation of the cell and for activity of the at least one exogenously introduced factor for a time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; and (d) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not.

[0018] The present invention also relates to a purified somatic cell population comprising at least 70% pluripotent cells derived from a reprogrammed differentiated somatic cell, the cell population being produced by a method comprising the steps of: (a) providing a differentiated somatic cell comprising at least one exogenously introduced factor that contributes to the reprogramming of the differentiated somatic cell to a pluripotent state; (b) maintaining the cell under conditions suitable for proliferation of the cell and for activity of the at least one exogenously introduced factor for a time sufficient to initiate reprogramming of the cell and to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; and (d) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not.

[0019] In another embodiment, the present invention provides a method of producing pluripotent cells from somatic cells, the method comprising the steps of: (a) providing one or more somatic cells, each comprising at least one exogenously introduced factor that contributes to reprogramming of one or more somatic cells to a pluripotent state, wherein said exogenously introduced factor is introduced using an inducible vector that has not been subjected to methylation-induced silencing; (b) maintaining said one or more cells under conditions suitable for proliferation of said cells and for activity of said at least one exogenously introduced factor for a sufficient time to initiate reprogramming of said one or more cells or to activate at least one endogenous pluripotency gene. (c) functionally inactivating said at least one exogenously introduced factor; (d) selecting one or more cells that exhibit markers of pluripotency; (e) generating a chimeric embryo utilizing said one or more cells that exhibit markers of pluripotency; (f) obtaining one or more somatic cells from said chimeric embryo; (g) maintaining said one or more somatic cells under conditions suitable for proliferation of said one or more somatic cells and for activity of said at least one exogenously introduced factor for a sufficient time to initiate reprogramming of said cells or to activate at least one endogenous pluripotency gene; and (h) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not. In certain embodiments, the method results in a purified population of somatic cells comprising at least 70% pluripotent cells derived from reprogrammed differentiated somatic cells.

[0020] The present invention also provides a method comprising the steps of: (a) providing one or more somatic cells, each comprising at least one exogenously introduced factor that contributes to the reprogramming of one or more somatic cells to a pluripotent state, wherein the exogenously introduced factor is introduced using an inducible vector that has not been subjected to methylation-induced silencing; (b) maintaining the one or more cells under conditions suitable for proliferation of the one or more cells and for activity of the at least one exogenously introduced factor for a sufficient time to initiate reprogramming of the one or more cells or to activate at least one endogenous pluripotency gene; (c) maintaining the one or more somatic cells under conditions suitable for proliferation of the one or more cells and for activity of the at least one exogenously introduced factor for a sufficient time to initiate reprogramming of the one or more cells or to activate at least one endogenous pluripotency gene; (d) functionally inactivating one exogenously introduced factor; (d) selecting one or more cells that exhibit markers of pluripotency; (e) generating a chimeric embryo using one or more cells that exhibit markers of pluripotency; (f) obtaining one or more somatic cells from said chimeric embryo; (g) maintaining said one or more somatic cells under conditions suitable for proliferation of said one or more somatic cells and for activity of said at least one exogenously introduced factor for a time sufficient to activate at least one endogenous pluripotency gene; and (h) distinguishing between cells that exhibit one or more markers of pluripotency and cells that do not.

[0021] In preferred embodiments of the invention, these methods result in purified somatic cell populations that contain at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%) pluripotent cells derived from reprogrammed differentiated somatic cells. In certain embodiments, the pluripotent cells are genetically homogenous.

[0022] The present invention also provides a method for identifying a reprogramming factor, comprising the steps of: (a) providing one or more somatic cells, each comprising at least one exogenously introduced factor that contributes to the reprogramming of one or more somatic cells to a pluripotent state, wherein each of the exogenously introduced factors is introduced using an inducible vector that is not subject to methylation-induced silencing and whose expression is controlled by a regulatory element that is induced by a separate inducer; (b) maintaining the one or more cells under conditions suitable for proliferation of the one or more cells and for activity of the at least one exogenously introduced factor for a sufficient time to reprogram one or more somatic cells or to activate at least one endogenous pluripotency gene; (c) functionally inactivating the at least one exogenously introduced factor; and (d) selecting one or more cells that exhibit markers of pluripotency. (e) generating a chimeric embryo using the one or more cells that exhibit markers of pluripotency; (f) obtaining one or more somatic cells from the chimeric embryo; (g) maintaining the one or more somatic cells under conditions suitable for proliferation of the one or more somatic cells and for activity of the at least one exogenously introduced factor, wherein the activity of the at least one exogenously introduced factor is insufficient by itself to activate at least one endogenous pluripotency gene; (h) contacting the somatic cells of (g) with one or more candidate reprogramming factors; and (i) identifying cells contacted with the one or more candidate reprogramming factors that exhibit one or more markers of pluripotency, wherein a candidate reprogramming factor that induces the somatic cell of (g) to exhibit one or more markers of pluripotency is identified as a reprogramming factor.

[0023] The present invention also relates to methods that utilize known inducible promoter systems. One example includes inducible promoters, such as DOX and tamoxifen-inducible lentiviral vectors. DOX-inducible retroviral vectors have been important for defining the minimum time of vector expression during sequential activation of pluripotency markers and reprogramming of mouse somatic cells. As described herein, the inventors have generated inducible lentiviral vectors that allow for the time-restricted expression of reprogramming factors. Following the same strategy used for mouse genes, the inventors have generated lentiviral vectors that transduce human OCT4, SOX2, KLF4, and C-MYC c-DNA either constitutively or under the control of DOX-inducible promoters. To generate DOX-inducible systems, the inventors have infected human fibroblasts with lentiviral vectors carrying rtTA transactivator.

[0024] To allow independent inducible control of the vectors, we also generated OCT4, SOX2, and C-MYC estrogen receptor (ER) fusion constructs by fusing the factors to the estrogen ligand binding domain to allow tamoxifen-dependent expression. Addition of tamoxifen to cells transduced with the SOX2-ER fusion construct leads to the translocation of SOX2 protein from the cytoplasm to the nucleus, as predicted for drug-induced activation. These results indicate that the DOX and ER fusion inducible systems can be used to independently control the expression of transduced factors.

[0025] One embodiment of the invention relates to the use of multiple, e.g., two different, controllable systems, each controlling the expression of a subset of factors. For example, one may place three factors under the control of a first inducible (e.g., dox-inducible) promoter, and a fourth factor under the control of a second inducible (e.g., tamoxifen-inducible) promoter. iPS cells can then be generated by inducing expression from both promoters, mice can be generated from the iPS cells, and fibroblasts (or any other cell type) can be isolated from the mice. These fibroblasts are genetically homogenous and can be reprogrammed without the need for viral infection. One then attempts to reprogram fibroblasts in the presence of different small molecules that can potentially substitute for the fourth factor under conditions where only the first promoter is active, in order to identify small molecule "reprogramming factors" or to optimize transient transfection or other protocols for introducing the fourth factor. Many variations are possible; for example, one may stably induce expression of the three factors, transiently induce expression of the fourth factor, and so on. Any combination of factors can be evaluated using the methods described, and the expression levels of the factors can be modulated by using different concentrations of the inducing agent.

[0026] Another approach is to place a gene encoding one of the factors between the sites for the recombinase, and then induce the expression of the recombinase to switch off the expression of that factor. For example, a heterologous sequence can be placed between the promoter sequence and the coding sequence, where the heterologous sequence is located between the sites for the recombinase; the heterologous sequence disrupts expression. The recombinase is introduced into the cell (e.g., by introducing an expression vector encoding the recombinase, e.g., adenovirus-Cre), causing the excision of the heterologous sequence, thereby allowing the expression of the transgene. The transgene can also be integrated into various non-essential loci (e.g., loci whose disruption does not significantly affect development, exemplified by the collagen I or Rosa26 loci).

[0027] These systems are useful for identifying reprogramming agents and for studying the requirements and events that occur during reprogramming, including discovering cell type specific differences. [Brief description of the drawings]

[0028] [Figure 1] Figures 1A-1D illustrate the generation of genetically homogenous cell cultures for epigenetic reprogramming. Figure 1A shows a scheme for infection of puromycin-resistant, Nanog-GFP, or Nanog-neo primary MEFs expressing reverse tetracycline transactivator (M2rtTA) with dox-inducible lentiviruses encoding the four reprogramming factors, followed by induction of reprogramming, primary iPS colony selection, dox withdrawal, chimera formation, and puromycin selection for iPS-induced secondary somatic cells. Figure 1B illustrates that NNeo secondary MEFs isolated from chimeras undergo complete epigenetic reprogramming. The dox-independent cultures express the pluripotency-associated genes alkaline phosphatase, SSEA1, and Nanog. Figure 1C shows that MEF-induced NNeo and NGFP2 secondary iPS cells form cells of all three germ layers in teratoma formation assays and contribute to chimera formation when injected into blastocysts, as indicated by the presence of iPS-induced agusticoat color on a dark background (Figure 1D). [Diagram 2]Figures 2A-2E illustrate that reprogramming kinetics and efficiency vary among MEFs from distinct iPS cell lines. As shown in Figure 2A, secondary MEFs from three "primary" iPS cell lines were treated with dox and reprogramming was monitored visually. Different MEF populations showed morphological changes 6 days after dox administration, but all formed colonies with ES cell morphology within 12 days (arrows). Figure 2B shows that colonies that were neomycin resistant and alkaline phosphatase positive were present in NNeo medium when the drug was added to the medium as early as 4 days after dox induction. Figure 2C illustrates flow cytometry analysis for reactivation of SSEA1 and Nanog-GFP reporter alleles (in NGFP2 and NGFP3 lines) over 18 days of dox culture. In Figure 2D, secondary NGFP2 MEFs were plated at densities varying from 0.025 to 500 cells / mm2, followed by dox addition. GFP+ colonies were counted after 4 weeks. As shown in Figure 2E, single secondary MEFs were plated onto 96-well plates containing γ-irradiated MEF feeder layers followed by dox induction. The percentage of single cells capable of growing sufficiently to form visible colonies on the MEF feeder layer (light grey bars) and the percentage of single cells capable of forming GFP+ or Neo-resistant secondary iPS colonies (dark grey bars) were scored after 4 weeks. [Diagram 3]Figures 3A-3F show the requirement and expression of the four factors in secondary MEFs. Figure 3A shows quantitative RT-PCR testing the induction of expression of the four reprogramming factors in response to 72 h of dox treatment relative to Gapdh levels. Figure 3B shows immunofluorescence detection of Oct4 and Sox2 in secondary MEF cultures 72 h after dox induction. As shown in Figure 3C, NGFP2 secondary MEFs were cultured in the presence of dox for the indicated times (5-22 days, red bars) followed by withdrawal of dox. Cultures were monitored daily for the first instances of GFP activation (green bars). Blue bars indicate the period during which GFP+ colonies appeared during dox treatment. Figure 3D shows that NGFP2 MEFs were cultured in the presence of dox for 10-15 days, at which point dox was discontinued and GFP+ colonies were scored at day 34. As illustrated in Figure 3E, NGFP2 MEFs were cultured in the presence of dox for 9 days (blue) or 22 days (red), and the appearance of GFP+ colonies was scored daily until day 29. Note that the appearance of GFP-positive colonies is late until day 15 after dox withdrawal. As illustrated in Figure 3F, NGFP3 secondary MEFs were cultured in the presence or absence of dox, dox+5-Aza, or dox+TSA, and GFP+ colonies were scored after 3 weeks. [Figure 4]Figures 4A-4N show the reprogramming of intestinal epithelial cells. As shown in Figure 4A, NNeo secondary intestinal epithelial crypt-villus structures were isolated from chimeras, and spheroids began to appear in suspension after 24 hours of culture in the presence of dox (Figure 4B, inset). Figure 4C illustrates that within 72 hours of dox culture, the suspended spheroids attached to a γ-irradiated feeder layer and adopted an ES-like morphology. As shown in Figure 4D, colonies continued to grow for 2 weeks after dox treatment, but differentiated and became indistinguishable from the feeder layer upon dox withdrawal (Figure 4E). Figure 4F shows that sox-dependent intestinal epithelial colonies were neomycin-resistant for 2 weeks after dox administration. Figure 4G shows the bisulfite sequences of the endogenous Oct4 and Nanog promoters in freshly isolated NNeo secondary intestinal epithelium, partially reprogrammed dox-dependent cells, and fully reprogrammed NNeo iPS cells after infection with Sox2 and Klf4 viruses. As shown in Figure 4H, qRT-PCR analysis of the expression of the four factors and Nanog revealed that dox-dependent NNeo intestinal epithelial colonies expressed high levels of Oct4 and cMyc, but very little Sox2 and Klf4, in comparison to ES cells. Figure 4I shows that NGFP2 secondary intestinal epithelial cells formed spheroids in suspension within 24 h of dox addition and adopted an ES-like morphology within 72 h (Figure 4J). Figures 4K and 4L illustrate that NGFP2 intestinal epithelium gave rise to dox-independent secondary iPS colonies expressing GFP from the endogenous Nanog locus. As shown in Figure 4M, EDTA-DTT-based fractionation of intestinal villi from apical differentiated cells (fraction 1) to crypt progenitor cells (fraction 7), followed by dox induction for 4 days, demonstrates that the crypt fraction in both NNeo and NGFP2 secondary lineages is more efficient in early colony formation. As shown in Figure 4N , qRT-PCR analysis showed that, with the exception of Klf4, the transgenes were more efficiently induced in fraction 7 (crypts) than in fraction 1 (villus tips) of NNeo and NGFP2 intestinal epithelial cells. [Diagram 5]Figures 5A-5L show the reprogramming of other somatic cell types. Figures 5A and 5B show NNeo mesenchymal stem cells (MSCs) 3 weeks before and after dox administration. Figures 5C and 5D show NGFP2 MSCs 10 days before and after dox treatment forming ES-like colonies. Figures 5E and 5F show that NGFP2 MSCs gave rise to dox-independent iPS colonies expressing GFP from the endogenous Nanog locus. As shown in Figure 5G, colonies of skin keratinocytes from NNeo chimeras with typical epithelial morphology (inset) began to show ES cell morphology within 12 days of dox treatment (Figure 5H). These cells were fully reprogrammed to form neomycin-resistant secondary iPS colonies (Figure 5I). As illustrated in Figure 5J, after expansion in serum-free medium, plated NNeo-derived neurospheres readily differentiated into astrocytes in response to ES cell medium containing dox and serum. When plated neurosphere cells were expanded in adherent conditions with EGF and FGF2 for an additional 3 weeks and then exposed to dox-containing medium, iPS cell-like colonies appeared in both ES cell (Figure 5K) and serum-free medium (Figure 5L). [Figure 6] FIG. 6 shows that fully reprogrammed NGFP2 secondary MEFs reactivate the endogenous Nanog locus, express Oct4, AP, and SSEA1, and can be maintained in the absence of dox. [Figure 7]Figures 7A-7C show further analysis. Figure 7A shows qRT-PCR analysis of endogenous Oct4, Sox2, Klf4, and c-Myc transcripts in NGFP2 MEFs during the time course of reprogramming in response to dox treatment. Expression levels in two ES cell RNA preparations (V6.5 line) and NGFP2 iPS cell line are also shown. Figure 7B shows a comparison of the experimental variability of iPS colony formation efficiency between direct infection and secondary lines. 3 x 105 Oct4-neo MEF1 were infected on 10 cm plates with four factors encoded by Moloney-based retroviral vectors, neomycin selection was started on day 6, and resistant colonies were counted on day 20 (left - direct infection). 3 x 104 secondary NGFP2 MEFs were plated in 6-well plates, exposed to dox-containing medium, and GFP-positive colonies were counted 3 weeks later (right - secondary line). Bars represent colony numbers in each of four independent experiments. Figure 7C shows Southern analysis of secondary iPS lines NGFP3, NGFP2, and NNeo with Klf4, c-Myc, Sox2, and Oct4 cDNA probes. Endogenous bands are marked with arrows and proviral insertions are marked with wedges, with the exception of Oct4 in the NNeo line, a transgene targeted to the collagen I locus. [Figure 8] 8A-8D: FIG. 8A shows that NGFP2 secondary tail tip fibroblasts were successfully reprogrammed into dox-independent GFP+ iPS cells. FIG. 8B shows that iPS cells derived from NGFP2 secondary intestinal epithelium express endogenous Nanog and SSEA1. FIG. 8C shows that iPS cells derived from NGFP2 secondary mesenchymal stem cells express endogenous Nanog and SSEA1. As shown in FIG. 8D, primary mesenchymal stem cells harboring a reverse tetracycline transactivator at the Rosa26 locus and Oct4 coding sequence under the control of Tet operator 16 were infected with viruses encoding Sox2, c-Myc, and Klf4. Addition of dox to the infected MSCs resulted in fully reprogrammed dox-independent iPS cells expressing endogenous Nanog protein (immunofluorescence). [Figure 9] Figures 9A-9G show successful reprogramming of cell cultures derived from adrenal gland (Figure 9A), kidney (Figure 9B), muscle (Figure 9C), keratinocytes (Figure 9D), and neurospheres (Figure 9E) of NNeo secondary chimeras, as determined by dox-independence, neomycin resistance, and Nanog expression (red, immunofluorescence). Figure 9F shows secondary intestinal epithelium isolated from NNeo chimeras, cultured in the presence of dox for 8, 10, or 12 days, and stained for alkaline phosphatase activity. As shown in Figure 9G, NNeo secondary intestinal epithelial cells became dox-independent iPS cells after infection with additional Sox2 and Klf4 viruses. Immunofluorescence analysis (red, top row) revealed expression of Oct4, Sox2, Nanog, and SSEA1 in fully reprogrammed cells (blue, bottom row represents nuclear DAPI staining). [Figure 10] Figures 10A-10D show the homologous insertion of GFP into the OCT4 locus. H9 huES cells were electroporated with a GFP-puroR gene trap vector targeted to the 3'UTR of the OCT4 locus as shown in Figure 10A. Correctly targeted clones were identified by Southern analysis stained with GFP (Figure 10B) and were puro-resistant when undifferentiated (Figures 10C, 10D), but when differentiated the marker and drug resistance genes were silenced (not shown). [Figure 11]Figures 11A-11B show DOX and tamoxifen-inducible factor expression. As shown in Figure 11A, human fibroblasts were infected with a lentiviral vector carrying a DOX-inducible factor (Brambrink et al., Cell Stem Cell Feb 7,2(2):151-159 (2008)). When DOX was added to the cultures, analysis by qPCR detected strong factor expression, whereas in the absence of DOX, little, if any, transcript was seen. Also, iPS cells derived from infected fibroblasts showed DOX-dependent expression (right two panels). As shown in Figure 11B, fibroblasts were infected with a vector containing a SOX2-ER fusion construct. Addition of tamoxifen to the medium resulted in translocation of the cytoplasmic protein to the nucleus, indicating drug-dependent protein activation. [Figure 12] Figures 12A-12C show the generation of iPS cells from human fibroblasts. As shown in Figure 12A, the expression of OCT4 and NANOG was quantified by qPCR and shown to be in a similar range as in control huES cells. Figure 12B shows an example of iPS cells generated from adult human fibroblasts. Human iPS cells formed tight colonies and stained for SSEA4, TRA 160, and OCT4. Figure 12C shows a teratoma with differentiated cell types formed after injection of iPS cells into SCID mice. [Figure 13A]Figures 13A-13C show reprogramming of mouse fibroblasts after transduction of four factors via polycistronic retroviral vectors. Figure 13A shows a schematic of vectors carrying four transcription factors, Sox2, Oct4, Klf4, and c-myc, each separated by a 2A sequence or various combinations of three or two factors. As shown in Figure 13B, fibroblasts were co-infected with polycistronic vectors of the four factors indicated at the top of the panel and a single Oct4 virus. Reprogrammed iPS cells expressed alkaline phosphatase (AP), SSEA1, Nanog, and Oct4. Figure 13C shows the results of Southern blot analysis for proviral integration of three independent iPS lines. DNA was digested with SpeI, which cuts once in PBS (giving one band per provirus), and blots were sequentially probed with Sox2, Klf4, c-myc, and Oct4 probes. Lines 4FO#5 and #9 had one and line 4FO#14 had two polycistronic vectors (one of the latter was truncated and had lost the 5′ cMYC sequence), however, hybridization with an Oct4 probe revealed between 8 and 11 additional Oct4 proviruses. [Figure 13B]Figures 13A-13C show reprogramming of mouse fibroblasts after transduction of four factors via polycistronic retroviral vectors. Figure 13A shows a schematic of vectors carrying four transcription factors, Sox2, Oct4, Klf4, and c-myc, each separated by a 2A sequence or various combinations of three or two factors. As shown in Figure 13B, fibroblasts were co-infected with polycistronic vectors of the four factors indicated at the top of the panel and a single Oct4 virus. Reprogrammed iPS cells expressed alkaline phosphatase (AP), SSEA1, Nanog, and Oct4. Figure 13C shows the results of Southern blot analysis for proviral integration of three independent iPS lines. DNA was digested with SpeI, which cuts once in PBS (giving one band per provirus), and blots were sequentially probed with Sox2, Klf4, c-myc, and Oct4 probes. Lines 4FO#5 and #9 had one and line 4FO#14 had two polycistronic vectors (one of the latter was truncated and had lost the 5′ cMYC sequence), however, hybridization with an Oct4 probe revealed between 8 and 11 additional Oct4 proviruses. [Figure 13C]Figures 13A-13C show reprogramming of mouse fibroblasts after transduction of four factors via polycistronic retroviral vectors. Figure 13A shows a schematic of vectors carrying four transcription factors, Sox2, Oct4, Klf4, and c-myc, each separated by a 2A sequence or various combinations of three or two factors. As shown in Figure 13B, fibroblasts were co-infected with polycistronic vectors of the four factors indicated at the top of the panel and a single Oct4 virus. Reprogrammed iPS cells expressed alkaline phosphatase (AP), SSEA1, Nanog, and Oct4. Figure 13C shows the results of Southern blot analysis for proviral integration of three independent iPS lines. DNA was digested with SpeI, which cuts once in PBS (giving one band per provirus), and blots were sequentially probed with Sox2, Klf4, c-myc, and Oct4 probes. Lines 4FO#5 and #9 had one and line 4FO#14 had two polycistronic vectors (one of the latter was truncated and had lost the 5′ cMYC sequence), however, hybridization with an Oct4 probe revealed between 8 and 11 additional Oct4 proviruses. [Figure 14]Figures 14A-14E show the generation of mouse iPS cells using a single 4F2A polycistronic virus. Figure 14A shows the FUW lentivirus constructs tested by transient transfection (also shown in previous figures). A total of four 2A peptides (F2A, T2A, E2A, and P2A) were used. Figure 14B shows the transient transfection of 293 cells with FUW 2A lentivirus. Cells were harvested after 48 hours and analyzed by Western blot (WB). Efficient protein expression was observed in all constructs tested, indicating that the unique 2A peptide supports robust protein expression. Note: Sox2 protein is not detectable in ES cells because only a short exposure was used. Figure 14C shows a schematic diagram of 4F2A DOX-inducible lentivirus containing three types of 2A peptides (P2A, T2A, and E2A). Mouse cDNA for Oct4, Sox2, Klf4, and c-Myc. This particular sequence of factors and 2A peptide is subsequently referred to as "4F2A". Figure 14D shows RT-PCR analysis of mRNA induction in cells transduced with OSKM 4F2A+rtTA for 3 days. Total Oct4 or Sox2 induction was used to test the level of 4F2A induction compared to ES cells. E2A-cMyc primers were used to detect virus-specific transcripts. Error bars represent the standard deviation of the mean of triplicate reactions. Figure 14E shows the results of Western blot analysis of MEFs transduced with 4F2A+rtTA for 3 days. Cells infected with 4F2A DOX-inducible lentivirus+rtTA produce all four reprogramming factors upon addition of doxycycline, DOX. [Figure 15]Figures 15A-15C illustrate that 4F2A iPS cells express pluripotency markers. As shown in Figure 15A, immunostaining of Oct4 protein indicates that high titer infection can be achieved with 4F2A. MEFs were cultured in DOX medium for 2 days after transduction with 4F2A+rtTA. Figure 15B illustrates the morphology changes in NanogGFP-MEFs transduced with 4F2A+rtTA cultured in ES medium+DOX. Colonies appeared at approximately day 8, similar to cells infected with a single virus. NanogGFP+ colonies were observed by day 25 after removal of DOX medium on day 20. Two columns show typical colonies observed on a plate. FIG. 15C shows 4F2A iPS lines generated from Nanog-GFP MEFs or 14-week tail tip fibroblasts (TTFs) that stain positive for pluripotency markers AP, SSEA1, Oct4, and reactivated the endogenous Nanog locus (GFP+ for MEFs and immunostained for TTFs). [Figure 16] Figures 16A-16C illustrate that 4F2A iPS cells are pluripotent and contain between one and three proviral integrations. Figure 16A shows in vivo differentiation of 4F2A MEF-iPS lines #1, 2, and 4. Histological analysis of teratomas induced after subcutaneous injection into SCID mice shows that the iPS lines contribute to all three germ layers. Figure 16B shows moderate to high contribution to postnatal chimeric mice as detected by aguticote color from 4F2A iPS line #4. Figure 16C shows the results of Southern blot analysis of 4F2A proviral integration in MEF-iPS cell lines #1-4. iPS cell DNA was digested with BamHI. Hybridization of the same molecular weight fragment using all four probes indicates the presence of the 4F2A provirus. The red arrow highlights iPS line #4, which contained one proviral copy of 4F2A. * indicates the endogenous allele. [Figure 17]Figures 17A-17E show the generation of human iPS lines using a single 4F2A polycistronic virus. Figure 17A shows neonatal human foreskin keratinocytes (NHFK) transduced with 4F2A (with mouse cDNA) + rtTA. On day 22, single colonies were picked and expanded, resulting in colonies similar to hES. These colonies were picked and stable hiPS lines were established. Figure 17B shows Ker hiPS #1.1 immunostaining for pluripotency markers AP, Oct4, Nanog, SSEA-4, Tra1-60, and Tra1-81. DAPI staining is the bottom panel. Figure 17C illustrates that the karyotype of Ker hiPS #1.1 is normal 46XY. Figure 17D shows in vivo differentiation of Ker hiPS #1.1. Hematoxylin and eosin staining of teratoma sections generated by Ker hiPS #1.1. Figure 17E shows in vitro differentiation of Ker hiPS #1.1. (Left) Ker-iPS #1.1-derived neural progenitor cells exposed to differentiation conditions for 6 days give rise to terminally differentiated neurons as detected by anti-Tuj1 immunostaining (green). (Right) Ker-iPS #1.1 neural progenitor cells (NP) undergo spontaneous differentiation. NPs are detected by anti-Nestin immunostaining and differentiated neurons are detected by anti-Tuj1 (red). DAPI staining of DNA in both pictures is in blue. [Figure 18] Figures 18A-18B show Southern blots of MEF-induced iPS lines and dox withdrawal for 8 days is sufficient to generate iPS lines. Figure 18A shows Southern blot analysis of 4F2A MEF iPS lines. A second digestion (XbaI) was performed to determine proviral copy number. In this digestion, iPS lines #2 and #4 showed 1 proviral copy, however, only #4 had 1 proviral copy in both digests. Figure 18B shows that Dox withdrawal 8 days after infection of Nanog GFP MEFs with rtTA+OSKM generated two iPS lines. Both generated stable iPS lines that were generated in 1-2 generations. [Figure 19]Figure 19 shows the relative efficacy of reprogramming using 4F2A in MEFs. NanogGFP MEFs were infected with 4F2A+rtTA and cultured in ES medium (+ / - DOX) for 48 hours. Cells were fixed and stained for Oct4 protein. Estimated infection efficiency was approximately 70%. The same virus was also used to infect 0.2 5x10^6 NanogGFP MEFs, and cells were cultured on DOX for 20 days. After withdrawal of DOX on day 20, GFP+ colonies were counted on day 25, and GFP+ colonies were observed in three plates 10, 10, and 17. [Figure 20] Figures 20A-20B illustrate the infection efficiency and pluripotency analysis of keratinocyte-derived human iPS lines. Figure 20A shows the infection efficiency from two experiments as detected by Oct4 immunostaining in keratinocytes infected with 4F2A+rtTA and cultured in hES medium+DOX for 48 hours. The efficiency of infection was approximately 10-20% based on the fraction of cells positive for Oct4 protein. Figure 20B shows human iPS lines staining positive for pluripotency markers expressed in hES (Ker iPS #3 not shown). [Figure 21] Figures 21A-21B show the proviral copy numbers of keratinocyte-derived human iPS lines. Figure 21A shows Southern blot analysis of Ker-iPS line. 10 mg of genomic DNA was collected and digested with XbaI. Hybridization of the same molecular weight fragment indicates the presence of 4F2A provirus. Probes for Sox2, Klf4, and c-Myc suggested 2 (#1.1) and 1 (#3) proviral copies. The common band observed between the two iPS lines was not a viral integration because these were derived from independent infections. Figure 21B shows Southern blot analysis of Ker-iPS line. 10 mg of genomic DNA was collected and digested with BamHI. Hybridization of the same molecular weight fragment indicates the presence of 4F2A provirus. Probes for Oct4 and c-Myc indicate 3 (#1.1) and 2 (#3) proviral copies. [Figure 22] FIG. 22 illustrates a strategy for generating iPS cells using a single polycistronic construct at a defined genomic location. [Diagram 23] Figures 23A-23B show the generation of secondary fibroblasts with DOX-inducible vectors that allow reprogramming without viral transduction. As illustrated in Figure 23A, "primary" fibroblasts carrying GFP at the OCT4 locus were transduced with all four factors using DOX-inducible vectors, as well as a vector carrying the tet rtTA transactivator, and "primary" iPS cells were generated after DOX induction. The cells were differentiated into "secondary" fibroblasts carrying the same vector combination that allowed the deviation of primary iPS cells in the absence of DOX. As shown in Figure 23B, reprogramming secondary fibroblasts into secondary iPS cells requires only DOX induction of the provirus, instead of infection with a new virus. [Figure 24] Figure 24 shows reprogramming without vector-mediated factor introduction. Primary fibroblasts are derived from huES cells carrying a DOX-inducible polycistronic construct expressing the OCT4-GFP marker, the tet transactivator M2rtTA, and the three reprogramming factors described in Figure 13 (in this example, OCT4, SOX2, cMYC) inserted into the COLlA1 locus. These cells are infected with a vector flanked by 2Lox sites (Ventura et al., Proc Natl Acad Sci USA, Jul 13;101(28):10380-5(2004)) carrying the KLF4 cDNA. DOX treatment generates primary iPS cells, which delete the KLF4 vector after Cre expression. Secondary fibroblasts are derived, which allow screening for small molecules that replace the deleted KLF4 factor upon DOX treatment. [Diagram 25]Figure 25 shows a scheme for quantifying the efficiency of reprogramming by testing for different markers. Cells carrying GFP and puro markers at the OCT4 locus were transduced with three or four factors. The fraction of drug-resistant or GFP-positive colonies, and the appearance of cells staining for alkaline phosphatase (AP), SSEA4, TRA61, or Nanog were determined in the cell population at different time points after infection. [Figure 26] Figure 26 illustrates the use of secondary fibroblasts with independently inducible factors to screen small molecules. Primary fibroblasts with viral M2rtTA and OCT4-GFP markers are transduced with a tamoxifen-inducible vector that transduces three factors, and a DOX-inducible vector that transduces a fourth factor (in this case cMYC). Primary iPS cells are induced by culture in tamoxifen and DOX to induce secondary fibroblasts. These cells can be screened for small molecules that replace cMYC for reprogramming into secondary iPS cells when cultured in tamoxifen. [Figure 27] Figures 27A-27C show the characterization of DOX-induced hiPSCs derived from fibroblasts from PD patients. Figure 27A shows phase contrast photographs and immunostaining of hiPSC lines M3F-1 (non-PD hiPSC), PDA3F-1, PDB3F-5, PDC3F-1, PDD3F-1, and PDE3F-3 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG. Figure 28A shows quantitative RT-PCR for reactivation of endogenous pluripotency-related genes NANOG, OCT4, and SOX2 in independent hiPSC lines, hESCs, and primary fibroblasts. Relative expression levels were normalized to the expression of these genes in fibroblasts. Figure 28C shows methylation analysis of the OCT4 promoter region. Light grey boxes indicate unmethylated and black boxes indicate methylated CpGs in the OCT4 promoter in hiPSCs and parental primary fibroblasts. [Figure 28-1]Figures 28A-28C illustrate that PD patient-derived hiPSCs have low copy number viral integration. Figure 28A shows hematoxylin and eosin staining of teratoma sections generated from hiPSC lines A6 (non-PD hiPSC), PDA3F-1, PDB3F-1, PDC3F-1, PDD3F-1, and PDE3F-3, which show: top row panels: neuroepithelium with dye; second row panels: neural rosettes; third row panels: intestinal epithelium; fourth row panels: bone / cartilage; bottom row panels: smooth muscle. Figure 28B shows the results of Southern blot analysis of hESC line BG01, mouse embryonic fibroblast (MEF) feeder cells, and hiPSCs derived by the indicated PD patients (and non-PD hiPSC line M3F-1) for proviral integration of XbaI-digested genomic DNA using 32P-labeled DNA probes for OCT4, KLF4, SOX2, and c-MYC. Figure 28C is a table summarizing the approximate number of proviral integrations for the four reprogramming factors in hiPSCs based on the Southern blot analysis shown in 28B. [Figure 28-2]Figures 28A-28C illustrate that PD patient-derived hiPSCs have low copy number viral integration. Figure 28A shows hematoxylin and eosin staining of teratoma sections generated from hiPSC lines A6 (non-PD hiPSC), PDA3F-1, PDB3F-1, PDC3F-1, PDD3F-1, and PDE3F-3, which show: top row panels: neuroepithelium with dye; second row panels: neural rosettes; third row panels: intestinal epithelium; fourth row panels: bone / cartilage; bottom row panels: smooth muscle. Figure 28B shows the results of Southern blot analysis of hESC line BG01, mouse embryonic fibroblast (MEF) feeder cells, and hiPSCs derived by the indicated PD patients (and non-PD hiPSC line M3F-1) for proviral integration of XbaI-digested genomic DNA using 32P-labeled DNA probes for OCT4, KLF4, SOX2, and c-MYC. Figure 28C is a table summarizing the approximate number of proviral integrations for the four reprogramming factors in hiPSCs based on the Southern blot analysis shown in 28B. [Figure 29]Figures 29A-29C show the generation of hiPSCs induced by PD patients using loxP-excisable reprogramming factors. Figure 29A is a schematic of the DOX-inducible lentiviral construct FUW-tetO-loxP, the genomic locus after proviral integration (2lox) and after Cre recombinase-mediated excision (1lox). The FUW-TetO-loxP vector contains a tetracycline-responsive element (TRE) located 5' of the minimal CMV promoter and a unique MfeI site used for diagnostic Southern blot digestion. The reprogramming factors are flanked by EcoRI restriction sites. The 3'LTR of this lentiviral vector contains a single loxP site, which is duplicated during proviral replication into the 5'LTR. This duplication results in a transgene that is flanked by 2loxP sites after genomic integration of the provirus (2lox). This allows excision of the transgene in combination with the intact promoter sequence using Cre recombinase (1lox) (WRE = woodchuck response element). Figure 29B shows phase contrast photographs and immunofluorescence staining of hiPSC lines PDB2lox-17 and PDB-21 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG. PDB2lox-17 and PDB2lox-21 are induced by expression of the three reprogramming factors OCT4, SOX2, and KLF4 from the FUW-tetO-loxP virus shown in A. In these cells, all three reprogramming factors are flanked by loxP sites at their genomic integration site. Figure 29C shows hematoxylin and eosin staining of teratoma sections generated from PDB2lox-17 and PDB2lox-21 cells with excisable reprogramming factors. [Diagram 30]Figures 30A-30D show the generation and characterization of hiPSCs without reprogramming factors. Figure 30A is a schematic overview of Cre-mediated excision of transgenes to generate hiPSCs without reprogramming factors. IPS PDB2lox cells are derived using a FUW-tetO-loxP lentiviral vector transducing the three reprogramming factors OCT4, KLF4, and SOX2. Figure 30B shows Southern blot analysis of parental fibroblasts (PDB), proviral-harbored PDB2lox clones (PDB2lox-17 and PDB2lox-21), and the indicated PDB1lox clones after Cre recombinase-mediated excision of the transgene. Puro indicates PDB1lox clones, which were isolated by puromycin selection; GFP indicates PDB1lox clones isolated by FACS sorting for EGFP (as shown in 30A). Genomic DNA was digested with XbaI and probed for proviral integration using 32P-labeled DNA probes for OCT4, KLF4, and SOX2. PDB1lox clones shown in blue were ignored due to remaining transgene integration based on the MfeI digest shown in Figure 34. Figure 30C shows cytogenetic analysis of hiPSC line PDB1lox-17Puro-5, and PDB1lox-21Puro-12 shows normal karyotype after Cre-mediated excision of the transgene. Figure 30D is a summary of the generation of factor-free hiPSCs. [Figure 31-1]Figures 31A-31E show characterization of reprogramming factor-free hiPSCs. Figure 31A shows phase contrast photographs and immunofluorescence staining of reprogramming factor-free hiPSC lines PDB1lox-17Puro-5 and PDB1lox-21Puro-12 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG. Figure 31B shows quantitative RT-PCR for reactivation of endogenous pluripotency-associated genes NANOG, OCT4, and SOX2 in hESCs, fibroblasts (PDB), provirus-carrying PDB2lox clones (PDB2lox-17 and PDB2lox-21), and the indicated PDB1lox clones after Cre recombinase-mediated excision of the transgene. Relative expression levels were normalized to the expression of these genes in fibroblasts. FIG. 31C shows hematoxylin and eosin staining of teratoma sections generated from factor-free PDB1lox-17puro-5 and PDB1lox-21puro-26 cells. Figure 31D includes quantitative RT-PCR for residual transgene expression of OCT4, KLF4, and SOX2 in hESCs (BG01), primary fibroblasts (PDB), primary infected fibroblasts (PDD3F+ / -DOX), hiPSCs (M3F3-1), PD-induced hiPSCs (PDA3F-1, PDB3F-5, PDC3F-1, PDD3F-1, PDE3F-3), provirus-carrying PDB2lox clones (PDB2lox-17 and PDB2lox-21), and PDB1lox clones without reprogramming factors (PDB1lox-17Puro-5, PDB1lox-17Puro-31, PDB1lox-21Puro-12, PDB1lox-21Puro-20). Relative expression levels are normalized to DOX-induced expression in primary infected fibroblasts.FIG. 31E is a Venn diagram displaying the number of differentially expressed genes (p<0.05 as determined by a moderated t-test, corrected for false discovery rate) between provirus-carried PDB2lox lines (PDB2lox-5, PDB2lox-17, PDB2lox-21, PDB2lox-22) compared to hESCs (H9, BG01), or between PDBlox lines without reprogramming factors (PDB1lox-17Puro-5, PDB1lox-17Puro-10, PDB1lox-21Puro-20, PDB1lox-21Puro-26) compared to hESCs (H9, BG01), respectively. [Figure 31-2]Figures 31A-31E show characterization of reprogramming factor-free hiPSCs. Figure 31A shows phase contrast photographs and immunofluorescence staining of reprogramming factor-free hiPSC lines PDB1lox-17Puro-5 and PDB1lox-21Puro-12 for pluripotency markers SSEA4, Tra-1-60, OCT4, SOX2, and NANOG. Figure 31B shows quantitative RT-PCR for reactivation of endogenous pluripotency-associated genes NANOG, OCT4, and SOX2 in hESCs, fibroblasts (PDB), provirus-carrying PDB2lox clones (PDB2lox-17 and PDB2lox-21), and the indicated PDB1lox clones after Cre recombinase-mediated excision of the transgene. Relative expression levels were normalized to the expression of these genes in fibroblasts. FIG. 31C shows hematoxylin and eosin staining of teratoma sections generated from factor-free PDB1lox-17puro-5 and PDB1lox-21puro-26 cells. Figure 31D includes quantitative RT-PCR for residual transgene expression of OCT4, KLF4, and SOX2 in hESCs (BG01), primary fibroblasts (PDB), primary infected fibroblasts (PDD3F+ / -DOX), hiPSCs (M3F3-1), PD-induced hiPSCs (PDA3F-1, PDB3F-5, PDC3F-1, PDD3F-1, PDE3F-3), provirus-carrying PDB2lox clones (PDB2lox-17 and PDB2lox-21), and PDB1lox clones without reprogramming factors (PDB1lox-17Puro-5, PDB1lox-17Puro-31, PDB1lox-21Puro-12, PDB1lox-21Puro-20). Relative expression levels are normalized to DOX-induced expression in primary infected fibroblasts.FIG. 31E is a Venn diagram displaying the number of differentially expressed genes (p<0.05 as determined by a moderated t-test, corrected for false discovery rate) between provirus-carried PDB2lox lines (PDB2lox-5, PDB2lox-17, PDB2lox-21, PDB2lox-22) compared to hESCs (H9, BG01), or between PDBlox lines without reprogramming factors (PDB1lox-17Puro-5, PDB1lox-17Puro-10, PDB1lox-21Puro-20, PDB1lox-21Puro-26) compared to hESCs (H9, BG01), respectively. [Diagram 32]Figures 32A-32D show that 8 days of transgene expression is sufficient to reprogram human fibroblasts after primary infection. Figure 32A shows immunofluorescence staining of primary fibroblasts (PDB) transduced with the four reprogramming factors OCT4, KLF4, SOX2, and c-MYC. Cells were fixed and stained for expression of NANOG (red) and Tra-1-60 (green) at different time points after DOX-induced transgene expression (upper panel, day 8; lower panel, day 10). No NANOG / Tra-1-60 positive cells were detected before day 8 or in cultures not treated with DOX. NANOG and Tra-1-60 colonies were also detectable in all cultures stained at later time points (days 12, 14, 16, 18, and 20). Figure 32B shows immunofluorescence staining of pluripotency-associated markers SSEA4, TRA-1-60, OCT4, SOX2, and NANOG of hiPSC clones PDB4F-1 and PDB3F-12d. To determine the temporal requirements for transgene expression, primary fibroblasts (PDB) were infected with DOX-inducible lentiviruses carrying reprogramming factors. Transgene expression was induced by the addition of DOX. At different time points, the medium was replaced with hESC medium without DOX, and iPSCs were isolated 24 days after the initial DOX addition. The left panel shows hiPSC clone PDB4F-1 isolated from a culture transduced with four reprogramming factors and exposed to DOX for 8 days. The right panel shows hiPSC clone PDB3F-12d isolated from a culture transduced with three reprogramming factors and exposed to DOX for 12 days. Figure 32C shows quantitative RT-PCR for reactivation of pluripotency-related genes NANOG, OCT4, and SOX2 in the following lines: hiPSC lines PDB4F-1 and PDB4F-2, D4, A6, hESC, and primary fibroblasts. Relative expression levels were normalized to the expression of these genes in fibroblasts. PDB4F-1 and PDB4F-2 iPSCs were isolated 8 days after transgene expression of four reprogramming factors OCT4, SOX2, KLF4, and c-MYC.Figure 32D shows hematoxylin and eosin staining of teratoma sections generated from hiPSC lines PDB3F-12d and PDB4F-2. PDB3F-12d was derived by DOX-inducible transgene expression of three reprogramming factors OCT4, SOX2, KLF4 for 12 days. PDB4F-2 was derived by DOX-inducible transgene expression of four reprogramming factors OCT4, SOX2, KLF4, and c-MYC for 8 days. [Diagram 33] Figure 33 shows generation of hiPSCs with Cre recombinase-excisable viral reprogramming factors. Southern blot analysis of the indicated iPS PDB2lox clones for proviral integration of XbaI-digested genomic DNA using 32P-labeled DNA probes for OCT4, KLF4, and SOX2. All PDB2lox clones were derived by retroviral transduction with Cre recombinase-excisable lentiviral vectors (FUW-tetO-loxP) for the three reprogramming factors OCT4, SOX2, and KLF4. [Diagram 34]Figure 34 shows Southern blot analysis for excision of reprogramming factors in hiPSCs. Southern blot analysis of proviral integration of parental fibroblasts (PDB), proviral-carried PDB2lox clones (PDB2lox-17 and PDB2lox-21), and PDB1lox clones shown after Cre recombinase-mediated excision of the transgene. Puro indicates clones isolated by puromycin selection; GFP indicates clones isolated by FACS sorting for EGFP (similar to that shown in Figure 5A). Genomic DNA was digested with MfeI and probed for proviral integration using 32P-labeled DNA probes for OCT4, KLF4, and SOX2. Based on Southern blot analysis, the PDB1lox clones shown in blue (PDB1lox-17GFP-10, PDB1lox-17GFP-18, PDB1lox-21Puro35, and PDB1lox-21GFP-28) were considered to be partially deleted or mixed cell populations with partial deletion of the transgene. [Diagram 35] Figure 35 shows Southern blot analysis of FUW-M2rtTA. Southern blot analysis of parental fibroblasts (PDB), provirus-carrying PDB2lox clones (PDB2lox-17 and PDB2lox-21), and the indicated PDB1lox clones for proviral integration of FUW-M2rtTA. Puro indicates clones isolated by puromycin selection; GFP indicates clones isolated by FACS sorting for EGFP (as shown in Figure 30A). Genomic DNA was digested with MfeI and probed for proviral integration using a 32P-labeled DNA probe for FUW-M2rtTA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Table 1: Human iPS cells derived from factor-transduced embryonic or adult human fibroblasts. Fibroblasts were infected with constitutive or DOX-inducible lentiviral vectors transducing different combinations of factors. Between 50 and 100 clones were picked in each experiment. Southern blots for viral uptake showed that iPS lines were derived from independently infected fibroblasts (O=OCT4, S=SOX2, K=KLF4, M=C-MYC, L=LIN28, N=NANOG).

[0030] Table 2: Summary of transgenic human ES or iPS cell lines used in this proposal. DOX-inducible polycistronic vectors with different factor combinations are integrated into the 3'UTR of the COL1A1 locus, or GFP is inserted into the OCT4 locus or the indicated neural-specific genes. The table also indicates the specific purposes for which the cells are used.

[0031] Detailed Description of the Invention PCT Application Serial No. PCT / US08 / 004516, filed April 7, 2008, and U.S. Patent Application 10 / 997,146, filed November 24, 2004, are hereby incorporated by reference in their entireties. It is intended that the various embodiments and aspects of the invention described herein are applicable to all different aspects and embodiments of the invention. It is also intended that, wherever appropriate, any of these embodiments or aspects may be freely combined with one or more other such embodiments or aspects.

[0032] The study of induction of pluripotency is complicated by the need for infection with high titer retroviral vectors that result in genetically heterogeneous cell populations. We generated genetically homogeneous "second" somatic cells carrying reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentivirus, reprogramming by dox addition, selecting iPS cells, and producing chimeric mice. Cells derived from these chimeras efficiently reprogram upon dox exposure without the need for viral infection. Utilizing this system, we demonstrate that (i) reprogramming factors at various induction levels can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed, and (iv) different cell types require different induction levels. This system facilitates characterization of reprogramming and provides a unique platform for genetic or chemical screening to enhance reprogramming or replace individual factors.

[0033] mouse 1-4 and humans 5-8 It has recently been shown that fibroblasts can be reprogrammed to a pluripotent state through retroviral-mediated introduction of four transcription factors, Oct4, Sox2, Klf4, and c-Myc. Reprogramming also occurs with reduced efficiency. 9、10 , or in the absence of c-Myc. Nevertheless, using these approaches, only a very small fraction of cells infected with all four factors ultimately undergoes reprogramming. 11 Random virus infection generates genetic heterogeneity in infected cell cultures, which likely plays a prominent role in the observed low frequency of induced pluripotent stem (iPS) cell formation. Hence, cells that are steadily reprogrammed are likely to be reprogrammed by reactivation of endogenous pluripotency genes. 1-3 , or based on morphological criteria 11、12, must be selected for. This reprogramming process has been shown to require sustained transgene expression for approximately 10-12 days after viral transduction and follows the sequential activation of pluripotency markers with the initial activation of alkaline phosphatase and stage-specific embryonic antigen (SSEA1), followed by reactivation of the endogenous Oct4 and Nanog genes, after which these cultures can sustain a pluripotent state in the absence of transgene activity. 13、14 .

[0034] The cellular and genetic heterogeneity of randomly infected fibroblasts complicates the search for key molecular events occurring during reprogramming and limits the scalability required for high-throughput analysis. To overcome these problems, we developed a system to generate genetically identical cell populations that are susceptible to reprogramming without any additional genetic interference. To this end, primary fibroblasts were infected with doxycycline-inducible lentiviruses encoding the four reprogramming factors. After blastocyst injection, chimeric mice were generated consisting of tissue types clonally derived from the reprogrammed fibroblasts. From these mice, homogenous donor cell populations could be derived with preselected vector integrations that are permissive for reprogramming, allowing robust and simple doxycycline-inducible reprogramming of primary cell types without the need for direct viral transduction of reprogramming factors. This technology facilitates the generation of large amounts of genetically identical donor cells and presents a powerful platform for the application of genetic or chemical screens to improve reprogramming. In addition, the same approach has been applied to generate pluripotent reprogrammed fibroblasts. 15 Genetic deletion of one specific factor in C. elegans can be used to screen for small molecules that replace each of the four factors. Furthermore, this tool is not limited to fibroblast cultures but, in principle, can be applied to all other somatic cell types as well, providing an attractive method for introducing genes into cell types that are difficult to infect with retroviruses, such as lymphocytes or intestinal epithelial cells.

[0035] result Generation of genetically homogenous cell populations for drug-induced reprogramming To generate cell populations that are homogenous with respect to the number and location of proviral integrations, we used a doxycycline (dox)-inducible transgene system. 16、17 Utilizing the above, we constructed dox-inducible lentiviral vectors encoding the four reprogramming factors. Mouse embryonic fibroblasts (MEFs) containing both a reverse tetracycline transactivator and a puromycin resistance gene driven by the PGK promoter targeted to the ROSA locus (ROSA-M2rtTA) in addition to green fluorescent protein (GFP) targeted to the endogenous Nanog locus were infected with the four lentiviruses. Similarly, we also infected the Oct4 cDNA under the control of a tetracycline operator targeted to the type I collagen locus, and a neomycin resistance gene targeted to the endogenous Nanog locus. 1、18 Rosa-M2rtTA with (NNeo) MEFs were infected with dox-inducible lentiviruses encoding Klf4, Sox2, and c-Myc (Fig. 1a ).

[0036] After viral transduction, doxycycline was added to the culture medium to activate the transgene and initiate the reprogramming process. As expected, Nanog-GFP-positive and Nanog-neo-resistant iPS colonies emerged and clonal iPS cell lines were established. All iPS cells could be expanded in the absence of dox, exhibited alkaline phosphatase activity, and homogeneously expressed the pluripotency markers SSEA1 and Nanog (not shown). This indicated that these "primary" iPS cell lines had activated their endogenous pluripotency core transcriptional network and were no longer dependent on exogenous expression of the four reprogramming factors. 191 shows the results of the clonal iPS cell lineage. To generate somatic tissues composed of genetically homogeneous cells carrying the same proviral insertions known to achieve reprogramming in primary fibroblasts, we injected some of these clonal primary iPS lines into blastocysts. The resulting dox-inducible iPS cell chimeras were allowed to gestate until E13.5, at which point MEFs were isolated. Puromycin selection was then used to select against cells derived from the host blastocyst, leaving only iPS-inducible cells. We refer to such cells as "secondary" MEFs, because they are derived from primary iPS and therefore carry a specific set of proviral insertions capable of reprogramming somatic cells (FIG. 1A).

[0037] Secondary MEFs were isolated from chimeric iPS cell embryos generated from three distinct clonal primary iPS cell lines (one Nanog-neo line and two Nanog-GFP lines) and cultured in the presence of dox to determine whether the integrated lentiviral vectors retained the ability to mediate epigenetic reprogramming after differentiation in the developing embryos. Addition of dox to these cultures initiated dramatic morphological changes, and "secondary" iPS cell lines were efficiently isolated from these cultures by neo selection or GFP expression and subsequently expanded in the absence of dox. Immunofluorescence demonstrated that the secondary iPS cells reactivated ES cell pluripotency markers, alkaline phosphatase, SSEA1, and the endogenous Nanog gene (Figure 1B and Figure 6). The pluripotency of these cell lineages was confirmed by their ability to form cells of endodermal, ectodermal, and mesodermal lineages in teratoma formation assays, as well as by their ability to contribute to adult chimeric mice upon blastocyst injection (Figures 1C, 1D).

[0038] Transgene induction levels, reprogramming kinetics, and efficiency vary between secondary MEFs derived from distinct iPS cell lines While secondary MEFs derived from all three dox-inducible iPS cell lines underwent reprogramming to form secondary iPS cell lines, we noticed differences regarding their morphological changes and proliferation rates after dox treatment. Initially, MEFs from both Nanog-GFP lines proliferated to form confluent fibroblast monolayers after exposure to dox. Cells from Nanog-GFP line 3 (NGFP3) then underwent robust post-confluent proliferation, including proliferation of cells in suspension, whereas cells from NanogGFP line 2 (NGFP2) proliferated slower and formed modest, alkaline phosphatase-positive, ES-like colonies on the fibroblast monolayer (Figure 2A). Fibroblasts derived from the Nanog-neo line never formed a confluent monolayer upon dox addition, but generated large three-dimensional colonies. Twelve days after dox administration, iPS cell colonies with ES cell morphology were readily visible in all three cultures ( Fig. 2A , arrows).

[0039] To assess the kinetics of reprogramming in more detail, MEFs from the three lineages were cultured in dox-containing medium and flow cytometry analysis was utilized to monitor the reactivation of SSEA1 and GFP (Figure 2C). All three secondary MEFs showed a gradual increase in SSEA-1 positive cells over time, however, some timing differences were observed. NNeo MEFs showed an initial increase in SSEA1 positive cells of 1.3%-17.8% between days 8 and 11. NGFP2 MEFs showed a similar increase, but at a much later time point (4.4%-29%, between days 14 and 18). In contrast, MEFs from the iPS cell lineage NGFP3 showed a slower, gradual activation of SSEA1, reaching approximately 10% at day 14. The first GFP positive cells were detected as early as day 14 in NGFP2 and at day 18 in NGFP3 MEFs.

[0040] To monitor the timing of reactivation of the endogenous Nanog locus in NNeo secondary MEFs, we plated cells at various time points after dox treatment and initiated drug selection. In contrast to activation of the Nanog-GFP reporter gene at approximately 2 weeks after induction, NNeo MEFs were neomycin resistant when neo was added to the initial culture until day 4 (Figure 2B). This may reflect a faster reactivation of the Nanog locus, similar to what we observed for SSEA1 expression in this cell line (Figure 2C). Alternatively, neo-resistant colonies may emerge earlier because low levels of Nanog gene activation are sufficient to confer drug resistance, in contrast to GFP detection, which requires higher expression. 14、20 Although generation of secondary cells selected for a specific set of proviral integrations whose expression was capable of inducing the formation of primary iPS cell lineages, the overall kinetics of pluripotency marker activation was similar to that seen in direct infection of MEFs. 1、13、14 This supports the notion that the reprogramming process requires a series of sequential epigenetic changes. 11、20 .

[0041] Next, we compared the reprogramming efficiency of various secondary MEFs. To determine the optimal plating density, we plated them at 0.025–500 cells / mm in dox-containing medium. 2 Secondary NGFP2 MEFs were plated at densities ranging from 100 to 1000 μg / ml and GFP-positive colonies were counted after 4 weeks. As shown in Figure 2D, plating density had a profound effect on iPS formation. Remarkably, both high and low plating densities completely inhibited GFP-positive colony formation. We speculate that paracrine factors may be required first to facilitate proliferation and that essential cell proliferation would be hindered if cells were contact inhibited prior to transgene activation.

[0042] To rigorously determine reprogramming efficiency in secondary lines, we plated single fibroblasts from the NNeo and NGFP2 lines into 96-well plates containing γ-irradiated MEFs as feeder cells to provide optimal growth support. We observed that only nearly 14% and nearly 8% of seeded cells from NNeo and NGFP2 MEFs, respectively, proliferated sufficiently to form individual colonies after dox administration (light gray bars in Figure 2E). However, approximately one-quarter of these cells eventually became neomycin-resistant or GFP-positive after 4 weeks of culture, resulting in an overall reprogramming efficiency of nearly 4% for the NNeo line and nearly 2% for the NGFP2 line (dark gray bars in Figure 2E). This is 25-50 times more efficient than originally reported for drug resistance-based iPS selection. 1、12 , which is between 4-8 times more efficient than morphology-based iPS selection in cultures of primary infected fibroblasts. 11 .

[0043] Next, we compared the regeneration of secondary MEF lines with direct infection. We infected Oct4-neo MEFs with Moloney-based viruses encoding the four reprogramming factors and counted neo-resistant colonies on day 20. Four independent experiments revealed a high degree of inter-experimental variability in iPS formation using this method (Figure 7B). In contrast, we found that doxycycline-treated NGFP2 MEFs in four independent experiments showed no significant difference in iPS formation. When counting Nanog-GFP positive colonies from MEFs, we noticed a much smaller degree of variability in the secondary lines.

[0044] To correlate the phenotypic behavior of the three secondary MEF populations with transgene induction, equal numbers of secondary MEFs were plated in the presence or absence of dox for 72 h, at which time the overall transcript levels of the four factors were determined by quantitative RT-PCR. Strikingly, both Nanog-GFP lines induced Oct4 at much lower levels than the NNeo line, which expressed Oct4 from a transgene at the collagen 1A1 locus at levels similar to ES cells (Figure 3A). Conversely, Sox2 induction in the Nanog-GFP line reached levels very close to those of endogenous Sox2 in ES cells, whereas NNeo expressed Sox2 at significantly lower levels in response to dox. c-Myc expression was higher in uninduced MEFs compared to ES cells, and the addition of dox produced a dramatic induction of transcript levels in all three secondary MEF lines. In contrast, overall Klf4 levels were similar to those of ES cells in all three secondary MEF populations after transgene induction. The observation that the overall Oct4 levels in dox-treated NNeo secondary MEFs were most similar to ES cells may explain the faster and more efficient reprogramming kinetics observed in this lineage (see above). We then determined expression levels at later stages of reprogramming in NGFP2 MEFs. Sox2, Klf4, and c-Myc were always robustly induced with very little variation, whereas Oct4 expression increased slowly over time (Figure 7A). This may reflect the selection of cells with higher Oct4 induction over time in culture. Southern blot analysis showed genomic integration of 1-2c-Myc, 1-3 Oct4, 1-3 Sox2, and 3-4 Klf4 proviruses in the three lines studied (Figure 7C).

[0045] Despite their genetic uniformity, dox induction resulted in variable transgene activation at the single cell level, as determined by immunofluorescence analysis of Oct4 and Sox2 (Figure 3B). Because not all secondary MEFs induced the transgene equally in response to dox, we cannot exclude the possibility that a specific stoichiometry of transgene expression is required for reprogramming and occurs in only a subset of secondary MEPs.

[0046] Effect of transgene expression on efficiency and timing of reprogramming To study how long expression of the four reprogramming factors was required for stable reprogramming to occur, secondary NGFP2 MEFs were plated at optimal density (see above), exposed to doxycycline for various times ranging from 5 to 22 days, and monitored daily for GFP fluorescence. The minimum length of dox exposure that resulted in GFP+ colonies was 9 days, with the first GFP+ colonies appearing 7 days after dox removal on day 16 (Figure 3C). Remarkably, further exposure to dox did not accelerate the appearance of GFP+ colonies, with GFP appearing between days 16 and 18, regardless of the length of dox administration. Similarly, NNeo secondary MEFs were found to require 11 to 13 days of dox exposure, after which stable neomycin-resistant secondary iPS colonies could be established.

[0047] To correlate the duration of transgene expression with overall reprogramming efficiency, we exposed secondary NGFP2 MEFs to toxocycline for 10–15 days and quantified GFP-positive colonies on day 34. We found a surprising correlation between the length of transgene expression and the number of GFP-positive colonies. 14(Fig. 3D). We then monitored the appearance of newly developing GFP-positive colonies over time in the same dishes. Strikingly, MEFs exposed to doxycycline for only 9 days continued to generate GFP-positive colonies until day 25 (15 days after dox cessation) (Fig. 3E, blue line). Dox treatment for 22 days produced a much more pronounced increase in GFP-positive colony formation over time (Fig. 3E, red line). These findings are consistent with reprogramming being a stepwise stochastic process even in this genetically homogenous system, and are in agreement with previous conclusions based on primary infection. 11、13、14、20 Moreover, this reprogramming process continues and can be completed long after the four transgenes are downregulated in response to dox withdrawal.

[0048] We also tested whether secondary cells could be used to evaluate the effect of drugs on the efficiency of reprogramming. To this end, we explored the effects of the DNA dimethylation compound 5-aza-deoxycytidine (5-aza) and the histone deacetylase inhibitor trichostatin A (TSA). Due to their action on chromatin modifications, both small molecules are candidates for improving 5-aza programming efficacy. Figure 3F shows that the addition of 5-aza to the medium increased the reprogramming efficiency of MEFs from the NGFP3 lineage, whereas TSA treatment had no obvious effect on the number of colonies.

[0049] Reprogramming other cell types We sought to determine what range of tissue types were susceptible to reprogramming by isolating secondary cells from iPS cell chimeras generated from NNeo and NGFP2 lines, and tested the reprogramming capacity of multiple cell types derived from these chimeras. As summarized in Table 1, certain cell types could be readily reprogrammed when isolated from the NGFP2 line, whereas the same cell types isolated from the NNeo line did not give rise to iPS cells. This suggests that different cell types require different levels of transgene induction, which may result from different proviral integration sites between the lineages studied.

[0050] [Table 1] intestinal epithelial cells Purified intestinal epithelial cells from both secondary NGFP2 and NNeo chimeras responded remarkably rapidly to doxycycline treatment, forming spheroids in suspension within 48 h, which subsequently attached to a MEF feeder layer and adopted ES-like morphology within 3–4 days (Figures 4A–4C and 4I–4J). However, alkaline phosphatase activity was not detectable prior to 10–12 days of culture with dox (Figure 9F). Using a mechanical fractionation protocol (see Methods), we found that these colonies formed much more efficiently from fraction 7 (mostly crypt-derived cells) than from earlier fractions (viral end-derived cells) (Figure 4M). Cells derived from NGFP2 chimeras developed into dox-independent iPS cells that expressed endogenous Nanog after approximately 2 weeks of culture in the presence of dox (Figures 4K–4L, Figure 8B).

[0051] In contrast, cells derived from NNeo chimeras became neo-resistant after 2 weeks of dox culture, but were unstable and lost their ES-like morphology upon dox withdrawal (Figures 4D-4F). Bisulfite sequencing revealed some demethylation of the Nanog promoter, only minimal demethylation of the Oct4 promoter (Figure 4G), and when injected subcutaneously into SCID mice, these cells were unable to generate teratomas in the presence or absence of doxycycline. Quantitative RT-PCR showed that these cells failed to induce Nanog and expressed only very low levels of Sox2 and Klf4 and high levels of Oct4 and c-Myc (Figure 4H). Further infection with Sox2 and Klf4 led to the generation of fully reprogrammed dox-independent iPS cells that expressed pluripotency markers and showed complete demethylation of their Oct4 and Nanog promoters (Figures 4G and 9G).

[0052] Comparison of transgene induction levels in NGFP2 and NNeo intestinal epithelial cells 48 h after dox treatment revealed differences in induction levels similar to those observed in secondary MEFs from these lines (Figure 4N, compare with Figure 3A). Intestinal epithelial cells derived from crypts induced more transgenes more readily than cells from villi, providing an explanation for their increased colony formation rate. These findings indicate that proviral integration sites in the NNeo lineage, while permissive for reprogramming of MEFs, are not capable of mediating complete reprogramming in intestinal epithelial cells, in contrast to those present in NGFP2.

[0053] Mesenchymal stem cells and tail tip fibroblasts Next, we compared the reprogramming capacity of bone marrow-derived mesenchymal stem cells (MSCs) and tail tip fibroblasts (TTFs) isolated from NNeo and NGFP2 chimeras. These cells represent two mesenchymal populations that are susceptible to reprogramming by direct infection. 1、4、12(Supplementary Figure 8D). Similar to intestinal cells, secondary NGFP2 MSCs and TTFs were able to generate iPS cells in response to dox, whereas those derived from NNeo chimeras were not (Figures 5A-5F, Figures 8A, 8C).

[0054] Keratinocytes Cells isolated from the epithelium of NNeo chimeras were first grown in the absence of doxycycline in growth conditions optimized for keratinocytes. 21 Homogeneous epithelial cultures were obtained (Figure 5G) and doxycycline was added to the medium. Clusters of epithelial cells proliferated and changed their morphology over time. After 12 days, the medium was changed to doxycycline-containing ES cell medium (Figure 5H), and 7 days later neomycin was added. The neo-resistant cells growing in tight colonies resembled ES cells (Figure 5I) and were passaged onto γ-irradiated feeder cells, at which point the cultures were maintained in the absence of dox and expressed endogenous Nanog (Figure 9D).

[0055] Neural progenitor cells Brains from NNeo chimeras were dissected, and tissue blocks around the lateral ventricles were dissected into single cells and plated on uncoated culture dishes in EGF- and FGF2-containing serum-free medium (N3EF) in the presence of puromycin to select for secondary cells. After 4 weeks, neurospheres formed, which were subsequently plated on polyornithine / laminin-coated dishes in either ES cell medium or N3EF medium containing dox to activate the lentiviral transgene. As expected for neural progenitor cells, cells exposed to serum-containing ES cell medium differentiated into flat astrocytes and stopped dividing (Figure 5J). In contrast, cells plated in N3EF medium continued to grow robustly and resembled undifferentiated neuroepithelial cells. After 3 weeks, these proliferating cells were split and plated in either ES cell medium or N3EF medium containing doxycycline. Cells exposed to serum adopted a largely flat morphology, whereas in N3EF cells maintained a bipolar morphology. However, in contrast to previous passages, small ES-like colonies emerged in both conditions over the next 2 weeks (Figure 5K, 5L). Upon passage onto γ-irradiated feeder MEFs, neo-resistant, dox-independent iPS cell lines expressing endogenous Nanog were readily established (Figure S9E).

[0056] Other organizations In addition, we also successfully generated secondary iPS cell lines from cells expanded from the adrenal gland, kidney, and muscle of NNeo chimeras. These tissues were dissected, dissociated with trypsin, and plated in ES cell medium containing doxycycline. After 6-12 days in the presence of dox, colonies with ES cell morphology appeared, which eventually became neomycin-resistant, dox-independent, and activated Nanog (Figures 9A-9C).

[0057] Reprogramming somatic cell epigenomes to a pluripotent, embryonic state through ectopic expression of four transcription factors, Klf4, Sox2, c-Myc, and Oct4, is a slow and inefficient process. The current method for inducing reprogramming is through retroviral gene delivery, which results in heterogeneous cell populations with proviral integrations that vary in both number and genomic location, providing an explanation for the variability and inefficiency of direct reprogramming. Here, we describe a novel system for reprogramming genetically homogeneous cell populations. Reprogramming with doxycycline-inducible lentiviral vectors and subsequent chimera formation results in tissues composed of genetically homogeneous cells that have identical proviral integrations and re-express reprogramming factors upon exposure to doxycycline. This strategy selects for cells with the exact number of proviruses inserted at genomic loci that favor drug-induced activation, eliminating the heterogeneity inherent in de novo viral infection of target cells. Surprisingly, the timing of reprogramming in this system was similar to that in directly infected primary fibroblasts. The minimum time that dox was required to initiate reprogramming was 9-13 days. This time scale is consistent with the 10-14 day time frame observed in cells directly infected with vector. 13、14 We also observed that when dox was withdrawn from the cultures as early as day 9, GFP+ secondary iPS colonies continued to emerge over the next several weeks in the absence of doxycycline. These results support the notion that reprogramming is driven by a stochastic sequence of epigenetic modifications that requires a minimal period of transgene expression.

[0058] The observed reprogramming efficiency of secondary MEFs was as high as 4%, which is comparable to that of mature B cells. 22 , much higher than the 0.1% efficiency estimated using de novo infection and drug selection, and approximately 8-fold higher than that reported using morphological selection criteria. 1、11、12It is well documented that iPS cells derived from infected MEFs have, on average, 15 different proviral copies, suggesting a strong selection for a small fraction of infected cells that carry the "correct" number of proviruses or express the four factors in the proper stoichiometry for successful reprogramming. Thus, the reprogramming frequency of secondary MEFs is predicted to be higher, since these cells were clonally derived from infected cells carrying the "correct" combination of proviruses. If this is the case, why do 4% of dox-treated secondary cells give rise to secondary iPS cells and not most or all of these cells? We consider several non-reciprocal exclusive explanations: (i) It is well established that genetically identical subclones of directly infected MEFs are reprogrammed significantly different times or not at all. 11、20 As previously discussed, this suggests that reprogramming involves a sequence of stochastic events, such that cells with the same number of proviral copies activate endogenous pluripotency genes different times. (ii) Our data also show that dox treatment does not uniformly activate the provirus in all cells, but rather, there are differences in induction levels between individual cells. Because of these diversified expression levels, only a fraction of secondary MEFs can achieve sufficiently high expression levels of factors, or the correct relative expression levels between factors, and therefore are capable of generating secondary iPS cells.

[0059] While reprogramming is induced by viral delivery of the four factors, maintenance of the pluripotent state depends on reconstitution of an autoregulatory loop involving activation of four endogenous pluripotency factors, Oct4, Nanog, Sox2, and Tcf3, and silencing of exogenous factors. 20、23 Similarly, secondary MEFs could be fully reprogrammed to a pluripotent state that was maintained in the absence of transgene expression.

[0060] We also utilized the secondary system to test the reprogramming capacity of several additional adult somatic cell types. iPS cells could be derived from many tissues, including brain, epithelium, intestinal epithelium, mesenchymal stem cells, tail tip fibroblasts, kidney, muscle, and adrenal gland, through dox treatment, indicating that the provirus was appropriately activated in cell types other than MEFs. This demonstrates that the four reprogramming factors can mediate epigenetic reprogramming in cells with different developmental origins and epigenetic states, and highlights the utility of the secondary system for studying reprogramming in a wide range of cell types. Although special care was taken to avoid other contaminating cell types, we cannot categorically demonstrate the cell of origin of iPS cells from these various cell types. Genetic lineage tracing experiments have indeed demonstrated that iPS cells can be derived from liver and pancreas after transduction with Oct4, Sox2, c-Myc, and Klf4. 24、25 However, not all cell types are permissive for reprogramming with these four factors. We have shown that reprogramming of mature B cells, but not immature B cells, required transduction of additional factors (c / EBPα) or inhibition of the B cell-specific transcription factor Pax5. 22 It is possible that additional and as yet unknown factors are required to reprogram specific cell types. One practical advantage of the system described herein is that cell types such as intestinal epithelial cells can be studied, including those that may be refractory to ex vivo culture and retroviral infection.

[0061] The drug-inducible system described herein presents a novel reprogramming platform with predictable and highly reproducible kinetics that should facilitate the study of early molecular events leading to epigenetic reprogramming (see Supplementary Figure 7B). In addition, the genetic homogeneity of the secondary cell type offers the feasibility of chemical and genetic screening approaches to enhance reprogramming efficiency. As one example, we demonstrate that the DNA demethylating agent 5-aza-deoxycytidine substantially enhances reprogramming efficiency. Moreover, such screens can also be applied to identify compounds that replace the original reprogramming factors. Since the reprogrammed state is independent of exogenous factors, transgenes can be genetically excised and secondary cells can be generated by chimera formation lacking specific reprogramming factors. 15 . EXAMPLES

[0062] The teachings of all references cited herein are incorporated herein by reference in their entirety.

[0063] Example 1 Virus preparation and infection Constructs for lentiviral vectors containing Klf4, Sox2, Oct4, and c-Myc under the control of a tetracycline operator and a minimal CMV promoter have been described previously. 14 Replication-incompetent lentiviral particles were packaged in 293T cells with a VSV-G coat, as well as either a neomycin resistance or GFP allele targeted to the endogenous Nanog locus. 1、11 , R26 locus 17The four viruses were used to infect MEFs containing the M2rtTA and PGK-Puro resistance genes in vitro. Prior to application to MEFs, viral supernatants from cultures packaging each of the four viruses were pooled, filtered through a 0.45 μM filter, and mixed 1:1 with ES cell medium (DMEM supplemented with w / 10% FBS (Hyclone, Logan, UT), leukemia inhibitory factor, β-mercaptoethanol (SIGMA-Aldrich), penicillin / streptomycin, L-glutamine, and non-essential amino acids (all from Invitrogen, Carlsbad, CA)).

[0064] Primary iPS isolation, teratoma, and chimera formation Approximately 3 weeks after addition of dox (Sigma-Aldrich St. Louis MO. 2 μg / mL), GFP+ or neomycin-resistant iPS colonies were isolated and expanded in the absence of dox. The NanogGFP2 iPS line was cloned from the NanogGFP1 line ( 22 The NanogGFP3 line was derived from an independent experiment, whereas the iPS line was picked from the same plate as the MEF-iPS#1 line described in , whereas the NanogGFP3 line was derived from an independent experiment. The iPS line was injected into C57 / B6×DBA / 1 F1 blastocysts. The blastocysts were placed in a drop of DMEM with 15% FBS under mineral oil. A flat-tipped microinjection pipette with an inner diameter of 12-15 mm was used for iPS cell injection using a piezo micromanipulator. Approximately 10 iPS cells were injected into the blastocyst cavity, and the blastocysts were placed in KSOM (Specialty Media, Phillipsburg, NJ) and incubated at 37°C until they were transferred to recipient females. 15 injected blastocysts were transferred to the uterine horns of pseudopregnant C57 / B6×DBA / 1 F1 females 2.5 days after coitus. For teratoma generation, 2×10 6 Cells were injected subcutaneously into the flank of recipient SCID mice and tumors were isolated for histological analysis 3–6 weeks later. All animals were handled in accordance with institutional IACUC guidelines.

[0065] Isolation and culture of secondary somatic cells For MEF isolation, chimeric embryos were isolated at E13.5 and the head and internal (including reproductive) organs were removed. Remaining tissues were physically dissected and incubated in trypsin for 20 min at 37 °C, after which the cells were resuspended in MEF medium containing puromycin (2 μg / mL) and expanded for two passages and then frozen. Secondary MEFs used for the described experiments were thawed and plated 1-2 passages after thawing. Kinetics experiments (Figure 2) were performed using 4 × 10 cells per well in 6-well plates. 4 iPS cells were stained or analyzed for the indicated times. Cell density experiments were performed in 12-well plates and GFP+ iPS colonies were scored 4 weeks after dox induction. Single cell efficiency experiments were performed by plating single secondary MEFs on a layer of wild-type feeder MEFs in 96-well plates prior to dox induction (limiting dilution was used, which was confirmed by visual inspection in a replicate plate lacking feeder MEFs). iPS information was scored after 4 weeks. A representative experiment from 2-3 biological replicates is shown. For 5-Aza and TSA experiments, 1 × 10 6 Secondary MEF cells were plated in 6-well plates (approximately 100 cells / mm 2 ), 5-Aza (1 μM) or TSA (1 μM) for 48 h. After 48 h, secondary MEFs were cultured in ES cell medium lacking 5-Aza or TSA plus dox. MEFs were exposed to 5-Aza or TSA for a second 48 h period between days 8–10 after induction, followed by culture with dox alone until scoring on day 21 for GFP+ colonies.

[0066] Somatic organelles were isolated from 3- to 4-month-old chimeras. Epidermal keratinocytes were isolated and cultured as previously described. 21、26 Neural progenitor cells were isolated and cultured as previously described. 27Whole intestinal epithelium was dissociated using a solution of 3 mM EDTA and 0.05 mM DTT in PBS for 30 min at room temperature. Muscle tissue was discarded and purified crypts / villi were plated on gamma-irradiated feeder MEFs in the presence of dox. For the crypt-villus fraction, the same EDTA-DTT was used, but fractions were collected by gentle agitation for 10, 6, 5, 9, 10, and 25 min (corresponding to fractions 1–7, respectively, with 1 representing the villus tip and 7 representing the crypt). 28 After incubation as described in, 0.8 x 10 6 Epithelial cells were plated on MEF feeder layers in ES medium containing 2 μg / mL dox. No proliferation was observed in cultures lacking dox. Whole bone marrow was cultured from secondary chimeric mice (or Coll1-TetO-Oct4, Rosa26-M2rtTA mice for direct infection). 16 Mesenchymal stem cells were isolated from femurs and tibias following removal of the condyles in the growth plate (from the femoral bone marrow) by flushing with a syringe and 30-gauge needle containing DMEM + 5% fetal bovine serum (FBS) (Hyclone, Thermo Fisher Scientific). Mesenchymal stem cells were selected through differential plating on tissue culture plates for 72 h in α-MEM supplemented with 15% FBS (HyClone). Colony formation of MSCs in culture was determined by the addition of 4 × 10 cells from freshly isolated whole bone marrow. 6 This was performed by plating nucleated cells onto 10 cm plates and expanding them in the presence of puromycin for 5 days to eliminate host blastocyst-derived cells, after which dox was introduced to induce reprogramming. Cultures derived from adrenal gland, muscle, and kidney were dissected, mechanically dissociated, and digested in trypsin for 20 min at 37°C, then plated onto gelatin-coated culture dishes containing ES medium with dox.

[0067] antibody For flow cytometry analysis, we used APC-conjugated anti-mouse SSEA1 (R&D systems, Minneapolis, MN) and alkaline phosphatase substrate kit: Vector Red substrate kit (Vector Laboratories, Burlingame, CA). For immunofluorescence, cells were fixed in 4% paraformaldehyde, and we used mouse monoclonal antibodies against SSEA1 (Developmental Studies Hybridoma Bank), goat anti-Sox2 (R&D Systems), mouse anti-Oct4 (Santa Cruz), and rabbit anti-Nanog (Bethyl). Appropriate fluorophore-labeled secondary antibodies were purchased from Jackson ImmunoResearch.

[0068] Flow cytometry Cells were trypsinized, washed once in PBS, and resuspended in FACS buffer (PBS + 5% fetal bovine serum). 6 Cells were stained with 10 μl of APC-conjugated anti-SSEA1 antibody in a volume of 100 μl for 30 minutes, then cells were washed twice with PBS, cells were then washed once with wash buffer, and resuspended in FACS buffer for analysis on a FACS-calibur cell sorter.

[0069] Bisulfite sequencing and Southern blotting Bisulfite treatment of DNA was performed using the CpGenome DNA modification kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. The resulting modified DNA was amplified by nested polymerase chain reaction (PCR) using the following two forward (F) and one reverse (R) primers:

[0070] [ka] The PCR products were amplified using the 5'- ...

[0071] Quantitative RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). Five micrograms of total RNA was treated with DNase I using a DNA Free RNA kit (Zymo Research, Orange, CA) to remove potential genomic DNA contamination. One microgram of DNase I-treated RNA was reverse transcribed using a First Strand Synthesis kit (Invitrogen) and finally resuspended in 100 μl water. qPCR was performed on an ABI Prism 7000 (Applied Biosystems, Foster City, CA) using Platinum SYBR green qPCR SuperMix-UDG with ROX (Invitrogen). Quantitative PCR analysis was performed in triplicate using a 1 / 50 reverse transcription reaction at RT (Central Plant, Calif.). The primers used for amplification were as follows:

[0072] [ka] To ensure equal loading of cDNA into the RT reaction, GAPDH mRNA was amplified using:

[0073] [ka] Data were extracted from the linear portion of the amplification. qRT-PCR data in all graphs shown represent RNA samples that were DNase treated, reverse transcribed, and amplified in parallel to avoid the inherent variability in these procedures. Error bars represent the standard deviation of the mean of triplicate reactions.

[0074]

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[0077] 1. DOX and Tamoxifen-inducible Retroviral Vectors DOX-inducible retroviral vectors were important for defining the minimum time of vector expression during sequential activation of pluripotency markers and reprogramming of mouse somatic cells. We generated inducible lentiviral vectors that allow for the temporally restricted expression of reprogramming factors.

[0078] (a) DOX-inducible lentiviral vectors: Following the same strategy used for mouse genes, we generated lentiviral vectors transducing human OCT4, SOX2, KLF4, and C-MYC c-DNA either constitutively or under the control of a DOX-inducible promoter [Brambrink, 2008 #6877]. To generate a DOX-inducible system, we infected human fibroblasts with lentiviral vectors carrying the rtTA transactivator. Figure 11A shows high DOX-dependent expression of OCT4, SOX2, and KLF4 in fibroblasts transduced with the respective DOX-inducible vectors. Similarly, robust DOX-dependent transgene expression was observed in iPS cells derived from infected fibroblasts (right two panels of Figure 11A).

[0079] (b) Tamoxifen-inducible lentiviral vectors: To allow independent inducible control of the vectors, we also generated OCT4, SOX2, and C-MYC estrogen receptor (ER) fusion constructs by fusing the factors to an estrogen ligand binding domain to allow tamoxifen-dependent expression [Grandori, 1996 #6505]. As shown in Figure 11B, addition of tamoxifen to cells transduced with the SOX2-ER fusion construct results in the translocation of SOX2 protein from the cytoplasm to the nucleus, as predicted for drug-induced activation. These results indicate that the DOX and ER fusion inducible systems can be used to independently control the expression of transduced factors.

[0080] One key concept is the use of two different regulatable systems, each controlling the expression of a subset of functions. For example, one may place three factors under the control of a first inducible (e.g., dox-inducible) promoter, and a fourth factor under the control of a second inducible (e.g., tamoxifen-inducible) promoter. One can then generate iPS cells by inducing expression from both promoters, generate mice from the iPS cells, and isolate fibroblasts (or any other cell type) from the mice. These fibroblasts are genetically homogenous and can be reprogrammed without the need for viral infection. One then attempts to reprogram fibroblasts under conditions where only the first promoter is active, in the presence of a different small molecule that can potentially be replaced by the fourth factor, to identify small molecule "reprogramming factors" or to optimize transient transfection or other protocols to introduce the fourth factor. Numerous variations are possible; for example, one may stably induce expression of the three factors and transiently induce expression of the fourth factor, etc. Also, the expression level of the factor can be modulated by using different concentrations of the inducing agent.

[0081] Another approach is to place a gene encoding one of the factors between the sites for the recombinase and then induce expression of the recombinase to turn off expression of that factor. Recombinase expression can be induced by infection with a viral vector (e.g., adenovirus-Cre). Hanna et al., Science, 318, 1920-1923 (2007) describe such an approach, which was used to reduce the potential risk of tumor formation due to c-Myc transgene expression--cells were infected with retroviruses encoding the Oct4, Sox2, and Klf4 factors, and lentiviruses encoding the 2-lox c-Myc cDNA. iPS cells generated from these cells were infected with an adenovirus encoding the Cre recombinase to delete the lentivirus-transduced c-Myc copy.

[0082] These systems are useful, for example, to identify reprogramming factors and to study the requirements and events that occur in reprogramming, including discovering cell type-specific differences.

[0083] 2. Confirm that the inducible system works as predicted in humans as it does in mice Generation of human iPS cells A number of different strategies have been described to derive iPS cells from mouse or human somatic donor cells, including constitutive or inducible expression of the four transcription factors Oct4, Sox2, Klf4, and c-myc or a subset of these four factors, or alternative factor combinations [Lowry, 2008 #6827; Park, 2008 #6783; Takahashi, 2007 #6769; Yu, 2007 #6793]. The utility of the different vector systems described in Figure 11A for reprogramming human fibroblasts was compared. Table 1 shows that iPS cells were obtained with four or three (without c-MYC) constitutively expressed or DOX-inducible transcription factors. When DOX-inducible lentivirus was used, iPS clones appeared at similar frequencies and at the same time in infected cultures as published by other researchers [Takahashi, 2007 #6769]. Figure 12A shows that endogenous OCT4 and NANOG genes were expressed in the two iPS lines at levels similar to huES cells. The reprogrammed iPS cells grew as tightly packed colonies with morphology typical of human ES cells, and they expressed appropriate pluripotency markers (Figure 12B). To test pluripotency, the iPS cells were injected into SCID mice. Histological examination of the resulting tumors showed typical teratomas containing multiple differentiated cell types (Figure 12C).

[0084] B. Generation of Mouse and Human iPS Cells with Polycistronic Retroviral Vectors Many current protocols for generating iPS cells require the transduction of four transcription factors Oct4, Sox2, c-myc, and Klf4 by four different retroviral vectors. Reprogramming in this manner involves the selection of a small fraction of infected cells with multiple integrated vectors (up to 15 or more proviruses), raising concerns about cancer due to the use of strong oncogenes and / or retrovirus-induced insertional mutagenesis. To reduce the number of independent proviral integrations required for reprogramming, we have designed and used polycistronic vectors that can transduce any combination of factors with the goal of reducing the number of proviral integrations.

[0085] Internal ribosome entry sites (IRES) are widely used to express multiple genes from one promoter, however, this often results in non-stoichiometric expression of the genes. This self-cleaving 18-22 amino acid long 2A peptide mediates "ribosome skipping" between proline and glycine residues, inhibiting peptide bond formation without affecting downstream translation. These peptides allow multiple proteins to be encoded as polyproteins, which upon translation dissociate into component proteins. Use of the term "self-cleavage" is not intended to imply a proteolytic cleavage reaction.

[0086] Self-cleaving peptides are found in members of the Picornaviridae family of viruses, including aphthoviruses, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1) (Donnelly, ML et al., J. Gen. Virol., 82, 1027-101 (2001); Ryan, MD et al., J. Gen. Virol., 72, 2727-2732 (2001)), and cardioviruses, such as Theiloviruses (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses. 2 The A peptides are sometimes referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Aphthovirus 2A polypeptides are typically approximately 18-22 amino acids long and contain Dx1Ex2NPG (SEQ ID NO: 34), where x1 is often valine or isoleucine. As noted above, the 2A sequence is believed to mediate "ribosomal skipping" between proline and glycine, impairing normal peptide formation between P and G without affecting downstream translation. An exemplary 2A sequence is from FMDV:

[0087] [ka] where the underlined residues are conserved in many 2A peptides. The C-terminus of the cardiovirus 2A peptide is conserved and shows a high degree of similarity to the FMDV 2A peptide, which has also been shown to mediate autocleavage (Donnelly, ML, et al., J. Gen. Virol., 78, 13-21 (1997)). The FDMV 2A peptide has been shown to mediate cleavage of an artificial polyprotein (Ryan, MD and Drew, J., EMBO J., 13, 928-933 (1994)). The ability to efficiently and stoichiometrically express four proteins from one polycistron in vivo was recently demonstrated using a self-processing 2A peptide expressing four CD3 proteins (Szymczak et al., Nature Biotech. 5, 589-594, 2004). Polycistronic transgenes in which individual cDNAs are separated by the 2A peptide have been shown to promote polycistronic gene expression in transfected cells containing huES (Hasegawa, K., et al., Stem Cells. 2007). Jul;25(7):1707-12, 2007).

[0088] The present invention provides polycistronic nucleic acid constructs, expression cassettes, and vectors that are useful for generating induced pluripotent stem (iPS) cells. In certain embodiments, the polycistronic nucleic acid constructs include a portion that encodes a self-cleaving peptide. The present invention provides polycistronic nucleic acid constructs that include at least two coding regions, where the coding regions are each linked by a nucleic acid that encodes a self-cleaving peptide to form a single open reading frame, and the coding regions encode a first and a second reprogramming factor that can reprogram a mammalian somatic cell to pluripotency, either alone or in combination with one or more additional reprogramming factors. In some embodiments of the present invention, the constructs include two coding regions separated by a self-cleaving peptide. In some embodiments of the present invention, the constructs include three coding regions, each of which encodes a reprogramming factor, where adjacent coding regions are separated by a self-cleaving peptide. In some embodiments of the present invention, the constructs include four coding regions, each of which encodes a reprogramming factor, where adjacent coding regions are separated by a self-cleaving peptide. Thus, the present invention provides a construct encoding a polyprotein comprising two, three, or four reprogramming factors separated by a self-cleaving peptide. In an embodiment, the construct comprises an expression control element, e.g., a promoter, suitable for directing expression in a mammalian cell, where the portion of the construct encoding the polyprotein is operably linked to the expression control element. Thus, the present invention provides an expression cassette comprising a nucleic acid encoding a polyprotein alpha comprising reprogramming factors, where each reprogramming factor is linked to at least one other reprogramming factor by a self-cleaving peptide and operably linked to a promoter (or other suitable expression control element). The promoter drives transcription of a polycistronic message encoding reprogramming factors, where each programming factor is linked to at least one other programming factor by a self-cleaving peptide.The promoter may be a viral promoter (e.g., a CMV promoter) or a mammalian promoter (e.g., a PGK promoter). The expression cassette or construct may contain other genetic elements, for example, to enhance expression or stability of the transcript. In certain embodiments of the invention, any of the aforementioned constructs or expression cassettes may further comprise a coding region that does not encode a reprogramming factor, where the coding region is separated from adjacent coding regions by a self-cleaving peptide. In certain embodiments, the additional coding region encodes a selection marker.

[0089] Specific reprogramming factors that may be encoded by the polycistronic construct include transcription factors Oct4, Sox2, Klf4, c-Myc, and Nanog, which are further described herein and known in the art. The present invention encompasses all combinations of two or more of the aforementioned factors, each in a possible order. For the sake of brevity, not all of these combinations are individually listed herein. In some embodiments, the construct encodes Oct4, Klf4, and Sox2 separated by 2A peptide. In some embodiments, the construct does not encode c-Myc. In some embodiments, the construct comprises a coding region encoding Lin28. In some embodiments, the construct comprises a coding region encoding C / EBPα.

[0090] In some embodiments, the construct comprises one or more sites that mediate or facilitate integration of the construct into the genome of a mammalian cell. In some embodiments, the construct comprises one or more sites that mediate or facilitate targeting of the construct to a selected locus in the genome of a mammalian cell. For example, the construct can comprise one or more regions that are homologous to a selected locus in the genome.

[0091] In some embodiments, the construct contains sites for a recombinase that is functional in mammalian cells, where these sites are adjacent to at least a portion of the construct that contains the coding region for the factor (i.e., one site is located 5' and a second site is located 3' to the portion of the construct that encodes the polyprotein), so that the sequence encoding the factor can be excised from the genome after reprogramming. The recombinase can be, for example, Cre or Flp, where the corresponding recombinase sites are LoxP and Frt sites. In some embodiments, the recombinase is a transposase. It is understood that the recombinase sites need not be directly adjacent to the region encoding the polyprotein, but are positioned such that the region whose eventual removal from the genome is desired is located between the sites. In some embodiments, the recombinase sites are at the 5' and 3' ends of the expression cassette. The excision results in a residual copy of the recombinase remaining in the genome, which in some embodiments is the only genetic change resulting from the reprogramming process.

[0092] In certain embodiments, the construct contains a single recombinase site, which is copied during insertion of the construct into the genome, such that at least a portion of the construct encoding a polyprotein containing the elements (and optionally any other portions of the construct whose eventual removal from the genome is desired) is flanked by two recombinase sites after integration into the genome. For example, the recombinase site can be in the 3'LTR of a retroviral (e.g., lentiviral) vector (see, e.g., Example 4).

[0093] In one aspect, the present invention provides a vector comprising a polycistronic nucleic acid construct.In one embodiment, these vectors are retroviral vectors, for example, lentiviral vectors.In other embodiments, these vectors are non-retroviral vectors, for example, they can be viral (e.g., adenoviral) or non-viral.Exemplary polycistronic nucleic acid constructs, expression cassettes, and vectors are described in Example 3.

[0094] In some aspects, the invention provides cells or cell lines (e.g., somatic cells and cell lines, e.g., fibroblasts, keratinocytes, and other types of cells discussed herein) in which a polycistronic nucleic acid construct or expression cassette (e.g., any of the constructs or expression cassettes described herein) is integrated into the genome. In some embodiments, the cell is a rodent cell, e.g., a mouse cell. In some embodiments, the cell is a primate cell, e.g., a human cell.

[0095] In some embodiments, at least a portion of the construct encoding the polyprotein is flanked by sites for the recombinase. After the programmed cell is induced, the recombinase can be introduced into the cell, for example, by protein transduction, or a gene encoding the recombinase can be introduced into the cell, for example, using a vector, such as an adenoviral vector. The recombinase excises the sequence encoding the exogenous reprogramming factor from the genome. In some embodiments, the cell contains an inducible gene encoding the recombinase, where the recombinase is expressed upon induction and excises the cassette. In some embodiments, the inducible gene is integrated into the genome. In some embodiments, the inducible gene is on an episome. In some embodiments, the cell does not contain an inducible gene encoding the recombinase.

[0096] In some embodiments, the nucleic acid construct or cassette is targeted to a specific locus in the genome, for example, using homologous recombination. In some embodiments, the locus is dispensable for the normal development of most or all cell types in the mammalian body. In some embodiments, the locus does not affect the ability to derive pluripotent iPS cells from somatic cells that have an insertion at the locus. In some embodiments, the locus is one whose insertion does not disrupt the pluripotency of ES cells. In some embodiments, the locus is the COL1A1 locus or an AAV integration locus. In some embodiments, the locus comprises a constitutive promoter. In some embodiments, the construct or cassette is targeted such that expression of a polycistronic message encoding a polypeptide comprising a factor is driven from an endogenous promoter present at the locus to which the construct or cassette is targeted.

[0097] The present invention further provides pluripotent reprogrammed cells (iPS cells) generated from somatic cells carrying the nucleic acid construct or expression cassette in their genome. The iPS cells can be used for any purpose intended for pluripotent cells. Also provided are differentiated cell lineages (e.g., neural cells, hematopoietic cells, muscle cells, cardiac cells) derived from the pluripotent reprogrammed cells. Exemplary somatic cells and iPS cells generated therefrom are described in Example 3.

[0098] The present invention established that reprogramming factors have essential structural requirements to allow efficient processing of the 2A sequence when placed between reprogramming factors. This was an important discovery because cleavage was recognized to be a structure-based event (Szymczak, supra). The present disclosure establishes that transcription factors with approximately 17-21 more amino acids from the 2A peptide at their C-terminus retain the ability to enter the nucleus and perform their function. The present disclosure also establishes that reprogramming factors can tolerate the presence of approximately 17-21 more amino acids from the 2A peptide remaining at the C-terminus of an upstream protein and remain functional in reprogramming.

[0099] Although reprogramming by infection with high titer retroviral vectors to express the required reprogramming factors is highly reproducible, the process is relatively inefficient and the exact requirements regarding the timing and order of expression of the factors, as well as the absolute and relative levels of expression required, have remained incompletely understood. Furthermore, when iPS cells are generated in many current protocols by infecting cells with multiple viruses, each encoding a single factor, each virus has been shown to integrate between 2-6 positions, resulting in approximately 14-20 insertion events across the genome. This process creates genetically modified iPS cells, potentially including mutations resulting from unknown insertions. Furthermore, since only a small fraction of infected cells are reprogrammed, results obtained using these multiple virus protocols leave open the question as to whether the location of integration at which expression from the transgene occurs and / or the timing at which it occurs are important determinants of whether a cell is reprogrammed or not. The present invention establishes that essentially simultaneous expression of multiple factors, from polycistronic transcripts and with relative levels dependent on the efficacy of the 2A cleavage event, is effective to induce reprogramming. Moreover, the present invention establishes that a single copy of a factor is sufficient for strand programming. Since the four factors are expressed from a predefined location (e.g., a preselected location or a location determined after vector integration) in certain embodiments of the present invention, the polycistronic vector system may simplify the study of reprogramming mechanisms and facilitate vector excision. In some embodiments, such excision results in the removal of at least one exogenous sequence that codes for a reprogramming factor. In some embodiments, such excision results in iPS cells that do not have genetic modifications, which modifications are, in some embodiments, other than the remaining recombinase. In other embodiments, there are no more than two, three, four, or five recombinase sites.Without wishing to be bound by theory, reprogramming cells containing a single integration construct increases the possibility or ease of recovering transgene-free iPS cells using recombinase-based approaches. It is also contemplated that polycistronic vectors encoding two, three, or four factors may be used in combination with small molecules, proteins, or other agents that enhance reprogramming and / or replace one or more factors not encoded by the polycistronic vector.

[0100] Example 4 describes an experiment in which human induced pluripotent stem cells (hiPSCs) without reprogramming factors are derived using Cre recombinase excisable virus derived from fibroblasts from individuals with Parkinson's disease (PD). In one embodiment of the present invention, iPS cells without exogenous genes encoding reprogramming factors are derived as described in Example 4 or using a similar method, with the exception that a single vector containing a polycistronic nucleic acid construct encoding a polyprotein containing multiple factors (2, 3, or 4 factors) is used, rather than multiple vectors encoding a single factor. Of course, the method described in Example 4 can also be used with multiple vectors encoding individual vectors to obtain iPS cells without exogenous genes encoding reprogramming factors, where the resulting iPS cells have only a small number of remaining recombinase sites. Although fibroblasts from individuals with PD were used as an exemplary cell type in Example 4, these methods are applicable to derive iPS cells with minimal genetic alterations from normal somatic cells (e.g., fibroblasts or other cell types, e.g., keratinocytes, intestinal cells, blood cells) or from somatic cells from individuals with a disease of interest. In one embodiment, the gene encoding the transactivator is also flanked by recombinase sites so that it is similarly removed from the genome.

[0101] iPS cells and differentiated cells derived therefrom are useful for research purposes (e.g., as model systems for studying disease and / or identifying therapeutics for disease) and / or for the development of cell-based therapies, which in certain embodiments are patient-specific cell-based therapies.

[0102] C. Developmental Potential of Human iPS Cells and Their Derivation from Peripheral Blood An exciting potential of the iPS system is to induce patient-specific pluripotent cells. The work described herein describes a protocol that allows the study of complex human diseases in vitro using patient-specific iPS cells. For example, currently, patient-specific iPS cells are derived from deep skin biopsies. In an effort to establish a potentially simpler protocol for isolating iPS cells in a clinical setting, the procedure described herein uses peripheral blood as donor material to generate iPS cells.

[0103] D. Screens for Small Molecules The research described herein provides a high-throughput system for identifying small molecules that improve reprogramming efficiency. This allows the establishment of reprogramming methods that do not require genetic manipulation or the insertion of exogenous genetic elements, such as vector-mediated transduction of oncogenes such as C-MYC or KLF4.

[0104] II. Experimental Approach In the mouse system, the use of vectors that allow drug-inducible expression of transcription factors was crucial to define the molecular events that trigger reprogramming. These experiments showed that reprogramming involves sequential activation of ES cell markers such as alkaline phosphatase, SSEAl, Oct4, and Nanog, and that the transduced transcription factors needed to be expressed for at least 12 days to generate iPS cells [Brambrink, 2008 #6877]. The main goal of objective A is to generate tools that will aid in reprogramming somatic cells and allow genetic manipulation of human ES and iPS cells. These tools are important for objective B, which is focused on the mechanisms of human somatic cell reprogramming. The goal of objective C is to establish an experimental system to evaluate the potential of human iPS cells to differentiate into functional neural cells in vitro as well as in vivo in chimeric mice. In addition, we design a protocol to generate human iPS cells from human peripheral blood. Finally, the focus of Objective D is to screen for chemical compounds as alternatives to activating reprogramming pathways by genetic means.

[0105] A. Generation of Tools for Genetic Manipulation of Human ES and iPS Cells The ability to genetically alter endogenous genes by homologous recombination has revolutionized biology and, in combination with embryonic stem cells, holds great promise for molecular medicine. Although gene targeting is a routine procedure in mouse ES cells, transferring this technique to human embryonic stem cells has previously been difficult [Giudice, 2008 #6863]. Indeed, since the first isolation of human ES cells by Thomson ten years ago, only four publications have appeared reporting successful targeting of endogenous genes [Davis, 2008 #6860;Irion, 2007 #6857;Zwaka, 2003 #6223;Urbach, 2004 #6163]. The difficulties of genetically modifying endogenous genes need to be overcome to realize the full potential of human ES cells.

[0106] The focus of this study is to establish tools that allow efficient genetic manipulation of human ES and iPS cells. To generate huES cells with markers in lineage-specific genes, we used two different approaches: conventional homologous recombination was used to generate genetically modified human ES cells with markers in key developmental regulators. These markers inserted in lineage-specific genes are then used for the differentiation of iPS cells into specific neural cell lineages. An experimental system was also developed that allows efficient reprogramming of somatic cells in the absence of retrovirus-mediated factor transduction.

[0107] Lineage-specific gene targeting by homologous recombination The derivation of differentiated cells from undifferentiated ES cells is facilitated by markers inserted into lineage-specific endogenous genes that can be used to isolate the desired differentiated cell type.Our preliminary experiments have demonstrated the targeting of OCT4 and COL1A1 loci with GFP or drug resistance markers.The goal was therefore to generate ES and iPS cells with drug resistance markers and / or GFP (or other detectable markers) in genes that can be expressed in cells of neural or other lineages and used to screen or select differentiated cell types that suffer from diseases such as Alzheimer's and Parkinson's.

[0108] (i) Gene targeting of neural lineage-specific target genes by homologous recombination In contrast to mouse ES cells, human ES cells are usually passaged mechanically using only limited enzymatic digestion. This is because cell cloning selects for chromosomal abnormalities that enhance single-cell proliferation. This and the slow proliferation may be important reasons why gene targeting has been too inefficient in huES cells. Recently, it has been shown that application of the ROCK inhibitor Y-27632 to huES significantly reduces dissociation-induced apoptosis and increases cloning efficiency [Watanabe, 2007 #6549]. Therefore, all experiments are performed in the presence of this inhibitor.

[0109] For homologous recombination, targeting vectors containing GFP and neo resistance markers separated by 2A sequences are constructed from isogenic genomic DNA of BGO2 or H9 ES cells using routine procedures. DNA is electroporated according to published procedures [Costa, 2007 #6868], and DNA from drug-resistant colonies is isolated and analyzed for precise targeting. We target genes that are activated at different time points during neural differentiation and in different subsets of neurons as detailed below.

[0110] SOX1: The transcription factor SOX1 is the earliest known gene expressed exclusively in mouse neural progenitor cells [Aubert, 2003 #6841]. GFP inserted into this gene serves as a convenient marker for selecting neural progenitor cells derived from huES or iPS cells.

[0111] FOXG1: Expression of this gene has been demonstrated in proliferating telencephalic progenitor cells and in acetylcholinergic neurons of the basal forebrain, cells affected by Alzheimer's disease [Hebert, 2000 #6844].

[0112] PITX3: This homeodomain transcription factor is selectively expressed during terminal differentiation of tyrosine hydroxylase-positive neurons, and sorts of differentiated ES cells derived from PITX3-GFP transgenic mice have been shown to be enriched in dopaminergic neurons [Hedlund, 2008 #6845; Zhao, 2004 #6846].

[0113] LMX1: This homeodomain transcription factor appears to be a key determinant of dopaminergic progenitor cell proliferation [Andersson, 2006 #6840].

[0114] Marking of relevant lineage-specific genes with GFP has been shown to aid in establishing robust differentiation protocols that allow the isolation of enriched or even homogenous populations of differentiated cells. huES cells with GFP in four genes allow enrichment for progenitor cells as well as more differentiated cells, which is relevant for the study of iPS cells derived from patients with diseases such as Alzheimer's or Parkinson's.

[0115] The difficulty in establishing efficient methods of homologous recombination has greatly hindered the utility of huES cell lines. Preliminary data are promising, demonstrating that two endogenous loci, OCT4 and COL1A1, have been targeted using GFP and puromycin resistance cDNA (Figure 10). However, so far, only genes that are expressed in ES cells (OCT4, HPRT, ROSA26 [Irion, 2007 #6857; Zwaka, 2003 #6223; Urbach, 2004 #6163]) or that are easily expressed, such as MOXL1 [Davis, 2008 #6860], have been targeted in human ES cells. Also, the COL1A1 locus is highly recombinational in mouse cells [Beard, 2006 #6199], and targeting of this locus is not representative of other non-expressed genes. Thus, our intention was to target unexpressed genes by homologous recombination, an objective that poses challenges.

[0116] "Secondary" iPS cells with various combinations of reprogramming factors We have shown that mouse iPS cells may have 15 or more proviral insertions [Wernig, 2007 #6641], suggesting a strong selection for a small fraction of cells with multiple copies of each vector to achieve high levels of factor expression or a specific stoichiometry required for the initiation of the reprogramming process. Presented here is a system that circumvents the need for viral transduction and thus eliminates the need to select a small fraction of cells carrying the "right" proviral combination. Indeed, the generation of "secondary" fibroblasts clonally derived from "primary" iPS cells and carrying the appropriate number of DOX-inducible proviruses that achieved reprogramming in the first place allowed us to reprogram mature B cells to a pluripotent state [Hanna, 2008 #6842]. This approach applies to human cells and generated secondary fibroblasts carrying reprogramming factors either (i) as proviral vectors integrated into preselected chromosomal locations or (ii) inserted by homologous recombination into genomic expression loci. This system can be used to determine the mechanism of reprogramming and to screen small molecules that enhance reprogramming or replace either factor.

[0117] (i) Secondary fibroblasts with preselected provirus: A two-step protocol may be utilized to preselect cells with the "right" combination and number of retroviral copies. Figures 23A-23B outline this approach, which follows the same logic utilized to reprogram mouse B cells into iPS cells [Hanna, 2008 #6842]. First, ES or iPS cells carrying a GFP marker in the OCT4 gene, as well as a lentivirally transduced tet rtTA transactivator, are differentiated into fibroblasts. These "primary" fibroblasts are transduced with all four factors using a DOX-inducible vector, cultured in the presence of DOX, and screened for OCT4 activation to isolate reprogrammed "primary" iPS cells. These iPS cells are differentiated in the absence of DOX to generate "secondary" fibroblasts (Figure 23A). The rationale for this approach is that since the secondary fibroblasts were selected as "primary" iPS cells in the first step, they carry the "right" combination of vector copies. These secondary fibroblasts are genetically homogenous since they arise from a single iPS colony. Upon addition of DOX to such cultures, the integrated vector is reactivated, resulting in consistent generation of "secondary" iPS cells without the need for the introduction of new factors (Figure 23B). This can be used to generate human (or mouse, monkey, etc.) secondary iPS cells without going through the process of animal generation from primary iPS cells. Alternatively, DOX-inducible polycistronic vectors (Figures 13A-13C) can be used in place of single factor vectors for the generation of primary iPS cells. (ii) Secondary fibroblasts with reprogramming factors at the COL1A1 locus: In an effort to avoid all retroviral infection, secondary fibroblasts are produced that have all reprogramming factors at the COL1A1 locus or other non-essential loci, e.g., the ROSA26 or AAVS1 loci (specific loci that adeno-associated virus (AAV) integrates).In mouse ES cells, we have shown that the Col1a1 locus can be efficiently targeted, resulting in reproducible ubiquitous or inducible expression of inserted transgenes [Beard, 2006 #6199; Hochedlinger, 2005 #5758]. Dox-inducible rtTA transactivator and OCT4. In addition to the GFP reporter, reporter cells are constructed with a reporter polycistronic vector inserted into the COL1A locus that encodes all or a subset of the reprogramming factors under the control of the tet operator (Figure 24). In this figure, OCT4, SOX2, and cMYC are inserted into the COL1A1 locus. Primary fibroblasts are induced in vitro and infected with the KLF4 virus flanked by two Lox sites. Primary iPS cells are selected with the three factors induced by DOX as described above, the KLF4 virus is deleted by Cre transduction [Hanna, 2007 #6781], and vKLF4 is expressed in each of the secondary fibroblasts induced by in vitro differentiation. These cells can be screened for small molecules that replace the requirement for KLF4 in reprogramming (see below, Objective D) or for streamlining the transient transfection protocol (Objective B.2, 4).

[0118] Reprogramming selects for a small fraction of iPS cells with a higher number of proviral insertions. The experiments proposed in this objective seek to establish an experimental system that allows more efficient and reproducible reprogramming, since this process is independent of random proviral insertions that select for rare iPS cells. The aim is to generate secondary fibroblasts with any combination of 2 or 3 DOX-inducible factors, thus allowing to screen for small molecules that can enhance or induce reprogramming, replacing the missing factors for our purposes (Aim D). This system is also important for studying the molecular mechanisms of reprogramming (Aim B.4).

[0119] B. In vitro reprogramming of human somatic cells The DOX-inducible lentiviral system has been used to define the reprogramming kinetics of mouse fibroblasts. The work described herein uses the above tools to determine the kinetics and minimal vector expression for reprogramming of human somatic cells. In addition, we develop a method of reprogramming that minimizes or avoids genetic changes, and we use insertional mutagenesis to isolate additional genes that enhance reprogramming. Finally, we define the epigenetic state of iPS cells, as well as intermediate stages of reprogramming.

[0120] C. Developmental Potential and Derivation from Blood Donor Cells The most important application of patient-specific iPS cells is their potential use in studying complex human diseases in vitro. For this application, robust experimental approaches need to be established before this technology can be used in clinical settings. The work described herein establishes procedures that allow reproducible in vitro differentiation of iPS cells and huES cells, as well as evaluation of the in vivo potential of iPS cells. Isolation of iPS cells from human peripheral blood samples may also be performed.

[0121] 3. Donor B cells, T cells, and macrophages It is of interest to directly reprogram cells obtained from peripheral blood instead of from deep skin biopsies, because this would facilitate the generation of patient-specific iPS cells in a clinical setting. We have recently shown that immature and mature mouse B cells can be efficiently reprogrammed into pluripotent iPS cells, and that these cells have donor cell-specific gene rearrangements of the immunoglobulin locus [Hanna, 2008 #6842]. Surprisingly, the efficiency of reprogramming mature mouse B cells is 3%, which is substantially higher than that of adult fibroblasts in MEFs. The aim is to explore adapting the method used for reprogramming mouse lymphocytes to human peripheral blood samples.

[0122] Donor cells: Transduction with c / EBPa transactivator was required to render mature mouse B cells susceptible to the action of four reprogramming factors [Hanna, 2008 #6842]. We will isolate different cell populations from human peripheral blood and test their susceptibility to reprogramming.

[0123] (i) B and T cells: In an effort to adapt a protocol for mouse B cell reprogramming, we used an established procedure to stimulate proliferation of B and T cells [Mercier-Letondal, 2008 #6855] and infected the cells with vectors transducing the c / EBPa and rtTA transactivators. After several days of culture in cytokines, the cells were reprogrammed with DOX-induced The iPS cells were transduced with the sexual reprogramming factors OCT4, SOX2, C-MYC, and KLF4 and cultured in ES cell medium. Reprogrammed colonies were isolated by morphology and tested for expression of pluripotency markers such as TRA160, SSEA3 / 4, NANOG, and OCT4. To confirm the donor cell origin of the iPS cells, we analyze genomic DNA for the presence of Ig or TCR rearrangements.

[0124] (ii) Monocytes: Our results with mice suggested that an intermediate step in the reprogramming of mature B cells could be macrophage-like cells [Hanna, 2008 #6842]. Monocytes are isolated from the buffy coats of human volunteers by Ficoll gradient centrifugation and adherent cells are isolated. Cells are grown in IL4 and GM-CSF according to established procedures [Damaj, 2007 #6854]. We transduce cells with the four factors OCT4, SOX2, cMYC, and KLF4 as described above and continue the culture in ES cell medium in the presence of DOX. Colonies with iPS morphology are picked and analyzed for the expression of pluripotency markers as described above. The developmental potential of blood-derived iPS cells is assessed by standard procedures such as teratoma formation and in vitro differentiation.

[0125] Currently, the strategy of isolating patient-specific iPS cells envisages reprogramming donor cells derived from deep skin biopsy, which is a more complicated and painful procedure than collecting blood. For routine clinical application, it is of obvious interest to design a reproducible protocol for the routine isolation of patient-specific iPS cells from peripheral blood samples. We expect that the proposed experiment will be of assistance in establishing such a protocol.

[0126] Given the ease and efficiency of mouse B cell reprogramming, we are encouraged that this protocol should also be effective in samples derived from human peripheral blood. Because B cell or T cell derived iPS cells have gene rearrangements at the Ig or TCR loci, respectively, using macrophages or monocytes as donors may be advantageous for potential therapeutic applications, as they do not have genetic alterations. We do not know the mechanism by which c / EBPα renders mature B cells susceptible to reprogramming by OCT4, SOX2, cMYC, and KLF4, but this may involve the conversion of B cell identity to that of macrophages [Xie, 2004 #5447]. These considerations suggest that deriving iPS cells from human monocytes may be straightforward. However, if the procedure developed in mice fails to yield blood-derived human iPS cells, we will screen additional factors using established approaches.

[0127] D. Small Molecule Screening Induction of reprogramming by retroviral vector-mediated gene transfer, especially the transduction of oncogenes, represents a serious obstacle to the ultimate therapeutic application of this approach. For example, we and other researchers [Okita, 2007 #6542] have seen that tumor types in chimeras are produced using iPS cells due to v-myc c-Myc activation. It is therefore of interest to identify small molecules that can either improve reprogramming activity or activate relevant pathways, thus replacing the need to express certain factors such as c-MYC or KLF4. The goal of this end is to establish a high-throughput cell-based assay system for screening chemical libraries of such compounds.

[0128] D.1 Experimental design and reporter cells for small molecule library screening To detect reprogramming in a high-throughput screen, we require cells with a marker, such as GFP, inserted into the endogenous OCT4 or NANOG locus. Such cells do not express the marker but can be used to screen for compounds that activate any of the endogenous genes.

[0129] To set up a high-throughput screen for reprogramming, we consider two major constraints that limit the experimental design.

[0130] Heterogeneous cell population: The most critical limitation is that transduction of fibroblasts with the four factors produces a genetically heterogeneous population of cells. As discussed above in VA3, it is likely that only a small fraction of infected cells with a particular number of viral vectors that produce the "right" expression level or "right" combination of expression levels of the four factors are the ones selected when screening for reprogramming. Thus, infected cells in individual wells will differ with respect to viral integration and viral copy number, precluding meaningful comparison of wells exposed to different compounds in the screen.

[0131] Frequency of marker activation, sensitivity, and time constraints of the assay: Another important consideration for setting up a screen concerns the sensitivity of the detection system: how many cells need to express the OCT4-GFP reporter gene to be detectable in a given well? Reporter gene expression is an important constraint because the fraction of reprogrammed cells needs to be high enough to produce at least a single detectable reprogramming event in a well with an active compound. Furthermore, reprogrammed cells are found among the population of fibroblasts only 3-5 weeks after infection with the four factors. Thus, infected cells need to survive and grow in 96- or 384-well formats during this period, which limits the number of cells that can be plated.

[0132] To overcome these limitations, we generate fibroblast populations that are genetically homogenous because they (i) have the same number of vector integrations or (ii) have various combinations of reprogramming factors inserted into endogenous gene expression loci by homologous recombination.

[0133] (i) "Secondary" clonal fibroblasts with specific and predetermined proviral combinations: We have recently shown that "secondary" mouse iPS cells can be derived from "primary" iPS cells generated by infection of fibroblasts with a DOX-inducible lentivirus that transduces the four transcription factors Oct4, Sox2, c-myc, and Klf4 [Hanna, 2008 #6842]. Because the "right" combination and number of proviral copies was contained in the "secondary" fibroblasts, viral infection was not required to induce reprogramming of B cells into secondary iPS cells.

[0134] We follow a similar protocol to preselect cells with the "right" combination and number of retroviral copies. As shown in Figures 23A-23B, "secondary" fibroblasts are derived from "primary" iPS cells by in vitro differentiation without DOX. Instead of using multiple vectors transducing single factors, we alternatively use polycistronic constructs as described in Figures 13A-13C and 14A-14E for transduction of various factor combinations. As outlined in VI.A.3, this approach using "secondary" fibroblasts or B cells resulted in efficient and DOX-dependent activation of reprogramming factors leading to iPS formation without the need for any further viral infection [Hanna, 2008 #6842]. To evaluate the fraction of iPS cells arising in DOX-enriched fibroblasts, we plate 500-1000 cells per well in 96-well plates and approximately 100 cells per well in 384-well plates and evaluate the fraction of GFP-positive cells. Results in the mouse system showed that secondary iPS cells arise only at 2 or 3 weeks of DOX induction. Because cells can be cultured for only approximately 7 days in 96-well or 384-well plates, we pretreat secondary fibroblasts with DOX at various time points before plating.

[0135] (ii) Transgenic fibroblasts with DOX-inducible reprogramming factors at the COL1A1 locus: We have shown that transgenes inserted at the Col1a1 locus are highly expressed in transgenic mice and, when placed under the control of the tet operator, are reproducibly expressed in all tissues upon DOX application [Beard, 2006 #6199; Hochedlinger, 2005 #5758]. We insert polycistronic constructs expressing various combinations of three or all four reprogramming factors under the control of the tet operator into the COL1A1 locus of huES cells with a GFP marker at the OCT4 locus (Figure 10). In addition, the cells are infected with a lentiviral vector transducing the rtTA transactivator. The cells are differentiated into secondary fibroblasts that can be screened for compounds that enhance reprogramming or replace a given factor (see below, Figure 24).

[0136] D.2 Screening for compounds that enhance reprogramming efficiency To screen for compounds that increase reprogramming efficiency, we culture secondary iPS cells with the "right" combination of all four factors, or fibroblasts with all four factors at the COL1A1 locus, in the presence of DOX (Figure 24). In preliminary experiments, we determine the fraction of GFP-positive cell lines that can be detected in the screen. If the fraction of reprogrammed cells resulting from fibroblasts transduced with the four factors is low, it may be difficult or impossible to detect a single reprogrammed cell among the 1000 or 100 cells that can be plated per 96-well or 384-well plate, respectively, unless a given compound significantly increases the fraction of reprogrammed cells. However, this assay has a very low background that compensates for the inherently low signal.

[0137] In a pilot screen, we tested the fraction of GFP-positive cells that arise in four-factor reporter cells that were cultured in the presence of DOX and either treated or not with 5-AzadC or infected with DNMT1 siRNA, both of which are treatments that have been shown to reduce global DNA methylation levels and enhance reprogramming of mouse fibroblasts [Mikkelsen, 2008 #6891]. The fraction of GFP-positive cells under either of these conditions determines how many cells need to be plated per well to detect compounds that enhance the fraction of GFP-positive cells in a less stringent screen. A more stringent screen would be one that does not treat with 5-AzadC or infect DNMT1 siRNA. Cells infected with the siRNA vector are used because this allows non-sensitized cells to be monitored for compounds that activate the reporter more efficiently than above.

[0138] D.3 Screening for compounds that replace any of the four factors To screen for compounds that can replace any of the retrovirally transduced factors, we transduce cells with vectors that can be independently regulated. The concept of this approach is that three factors are under the control of one inducible system, and the fourth factor is under independent inducible control. We use two different strategies to generate cells used for screening.

[0139] (i) Tamoxifen-inducible vector: We generated a vector that transduces OCT4, SOX2, KLF4, and c-MYC estrogen receptor (ER) fusion construct [Grandori, 1996 #6505], whose expression is activated by the addition of tamoxifen to the medium (Figure 11B). As outlined in Figure 26, OCT4-GFP reporter primary fibroblasts are transduced with a retrovirus that expresses the three tamoxifen-inducible factors, along with a fourth factor expressed from a DOX-dependent vector. The infected cells are grown in medium containing tamoxifen and DOX, and "primary" iPS cells are selected by screening for GFP expression. As above, secondary fibroblasts are induced and exposed to tamoxifen to activate the three tamoxifen-dependent factors, and screened for small molecule compounds that activate the GFP reporter in the absence of DOX and cMYC expression.

[0140] (ii) Transgenic fibroblasts with various combinations of factors at the COL1A1 locus: We pursue an alternative strategy to avoid retroviral infection, as outlined in Figure 24. Primary fibroblasts are derived from huES cells carrying a polycistronic construct encoding any combination of three reprogramming factors at the COL1A1 locus in addition to the OCT4-GFP marker and the virally transduced tet M2rtTA transactivator [Beard, 2006 #6199; Hochedlinger, 2005 #5758]; compare Figures 13A-13C, 14A-14E). Fibroblasts are transduced with a Lox-flanked vector (KLF4 in Figure 24) carrying the missing fourth factor, and primary iPS cells are derived. After Cre transduction to delete the KLF4 vector, secondary fibroblasts are derived. DOX exposure activates three DOX-dependent factors inserted into the COL1A1 locus, and cells are screened for small molecules that activate the GFP reporter in the presence of the missing fourth factor (in this case, KLF4). To prime the screen, we use cells treated with 5-Aza-dC.

[0141] D.4 Screening Platform Screening of the small molecule library will be performed in collaboration with S. Ding's lab at Scripps (see letter by S. Ding). For example, the Ding lab has developed and optimized a cell-based phenotypic high-throughput screen [Xu, 2008 #6875] and identified the small molecule pluripotin, which sustains ES cell self-renewal in chemically defined medium and in the absence of LIF [Chen, 2006 #6871]. The screen was based on expression of the GFP marker driven by the Oct4 promoter. We will screen Oct4-GFP transgenic fibroblasts with various combinations of factors, similar to the above for GFP activation.

[0142] The activity of any compounds scoring positive in the screen was confirmed under defined culture conditions in primary tissues to investigate the molecular pathways involved in the reprogramming process.

[0143] Potential Results and Interpretations: We expect that screening for activation of the OCT4 gene will identify compounds that facilitate the transition from the epigenetic state of somatic cells to one characteristic of pluripotent cells, thus making the reprogramming process more efficient. Another important goal of these experiments is to discover small molecule compounds that can replace the need for genetic manipulation, including the transduction of genes encoding oncogenes such as MYC, OCT4, or KLF4.

[0144] The two most prominent potential problems for high-throughput screening are (i) the time required for reprogramming to occur, and (ii) whether rare reprogramming events can be detected with the limited number of cells that can be plated per well of a 96- or 384-well plate. As discussed above, we preconditioned the cells to have the "right" number and combination of factors, and further sensitized the cells to increase the frequency of reprogramming-induced activation of various reporter genes. Once compounds that increase reprogramming efficiency are identified, they are used as sensitizers in subsequent screens for additional components that can further enhance iPS cell formation.

[0145] Significance: Current strategies to induce reprogramming rely on the transduction of potent oncogenes, which is an obstacle to any therapeutic application. The aim is to seek to identify small molecules that can activate relevant pathways, thus improving the efficiency and possibly minimizing the genetic changes required to induce reprogramming.

[0146] Significance and Long-Term Implications

[0147] Methods for in vitro generation of pluripotent iPS cells promise to revolutionize the study of complex human diseases, with significant implications for the eventual treatment of degenerative diseases; in vitro reprogramming of mouse somatic cells to a pluripotent state has been shown to be reasonably efficient, and the molecular mechanisms underlying this process are being actively investigated. However, reprogramming of human cells has proven to be more laborious and difficult, and major technical issues need to be resolved before this technology can be adapted for clinical use. The work described herein seeks to define the molecular mechanisms that trigger the conversion of human somatic cells to a pluripotent state, devise strategies to evaluate the developmental potential of human iPS cells, and achieve reprogramming without the need for genetic manipulation. The work described herein contributes to resolving some of the critical obstacles that currently prevent the application of this technology to study human diseases, and its eventual use for transplantation therapy of degenerative diseases.

[0148] Example 3 Reprogramming mouse and human somatic cells using a single polycistronic vector material and method Virus preparation and infection The construction of 4F2A lentiviral vectors containing Oct4, Sox2, Klf4, and c-Myc under the control of a tetracycline operator and a minimal CMV promoter was generated after EcoRI cloning from the FUW lentiviral backbone. All constructs were generated using unique restriction sites after amplification by PCR to place the individual elements between the respective 2A peptides (1st XbaI-NheI; 2nd SphI; 3rd XhoI; 4th AscI). The respective 2A sequences are as follows:

[0149] [ka] Replication-incompetent lentiviral particles (4F2A and M2rtTA) were packaged in 293T cells with VSV-G coat and used to infect MEFs containing a GFP allele targeted to the endogenous Nanog locus (25) (7). 14-week-old tail tip fibroblasts were derived from mice as previously published (12). Human keratinocytes (NHFK) were obtained from the Coriell Institute for Medical Research Camden, NJ. Supernatants from cells packaging each of the two viruses were pooled, filtered through a 0.45 μM filter, and subjected to ultracentrifugation for concentration. Viral pellets were resuspended in ES cell medium (DMEM (Hyclone) supplemented with 10% FBS, leukemia inhibitory factor, β-mercaptoethanol (Sigma-Aldrich), penicillin / streptomycin, L-glutamine, and non-essential amino acids (all from Invitrogen)) and then applied to the cells for 24 h.

[0150] Western blot 2% SDS, 10 mM dithiothreitol, 10% glycerol, 12% urea, 10 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor mixture (Roche), 25 μM MG132 100 μl of lysis buffer containing proteosome inhibitors was boiled for 5 min. Proteins were then quantified using Bradford reagent (Pierce) and read spectrophotometrically at 590 nm. Concentrations were estimated against a standard curve generated using bovine serum albumin. Total protein (5 μg) was subjected to electrophoresis in a denaturing 10% polyacrylamide gel containing 10% SDS. Proteins were then transferred to Immobilon-P membranes (Millipore) using a semi-dry transfer apparatus. Membranes were blocked with 0.01% Tween 20 in PBS with 2% non-fat powdered milk (Bio-Rad). Proteins were detected by incubation with antibodies at a concentration of 50 ng / ml in blocking solution. The antibodies used were Oct4 (h-134 Santa Cruz Biotechnology); Sox2 (mouse monoclonal R&D Biosystems); c-Myc (06-340 Upstate); Klf4 (H-180 Santa Cruz Biotechnology); GAPDH (sc-25778 Santa Cruz Biotechnology).

[0151] Quantitative RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen). 5 μg of total RNA was treated with DNase I using a DNA-free RNA kit (Zymo Research) to remove potential genomic DNA contamination. 1 μg of DNase I-treated RNA was reverse transcribed using the First Strand Synthesis kit (Invitrogen) and finally resuspended in 100 μl water. Quantitative PCR analysis was performed in triplicate using 1 / 50 of the reverse transcription reaction with Platinum SYBR green qPCR SuperMix-UDG with ROX (Invitrogen) on an ABI Prism 7000 (Applied Biosystems). Equal loading was achieved by amplifying GAPDH mRNA, and all reactions were performed in triplicate. The primers used for amplification were as follows:

[0152] [ka] Error bars represent the standard deviation of the mean of triplicate reactions.

[0153] Southern blotting Ten micrograms of BamHI-digested genomic DNA was separated on a 0.7% agarose gel, transferred to a nylon membrane (Amersham), and subjected to PCR amplification for OCT4 (EcoRI-PstI fragment of the pFUW-tetO-OCT4 plasmid), KLF4 (full-length KLF4 cDNA), c-MYC (full-length c-MYC cDNA), and SOX2 (full-length fragment of the pFUW-tetO-SOX2 plasmid). 32 P random primers (Stratagene) were hybridized with labeled probes.

[0154] Immunofluorescence staining Cells were fixed in 4% paraformaldehyde for 20 min at 25°C, washed three times with PBS, and blocked for 15 min with 5% FBS in PBS containing 0.1% Triton-X. After 1 h incubation with primary antibodies against Oct4 (Santa Cruz h-134), Sox2 (R&D Biosystems), Nanog (anti-ms R&D and anti-h), Tra-1-60, (mouse monoclonal, Chemicon International); hNANOG (goat polyclonal R&D Systems); mNANOG (Bethyl A300-398A), Tral-81 (mouse monoclonal, Chemicon International), SSEA4 and SSEA1 (mouse monoclonal, Developmental Studies Hybridoma Bank), in 1% FBS in PBS containing 0.1% Triton-X, cells were washed three times with PBS and blocked for 15 min with 5% FBS in PBS containing 0.1% Triton-X. Following incubation with appropriate fluorophore-labeled secondary antibodies purchased from Immunoresearch, samples were analyzed under an Olympus fluorescence microscope and images were captured with a Zeiss Axiocam camera.

[0155] Mouse chimeras and teratoma formation Diploid blastocysts (94–98 h after hCG injection) were placed in a drop of Hepes-CZB medium under mineral oil. A 16 μm inner diameter flat-tip microinjection pipette was used for iPS cell injection. Each blastocyst received 8–10 iPS cells. After injection, blastocysts were cultured in potassium simplex optimized medium (KSOM) and placed at 37 °C until transfer to recipient females. Approximately 10 injected blastocysts were transferred into each uterine horn of pseudopregnant B6D2F1 females 2.5 days after mating. Pups were collected on day 19.5 and, if necessary, fostered by lactating B6D2F1 mothers. Teratoma formation was initiated by injecting 2 × 10 blastocysts into the flanks of recipient SCID or Rag2− / − mice. 6 Cells were allowed to settle and tumors were isolated after 3-6 weeks for histological analysis.

[0156] Human teratoma formation and analysis hiPSCs were harvested by collagenase treatment (1.5 mg / ml) and separated from feeder cells by subsequent washing with medium and settling of iPSC colonies. iPSC aggregates were harvested by centrifugation and plated at 10 6 The iPSCs were resuspended at a 1:1 cellular ratio and injected subcutaneously into the backs of SCID mice (Taconic) through a 21-gauge needle. Tumors developed within 6 weeks, and animals were sacrificed before tumor size exceeded 1.5 cm in diameter. Teratomas were isolated after mouse sacrifice and fixed in formalin. After sectioning, teratomas were diagnosed based on hematoxylin and eosin staining. Karyotype analysis was performed using CLGenetics (Madison, WI).

[0157] In vitro differentiation of human iPS cells into neural progenitor cells Human keratinocyte iPSCs were grown in culture without passaging for 2 weeks with daily medium changes. On day 15 after passaging, distinct neural rosettes were observed and collected by mechanical picking with a glass pipette (26). Rosettes were replated on dishes precoated with 15 μg / ml polyornithine / 10 μg / ml laminin (Po / Lam) in N2B27 medium supplemented with FGF2 (20 ng / ml) EGF (20 ng / ml) (all from R&D Systems). After 5–7 days, cells were dissociated by scratching with a cell lifter and by pipetting single cells into N2B27 medium and replated on Po / Lam culture dishes.

[0158] Differentiation and immunocytochemistry Induction of neural progenitor cell differentiation was performed by withdrawal of FGF2 and EGF from the culture medium for 5 days. Cells were fixed in 4% paraformaldehyde for 20 min and stained for human nestin (Chemicon; 1:100) and Tuj-1 (1:100), followed by washing three times with PBS and incubation with appropriate fluorophore-labeled secondary antibodies purchased from Jackson Immunoresearch. Samples were analyzed on an Olympus fluorescent microscope and images were acquired using a Zeiss Axiocam camera.

[0159] result Vectors are constructed with various combinations of two, three, or all four reprogramming factors from one promoter. The aim was to generate polycistronic viral vectors expressing multiple reprogramming genes from a single promoter using the 2A peptide. For this, one, two, or three 2A oligopeptides containing unique restriction sites were ligated into the FUW lentiviral (18) backbone, allowing efficient cloning of Oct4, Sox2, c-Myc, and Klf4, each separated by a different 2A sequence. Vectors carrying four, three, or two factors constitutively bearing different combinations of F2A, T2A, E2A, or P2A sequences (Figure 13A and Figure 14A) were tested for their ability to express individual factors by transient transfection in human 293 cells. Western blot analysis demonstrated that the 2A peptide supported efficient expression of two, three, or all four cistrons from a single polycistronic vector (Figure 14B).

[0160] To test the usefulness of polycistronic vectors for reprogramming, we first transduced MEFs with retroviral vectors carrying various combinations of two or three reprogramming factors, and showed that these constructs could be combined with vectors carrying additional single factor-cDNAs to generate iPS cells. Importantly, polycistronic vectors carrying all four factors were able to generate iPS cells. In this preliminary experiment, we co-infected Oct4-GFP fibroblasts with polycistronic Sox2-Oct4-Klf4-myc vectors and additional Oct4 vectors (to account for the possibility that relatively more Oct4 protein may be required for reprogramming; FIG. 13B). FIG. 13B shows that iPS cells were obtained that expressed AP, SSEA1, Nanog, and Oct4. Furthermore, adult chimeras were generated from iPS lines infected with four factor 2A vectors and Oct4 Moloney virus. To determine the number of proviral integrations, Southern blots were hybridized sequentially with Sox2, Klf4, c-myc, and Oct4 probes. Figure 13C shows that a single polycistronic vector was integrated in two or three different tested iPS lines, and two proviruses were included in the third line (in this line, 4FO#14, the c-myc sequence was deleted in one of the proviruses). Surprisingly, an additional 8 to 11 Oct4 proviruses were included in each of the iPS lines, suggesting strong selection for multiple integrations of Oct4 proviruses. Since we did not see more than 4 or 5 Oct4 proviruses in iPS cells induced by the four separately transduced vectors, it is unlikely that the selection was for high Oct4 expression, although we cannot exclude the possibility. An alternative interpretation is that the selection for multiple proviruses was due to selection for insertional activation of unknown cellular genes.These initial data suggested that at least three reprogramming factors could be expressed from a single polycistronic provirus to induce reprogramming. As described further below, we went on to successfully generate mouse iPS cells using only a polycistronic vector carrying the four factors, and also used a polycistronic vector to generate human iPS cells with minimal genetic alterations.

[0161] Tetracycline-inducible lentiviral vectors were constructed where expression of genes was controlled from a tetracycline operator minimal promoter (tetOP; Figure 14C). To test whether all four genes of a single four-factor (Oct4 / Sox2 / Klf4 / c-Myc) virus could be expressed upon DOX addition, MEFs were infected with a polycistronic vector (hereafter referred to as "4F2A") as well as a constitutive FUW lentivirus carrying a tetracycline-controllable transactivator (M2rtTA; abbreviated as rtTA). Two independent experiments were performed and drug-inducible expression of the virus was tested by qRT-PCR 3 days after infection. Using primers for a virus-specific transcript (E2A-cMyc), robust induction was observed in cells cultured with DOX (7- to 10-fold) compared to control media (Figure 14D). To test the relative induction compared to ES cells, Oct4 and Sox2 primers that cannot distinguish between viral or endogenous transcripts were utilized, and in both experiments, DOX-induced MEFs infected were significantly higher than ES cells (~3.5- and ~17-fold relative to ES levels, respectively). Western blot analysis of cells isolated 3 days after infection demonstrated that when cells were cultured without DOX, little or no protein was expressed, whereas in the presence of DOX, robust expression was seen, with Oct4 and Sox2 proteins similar to those in ES cells (Figure S14E).

[0162] To test whether the 4F2A vector was able to reprogram somatic cells to a pluripotent state, MEFs containing a GFP promoter driven by the endogenous Nanog promoter were infected with the virus (4F2A+rtTA). 85-90% of cells were stained 48 h after transduction, indicating high titer infection (Figure 15A). Morphological changes were observed several days after addition of DOX (data not shown), and distinct colonies appeared approximately 8 days after DOX induction, and Nanog-GFP+ cells approximately 25 days later (Figure 15B). After mechanical isolation and subsequent passaging, the cells had a morphology typical of ES cells and proliferated independently of DOX. Four independent 4F2A iPS cell lines were established, which were positive for the pluripotency markers AP, SSEA1, and Nanog-GFP (Figure 15C).

[0163] To examine whether adult somatic cells could be reprogrammed using the 4F2A vector, we infected tail tip fibroblasts (TTF) from 14-week-old mice with the 4F2A+rtTA vector. Similar to MEFs, typical morphological changes were observed several days after the addition of DOX medium. Colonies appeared at about day 8 and continued to expand until they were picked based on morphology (day 16). After several passages, four stable iPS cell lines were established, which stained positive for all pluripotency markers (Nanog, Oct4, SSEAl, AP) (Figure 15C). MEF iPS cell lines were subcutaneously injected into SCID mice, and were shown to induce teratomas that contained differentiated cells of all three germ layers (Figure 16A). Finally, injection of MEF iPS cells (#4) into blastocysts generated postnatal chimeras (FIG. 16B), demonstrating that a single 4F2A polycistronic virus can reprogram MEFs to a pluripotent state.

[0164] To determine the number of proviruses contained in the 4F2A iPS cell line, DNA was extracted and subjected to Southern blot analysis using enzymes that do not cut in the vector sequence. Using Oct4, Sox2, c-Myc, and Klf4 probes for hybridization, we detected bands of identical molecular weight, confirming that the sequences of the factors were carried in one provirus. The total number of proviruses was between 1 and 3, with iPS cell line #4 having a single viral insertion (Figure 16C). One of the two integrations from iPS cell line #1 failed to produce a band after c-Myc hybridization, suggesting that a 3' deletion of the c-Myc sequence may have occurred. A second digest confirmed the provirus copy number (Figure 18A).

[0165] To estimate reprogramming efficiency, MEFs were infected with 4F2A and rtTA vectors at 0.25 × 10 cells / 10 cm dish. 6 MEFs were plated at 100°C for 20 min at 4°C. Approximately 70% of MEFs were infected as estimated by immunostaining for Oct4 48 h after infection (Figure 19A). Cells were cultured in ES medium containing DOX for 20 days and subsequently transferred to ES cell medium until GFP+ colonies were counted on day 25. On average, ∼14.7 ± 4 colonies were detected in three independent dishes (10 + 10 + 17), indicating a relative efficiency of 0.0001%, which is 1 to 2 orders of magnitude lower than that of "primary" infected fibroblasts (3, 7).

[0166] To test the kinetics of reprogramming using the 4F2A virus, we performed dox withdrawal experiments, in which the DOX-containing medium was replaced with ES medium for specific days (i.e., 2 days, 4 days, 8 days, 12 days, etc.), and the number of Nanog-GFP+ colonies was counted on day 25. Using separate drug-inducible viruses to deliver the four factors, it was reported that approximately 9-12 days was the minimum time required for the generation of stable iPS cells (20, 21). To minimize overlap of reprogramming events, cells were not passaged during this time. Two independent experiments were performed, and in both cases, a single Nanog-GFP+ colony was present on the plate cultured in DOX medium for 8 days, which remains the minimum time required using separate viruses (Figure 14B).

[0167] These data demonstrate that a single polycistronic virus containing four factors linked by three 2A peptides allows sufficient factor expression to generate iPS cells from polycistronic or adult somatic cells. Importantly, our results also show that a single polycistronic proviral copy is sufficient to reprogram somatic cells to pluripotency.

[0168] Generation of human iPS cells using a single polycistronic virus To examine whether human cells can be reprogrammed with polycistronic vectors, neonatal human forskolin keratinocytes (NHFKs) were transduced with both the constitutive rtTA vector and the DOX-inducible 4F2A vector. The fraction of infected cells was 10% as determined by staining for Oct4 48 hours after transduction (Figure 20A). Cells were incubated in keratinocyte medium + DOX and grown for 6 days until they were passaged and cultured in hESC medium + DOX on gelatinized plates. Colonies were first detected on day 12, many showing distorted morphology, with a few colonies showing a distinct appearance resembling hESC-like morphology. Two such colonies generated in independent infections were picked between days 22 and 35 post-infection and found to expand as distinct colonies with morphology similar to hESC (Figure 17A). These cells were expanded in the absence of DOX and after an additional 2-5 passages gave rise to homogenous populations identical to hESCs (Ker-iPS). These cells stained for pluripotency markers AP, Oct4, Nanog, Sox2, SSEA4, Tral-60, Tral-81 (Fig. 17B, Fig. 10B) and had a normal karyotype (Fig. 17C). DNA fingerprinting ruled out the possibility that such Ker-iPS cell lines were contaminants from previously established human iPS or hES cell lines from our laboratory (data not shown). To determine proviral copy numbers in the Ker-iPS cell lines, genomic DNA was extracted and subjected to Southern blot analysis using an enzyme that does not cut in the vector sequence. Probes for all four reprogramming factors again showed hybridization to similar molecular weight bands, indicating that they were on a single virus. Two different digests (XbaI and BamHI) indicate that the 4F2A proviral copy numbers are 3 (#1.1) and 2 (#3), respectively (Figures 21A-B).

[0169] To test pluripotency, one line, Ker-iPS#1.1, was subcutaneously injected into SCID mice. These cells induced teratomas and differentiated into cells of all three germ layers after histological examination (Figure 17D). In addition, when Ker-iPS#1.1 cells were subjected to an in vitro neural differentiation protocol, they produced a nestin+ neural progenitor population as well as Tujl+ postmitotic neurons, as detected by immunostaining (Figure 17E).

[0170] Consideration The above experiments demonstrate that up to four different reprogramming factors inserted into a polycistronic vector separated by 2A sequences can be expressed at sufficient levels to achieve reprogramming. Embryonic and adult mouse fibroblasts and neonatal human keratinocytes were induced to form pluripotent iPS cells when infected with FUW rtTA and 2A vectors transduced Oct4, Sox2, Klf4, and c-Myc.

[0171] We observe a significantly lower reprogramming efficiency than in previous experiments using a single vector to transduce each of the four factors (Figure 19B and Table 3).

[0172] [Table 3] It is possible that the lower reprogramming efficiency is due to the stoichiometry of expression of factors from polycistronic vectors, which may be suboptimal for inducing reprogramming. Transduction with separate vectors allows for integration of a variable number of proviruses for each factor, and therefore reprogramming may select for a particular set of proviral integrations that result in high expression or optimal stoichiometry between different factors. However, the 2A system has been reported to support near equimolar protein expression in vivo (17). Also, when separate vectors transducing each of the four factors were used for the derivation of iPS cells, Nanog-GFP positive cells were detected as early as day 16 after DOX induction, in contrast to the GFP positive cells observed 22–25 days after transduction with the 4F2A vector, consistent with suboptimal reprogramming. Furthermore, whereas iPS cells frequently harbor multiple Oct4 or Klf4 proviruses, we consistently found few Sox2 proviruses, suggesting that high levels of Sox2 expression may possibly not be favorable for reprogramming (24).

[0173] In another embodiment, the flp-in transgenic system is used to generate multiple mouse cell lines containing 4, 3, and 2 factor 2A constructs at the collagen gene locus (Figure 22) (20). This system contains two components: a tetracycline-controlled transactivator (rtTA) and a tetracycline operator minimal promoter (tetOP) to drive the gene of interest. After addition of medium containing doxycycline, the transactivator drives expression of the transgene at the collagen locus. If desired, insertion of a GFP reporter construct at the Nanog gene allows detection of complete reactivation of the Nanog locus, acting as a marker of genome-wide epigenetic reprogramming.

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[0178] For Cre recombinase-mediated vector excision, hiPSC lines are cultured in Rho Kinase (ROCK) inhibitor (Calbiochem; Y-27632) 24 hours prior to electroporation. Cells are harvested using 0.05% trypsin / EDTA solution (Invitrogen) and suspended in PBS at 1 × 10 7 Cells were either transfected with pCre-PAC (50 μg; Taniguchi et al., 1998) or co-transfected with pTurbo-Cre (40 μg; Genbank accession number AF334827) and pEGFP-N1 (10 μg; Clontech) by electroporation as previously described (Costa et al., 2007; Gene Pulser Xcell System, Bio-Rad: 250V, 500 μF, 0.4 cm cuvettes). Cells were then plated on a MEF feeder layer (DR4 MEFs for puromycin selection) in hESC medium supplemented with ROCK inhibitor for the first 24 hours. Cre recombinase-expressing cells were selected using one of the following methods: 1) addition of puromycin (2 μg / ml) for 48 hours 2 days after electroporation, 2) FACS sorting (FACS-Aria; BD-Biosciences) of single cell suspensions for EGFP-expressing cells 60 hours after electroporation, followed by replating at low density in hESC medium containing ROCK inhibitor. Individual colonies were picked 10-14 days after electroporation.

[0179] Viral constructs Lentiviral vectors containing the FUW-M2rtTA lentiviral vector and human DNA for KLF4 (FUW-tetO-hKLF4), OCT4 (FUW-tetO~hOCT4), SOX2 (FUW-tetO-hSOX2), and c-MYC (FUW-tetO-hMYC) under the control of a tetracycline operator and minimal CMV promoter have been previously described (Hockemeyer et al., 2008). To generate Cre recombinase excisable DOX-inducible lentiviral vectors, a NotI / Bsu36I fragment containing the tetracycline operator / minimal CMV promoter and human cDNA for either KLF4, OCT4, or SOX2 were subcloned from the FUW-tetO vector into the NotI / BSU36I sites of FUGW-loxP, which contains a loxP site in the 3'LTR (Hanna et al., 2007).

[0180] Lentiviral infection and hiPSC induction VSVG-coated lentiviruses were produced in 293 cells as previously described (Brambrink et al., 2008). Briefly, culture medium was replaced 12 h post-transfection and virus-containing supernatants were collected 60–72 h post-transfection. Viral supernatants were filtered through a 0.45 μm filter. Virus-containing supernatants were pooled for triple and quadruple infections and supplemented with FUW-M2rtTA virus and an equal volume of fresh culture medium. 1 × 10 6 Human fibroblasts were seeded in T75 flasks 24 h prior to transduction. Four successive infections in the presence of 2 μg / ml polybrene were performed over a period of 48 h. The culture medium was changed 12 h after the last infection. Five days after transduction, fibroblasts were passaged using trypsin and plated on 10 cm plates on gelatin-coated dishes. 2 5 x 10 per 4 From 2 × 10 5The cells were replated at different densities between 1 × 10 and 2 × 10. To induce reprogramming, the culture medium was replaced after 48 h by hESC supplemented with DOX (Sigma-Aldrich; 2 μg / ml). HiPSC colonies were manually picked based on morphology between 3 and 5 weeks after DOX induction, and were manually maintained and passaged according to the hESC protocol in the absence of DOX. To determine the reprogramming efficiency, 1 × 10 5 Human fibroblasts are 10 cm 2 The cells were seeded onto gelatin-coated dishes and reprogramming efficiency was calculated after 20 days based on immunocytochemistry for the pluripotency markers Tra-1-60 and NANOG.

[0181] Microarray gene expression analysis RNA was isolated from hESCs and iPSCs, which were mechanically separated from feeder cells using the RNeasy Mini Kit (Qiagen). 2 μg of total RNA was used to prepare biotinylated cRNA according to the manufacturer's protocol (Affymetrix One Cycle cDNA Synthesis Kit). Briefly, the method involves Superscript II-directed reverse transcription using a T7-Oligo(dT) promoter primer to generate first-strand cDNA. RNase H-mediated second-strand cDNA synthesis is followed by T7 RNA polymerase-directed in vitro transcription, which incorporates biotinylated nucleotide analogs during cRNA amplification. Samples for hybridization were prepared using 15 μg biotinylated cRNA in 1× hybridization cocktail according to the Affymetrix hybridization manual. GeneChip arrays (Human Ul33 2.0) were hybridized in a GeneChip Hybridization oven at 45° C. for 16 hours at 60 RPM. Washing was performed using a GeneChip Fluidics Station 450 and Affymetrix GeneChip Hybridization, Wash and Stain according to the manufacturer's instructions. The analysis was performed using buffers provided in the kit. Arrays were scanned on a GeneChip Scanner 3000 and images were extracted and analyzed using GeneChip Operating Software v1.4. U133 Plus 2.0 microarrays (Affymetrix) were processed using the MAS5 algorithm and the absent / present calls for each probe set were determined using standard Affymetrix algorithms, both implemented in Bioconductor. Probe sets that were absent in all samples were removed for subsequent analysis. Differential expression was determined as moderate using t-tests using the "limma" package in R (corrected for false discovery rate) or fold change. When genes were represented by multiple probe sets (based on annotation from Affymetrix), log ratios and p-values ​​of gene expression were calculated as the mean and minimum of these probe sets, respectively. Hierarchical cluster analysis was performed on log-transformed gene expression ratios using uncentered Pearson correlation and pairwise mean linkage. Correlations were compared using Fisher's Z transformation. Confidence levels for hierarchical cluster analysis were calculated using multiscale bootstrap resampling with the R package “pvclust.”

[0182] Reverse transcription of total RNA and real-time PCR RNA was isolated from EBs or hESCs and iPSCs mechanically dissociated from feeder cells using either the RNeasy Mini Kit (Qiagen) or Trizol extraction followed by ethanol precipitation. Reverse transcription was performed on 1 μg of total RNA using oligo-dT priming and Thermoscript reverse transcriptase (Invitrogen) at 50°C. Real-time PCR was performed on an ABI Prism 7000 (Applied Biosystems) with Platinum SYBR green pPCR SuperMIX-UDG with ROX (Invitrogen) using primers partly as described previously (Hockemeyer et al., 2008; Yu et al., 2007) and partly as described in Soldner et al., 2009, supplementary experimental procedures.

[0183] Teratoma formation and analysis hiPSCs were harvested by collagenase treatment (1.5 mg / ml) and separated from feeder cells by subsequent washing with medium and sedimentation of iPSC colonies. hiPSC aggregates were harvested by centrifugation and resuspended in 250 μl of phosphate-buffered saline (PBS). hiPSCs were injected subcutaneously into the backs of SCID mice (Taconic). Tumors generally developed within 4–8 weeks, and animals were sacrificed before tumor size exceeded 1.5 cm in diameter. Teratomas were isolated after mouse sacrifice and fixed in formalin. After sectioning, teratomas were diagnosed based on hematoxylin and eosin staining.

[0184] Methylation analysis Genomic DNA was collected from hESCs and hiPSCs by mechanical separation from feeder cells. DNA was proteinase K treated, phenol-chloroform treated, and 1 μg of DNA was subjected to transformation using the Qiagen EpiTect Bisulfite kit. The promoter region of OCT4 was amplified using primers previously described (Yu et al., 2007).

[0185] [ka] The PCR products were cloned using pCR2.1-TOPO vector using M13 forward and reverse primers.

[0186] immunocytochemistry Cells were fixed in 4% paraformaldehyde in PBS and incubated with the following primary antibodies: SSEA4 (mouse monoclonal, Developmental Studies Hybridoma Bank); Tra1-60, (mouse monoclonal, Chemicon International); hSOX2 (goat polyclonal, R&D Systems); Oct-3 / 4 (mouse monoclonal, Santa Cruz Biotechnology); hNANOG (goat polyclonal R&D Systems); and appropriate Molecular Probes Alexa Fluor® dye-conjugated secondary antibodies (Invitrogen).

[0187] Southern blotting XbaI-, EcoRI-, or MfeI-digested genomic DNA was separated on a 0.7% agarose gel, transferred to a nylon membrane (Amersham), and subjected to PCR amplification for OCT4 (EcoRI-PstI fragment of pFUW-tetO-hOCT4 plasmid), KLF4 (full-length hKLF4 cDNA), c-MYC (full-length c-MYC cDNA), SOX2 (FspI-EcoRI fragment of pFUW-tetO-hSOX2 plasmid), and M2rtTA (380 bp C-terminal fragment of M2rtTA c-DNA). 32 P random primers (Stratagene) were hybridized with labeled probes.

[0188] Accession number The microarray data are available in the NCBI Gene Expression Omnibus database under the serial accession number GSE14711.

[0189] Overview Here we show that fibroblasts from five patients with idiopathic Parkinson's disease (PD) can be efficiently reprogrammed. Furthermore, we used a Cre recombinase-excisable virus to derive human induced pluripotent stem cells (hiPSCs) that do not contain reprogramming factors. Factor-free iPSCs maintain a pluripotent state and display an overall gene expression profile that is more closely related to hESCs than transgene-bearing hiPSCs. Our results suggest that residual transgene expression in virus-bearing hiPSCs can affect their molecular properties, and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling human disease.

[0190] result Reprogramming fibroblasts from PD patients with DOX-inducible lentiviral vectors Dermal fibroblasts from five patients with idiopathic PD (age at biopsy, ranged from 53 to 85 years) and two unaffected subjects were obtained from the Coriell Institute For Medical Research (see Table 4). 6 Fibroblasts were infected with a constitutively active lentivirus expressing reverse tetracycline transactivator (FUW-M2rtTA) together with DOX-inducible lentiviruses transducing either four (OCT4, SOX2, c-MYC, KLF4) or three (OCT4, SOX2, KLF4) reprogramming factors. 4F hsPSC lines driven by transduction of the four factors as 3FThe present study refers to hsPSC lines obtained by transduction of three factors as DOX-induced pluripotency markers (Figure 27A). Colonies with well-defined hESC-like morphology were selected and manually picked after 3-5 weeks of DOX-induced transgene expression. All fibroblasts obtained from PD and non-PD patients gave rise to stable hiPSCs, which were maintained for more than 30 passages in the absence of DOX. At least one cell line from each donor fibroblast was analyzed in detail (Table 4). As determined by immunocytochemistry, all of these hiPSCs uniformly expressed the pluripotency markers Tra-1-60, SSEA4, OCT4, SOX2, and NANOG (Figure 27A). In addition, all hiPSC lines analyzed by quantitative RT-PCR showed reactivation of the endogenous pluripotency-associated genes OCT4, SOX2, and NANOG at levels of expression similar to those seen in hESCs (Figure 27B). As expected for hiPSCs, the OCT4 promoter region of hiPSCs derived from PD patients was found to be hypomethylated, in contrast to its hypermethylated state in parental fibroblasts (Figure 27C). To test for pluripotency, hiPSCs isolated from each donor fibroblast line were injected into SCID mice. All hiPSC-formed teratomas were composed of tissues developing from all embryonic germ layers, including cartilage, bone, smooth muscle (mesoderm), neural rosettes, pigmented neuroepithelium (ectoderm), and intestinal epithelium (endoderm) with goblet and Paneth-like cells (Figure 28A).

[0191] Cytogenetic analysis of PD-specific hiPSC lines revealed normal karyotypes in 11 of 12 lines (see Supplementary Figure 1 in Soldner, 2009). 4F Only one of three clones derived from the fibroblast line PDD transduced with iPSC-5 showed an unbalanced translocation between the long arm of chromosome 18 and the long arm of chromosome 22, resulting in a derivative chromosome 18 and a single copy of chromosome 22. 3F -1 and iPSCs 3FTwo independent hiPSCs derived from PD patients (Fig. 2A-2) showed balanced translocations between the short and long arms of chromosomes 4 and 7, suggesting that the 4;7 translocation was already present in the donor fibroblasts (Soldner et al., 2009, see Supplementary Figure 1). DNA fingerprinting of PD patient-derived hiPSCs and parental fibroblasts was performed to confirm the origin of the hiPSCs and to exclude cross-contamination with pre-existing pluripotent cell lines (data not shown). Southern blot analysis probing for lentiviral integration showed distinguishable patterns for each of the hiPSC lines, confirming that each analyzed line was derived from independently infected fibroblasts with a total of 4-10 proviral copies (Fig. 28B, 28C).

[0192] To further characterize the utility of this system, we determined the reprogramming efficiency for one fibroblast lineage (PDB) in detail. The reprogramming efficiency was calculated after 20 days based on immunocytochemistry for the resulting pluripotency markers Tra-1-60 and NANOG. hiPSCs were generated with a potency of about 0.005% after transduction with three factors and about 0.01% after transduction with four factors, which is comparable to previously reported potencies using Moloney-based retroviral vectors or constitutively active lentiviral vectors (Nakagawa et al., 2008; Takahashi et al., 2007; Yu et al., 2007). Immunohistochemistry for NANOG and Tra-1-60 at different time points after DOX addition showed that small pluripotent colonies could be detected in four-factor transduced fibroblasts as early as 8 days after transgene induction (Figure 32A). We also determined the temporal requirements for expression of reprogramming factors by varying the time of DOX-induced transgene expression in fibroblasts transduced with either the four or three reprogramming factors. After 24 hours, we were able to isolate hiPSC colonies from fibroblasts transduced with the four factors that had been exposed to DOX for only 8 days (PDB 4F-1, 2, 3), hiPSCs transduced with the three factors could only be isolated after exposure to DOX for at least 12 days (PDB 3F Although the reprogramming factors were expressed for only a limited period of time, all of the picked cells gave rise to fully reprogrammed hiPSCs, which stained for pluripotency markers (Figure 32B), reactivated endogenous OCT4, NANOG, and SOX2 genes (Figure 32C), and the formed teratomas were composed of cells derived from the three developmental germ layers (Figure 32D). Our results suggest that reprogramming with three factors is less efficient and takes longer than reprogramming with four factors, consistent with previous observations (Nakagawa et al., 2008; Wernig et al., 2008). However, we find that derivation of hiPSCs using three factors is more practical. This is because the infected fibroblast cultures are not overpopulated with granular, fast-growing non-hiPSCs, as previously described for cultures infected with four factors (Nakagawa et al., 2008; Takahashi et al., 2007).

[0193] The results described so far show that DOX-induced delivery of reprogramming factors can efficiently generate hiPSCs from skin biopsies obtained from PD patients, in the absence of c-MYC, with kinetics and efficiency similar to those previously reported using other approaches. Importantly, 8 of 13 triple-factor hiPSCs had only 3-5 proviral integrations in total (Figures 28B, 28C), which is significantly fewer than observed in proviral studies (Wernig et al., 2007).

[0194] Generation of PD patient-derived hiPSCs without viral reprogramming factors To derive provirus-free hiPSCs, we generated lentiviral vectors that could be excised after integration using Cre recombinase. The human ubiquitin promoter of the FUGW-loxP lentivirus, which contains a loxP site in the 3'LTR (Hanna et al., 2007), is replaced with a DOX-inducible, minimal CMV promoter followed by human cDNA for OCT4, KLF4, or SOX2. Upon proviral replication, the loxP site in the 3'LTR overlaps with the 5'LTR, resulting in an integrated transgene flanked by loxP sites in both LTRs (Figure 4A). 6 Fibroblasts (PDB) express these three viruses, as well as the reverse tetracycline transactivator (FUW-M2rtTA). 24 hiPSC lines (PDB2 lox 1–24) were isolated 3–4 weeks after DOX addition with similar kinetics and efficiency as above. Southern blot analysis of 12 cell lines confirmed the presence of 4 PDB 2lox System (PDB 2lox -5, PDB 2lox -17, PDB 2lox -21, PDB 2lox -22) contained only 5–7 incorporation of reprogramming factors (Figure 33). 2lox The cell lines were maintained in the absence of DOX for more than 20 passages and showed all of the hallmarks of hiPSCs, including expression of pluripotency-associated marker proteins Tra-1-60, SSEA4, OCT4, SOX2, and NANOG (Figure 29B), as well as reactivation of endogenous associated genes OCT4, NANOG, and SOX2 (included in Figure 31B). Furthermore, all tested PDB 2lox Clone (PDB 2lox -5, PDB 2lox -17, PDB 2lox -21, PDB 2lox -22) demonstrated in vitro multilineage differentiation in EBs (data not shown) and formed teratomas with contribution to all three embryonic germ layers after subcutaneous injection into SCID mice (Figure 29C).

[0195] We focused on two clones, 5 (PDB 2lox -21) or 7(PDB 2lox -17) with either the total integration of reprogramming factors to test whether excision of the lentiviral vector flanked by lox sites would generate transgene-free cells. Two different strategies for Cre-mediated vector excision were used (Figure 30A): (1) transient expression of a vector (pCre-PAC) encoding Cre recombinase and a puromycin resistance gene. After electroporation, cells were selected with puromycin for 48 h to enrich for cells that expressed the transiently expressed Cre recombinase and puromycin resistance gene. (2) co-transfection of Cre recombinase with an EGFP-expressing plasmid, followed by FACS sorting for EGFP-positive and Cre-expressing cells 60 h after transfection. Using these two methods, we isolated a total of 180 clones 10-14 days after electroporation (Figure 30A). Initial Southern blot analysis to screen for excision of KLF4 (highest number of integrations) using an internal EcoRI digest showed that 48 clones were negative for KLF4 lentiviral integration (data not shown). Subsequent Southern blot analysis for KLF4, OCT4, SOX2 proviral integration using an external XbaI restriction digest was performed using the PDB 2lox Seven clones derived from -17, and PDB 2lox The results revealed that 9 clones derived from -21 did not have any integration of reprogramming clones (Figure 30B, PDB 1lox The excision of all reprogramming factors was confirmed by further Southern blotting using different restriction digests (Figure 34). Furthermore, PCR of genomic DNA using primers specific for Cre recombinase identified the genomic DNA fragments identified in the PDB. 1loxIt was confirmed that none of the clones had stably integrated the electroporated plasmid (data not shown). Southern blot analysis for integration of the reverse transactivator M2rtTA was performed using the gene PDB. 2lox -17 1 integration, lineage PDB 2lox Two integrations were shown for -21 (Figure 35), which means that the total number of proviral integrations includes the transactivator in line. PDB 2lox -21 is PDB 2lox The results were the same as the excised transgene from -17, suggesting that excision of all transgenes, including transactivators, should be possible. Cytogenetic analysis demonstrated that 14 of 14 clones analyzed exhibited a normal karyotype after Cre-mediated transgene excision (Figure 30C and data not shown).

[0196] All virus-free clones retained hESC-like morphology upon long culture for more than 15 passages, maintained all of the characteristics of hiPSCs, such as expression of the hESC-associated marker proteins Tra-1-60, SSEA4, OCT4, SOX2, and NANOG, as shown by immunocytochemistry (Figure 31A), and maintained expression of the endogenous pluripotency-associated genes OCT4, SOX2, and NANOG at levels comparable to hESCs and parental hiPSCs before transgene excision (Figure 31B). 1lox Clones maintain pluripotency after ablation of reprogramming factors and are isolated from an independent PDB 1lox Clones were differentiated in vitro by EB formation or injected subcutaneously into SCID mice. 1lox Clones exhibited multilineage differentiation in vitro and developed into teratomas with contributions to all three embryonic germ layers (FIG. 31C).

[0197] To compare the residual transgene expansion between distinguishable hiPSCs and hiPSCs without integrated transgenes and factors, we performed quantitative RT-PCR using transgene-specific PCR primers. As previously reported, using either lentivirus or Moloney-based retroviral vectors (Dimos et al., 2008; Ebert et al., 2008; Hockemeyer et al., 2008; Park et al., 2008a; Yu et al., 2007), we detected residual expression of reprogramming factors for the majority of transgenes in all cell lineages. In hiPSCs with integrated virus but uninfected fibroblasts, hESCs, and PDBs, we found that the expression of reprogramming factors was significantly increased in hiPSCs with integrated virus but not in uninfected hiPSCs with integrated virus. 1lox (Figure 31D). Our results demonstrate that the use of loxP-flanked vectors for reprogramming following Cre-mediated excision can efficiently generate hiPSCs that do not contain reprogramming factors.

[0198] To address whether remaining transgene expression can affect global gene expression in reprogrammed cells, we compared hESCs, parental fibroblasts, and hiPSCs before and after transgene excision by genome-wide gene expression analysis. Initial correlation analysis based on all genes showing at least a 6-fold expression difference between fibroblasts and hESCs confirmed that all hiPSCs were closely related to hESCs, whether the transgene was removed or not (Soldner et al., 2009, see Supplementary Figure 7). Despite the similarity between hESCs and hiPSCs, the correlation with hESCs was poorly correlated with PDB 1lox and PDB 2lox Statistical analyses comparing cells are available in PDB1 lox But the parent PDB 2lox The results showed that the expression of hiPSCs with and without the transgene resembled hESCs more than that of hiPSCs with the transgene (Soldner et al., 2009, Supplementary Figure 7). Notably, correlation analysis based on all genes showing at least a 2-fold difference in expression between hiPSCs with and without the transgene included each individual PDB 1lox Gene expression profiles of the strains are available in the PDB 2loxThe remaining differentially expressed genes in factor-free hiPSCs are likely due to the diverse genetic backgrounds of hESCs and hiPSCs, or the expression of transactivators or genetic memory of the original somatic cells that were reprogrammed. A detailed list of differentially regulated genes is shown in Supplementary Table 1 of Soldner et al., 2009.

[0199] Consideration In the study described in this example, we derived hiPSCs from skin biopsies obtained from patients with idiopathic PD. We developed a robust reprogramming protocol that allowed for the reproducible generation of patient-specific hiPSCs with a low number of proviral vector integrations. The use of modified lentivirus with loxP sites flanking the integrated provirus allowed for the efficient removal of all transgene sequences and generated hiPSCs that were free of reprogramming factors. The factor-free hiPSCs were pluripotent and, using molecular criteria, more similar to embryo-derived hESCs than to conventional vector-containing parental hiPSCs. Efforts to understand the underlying pathophysiology of many neurodegenerative diseases, such as PD, are hampered by the lack of authentic in vitro models. Using hiPSC technology, we established hiPSC lines from five patients with idiopathic PD using DOX-inducible lentiviral vectors transducing either three or four reprogramming factors. These cells have been shown to possess all of the characteristics of pluripotent ES cells, including the ability to differentiate into cell types of all embryonic germ layers.

[0200] Our results show that removal of integrated transgenes by Cre / lox-mediated recombination can lead to vector-free hiPSCs. Previous reports failed to excise transgenes flanked by loxP sites (Takahashi and Yamanaka, 2006). Without wishing to be bound by theory, this is likely due to the large number of retroviral integrations (greater than 20), which precluded complete removal of all proviruses or caused castrophic genomic instability. Our results show that hiPSCs with as few as three or four viral integrations can be generated based on DOX-induced lentiviral transduction. Using DOX-inducible lentiviral vectors with loxP sites in the 3'LTR, we derived PD-specific, reprogramming factor-free hiPSCs after Cre recombinase-mediated cleavage of the transgene. Removal of promoter and transgene sequences in self-inactivating (SIN) lentiviral vectors is expected to significantly reduce the risk of oncogene transformation due to virus-mediated oncogene activation and / or re-expression of introduced transcription factors (Allen and Berns, 1996; von Kalle et al., 2004). The remaining risk of gene disruption can be eliminated by targeting reprogramming factors as polycistronic single expression vectors flanked by loxP sites to safe loci in the genome.

[0201] Factor-free hiPSCs maintain a pluripotent ESC-like state Although silencing of transgene expression has been reported for some hiPSCs, all hiPSCs generated to date (including the lines described in this example prior to removal of reprogramming factors) have sustained weak but detectable transgene expression (Dimos et al., 2008; Ebert et al., 2008; Hockemeyer et al., 2008; Park et al., 2008a; Yu et al., 2007). The question of whether hiPSCs depend on expression of reprogramming factors to maintain a pluripotent ESC-like state is therefore not conclusively resolved. The observation that factor-free hiPSCs were morphologically and biologically indistinguishable from parental hPSCs and maintained all the characteristics of hESCs demonstrates that human somatic cells can be reprogrammed to a self-sustaining pluripotent state, and that this state can be maintained even in the complete absence of exogenous reprogramming factors. These results provide further evidence that hiPSCs re-establish pluripotency-associated autoregulatory loops that have been proposed to rely on the activation of four endogenous transcription factors, OCT4, NANOG, SOX2, and TCF3 ( Jaenisch and Young, 2008 ).

[0202] Residual transgene expression from partially silenced viral vectors perturbs the transcriptional profile of hiPSCs Since the genomic integration site of a particular provirus influences the silencing of the provirus and its risk of reactivation, hiPSCs with identical and predictable properties cannot be generated by approaches that rely on stochastic silencing. Residual transgene expression may affect the differentiation properties of iPSCs. Indeed, striking differences have been observed between mouse ES and iPSCs in their ability to differentiate into cardiomyocytes (K. Hochedlinger, personal communication) as well as partially blocked EB-induced differentiation accompanied by incomplete OCT4 and NANOG downregulation in distinguishable hiPSC clones. These observations are consistent with the possibility that fluctuating base transcription of only partially silenced vectors may affect the generation of functionally differentiated cells.

[0203] In an effort to assess whether removal of the vector affects the properties of hiPSCs, we compared gene expression patterns in parental provirus-containing hiPSCs, factor-free hiPSCs, and embryo-derived hESCs. As previously reported (Park et al., 2008b; Takahashi et al., 2007; Yu et al., 2007), provirus-containing and factor-free hiPSCs clustered closely with hESCs when compared to donor fibroblasts. However, a more detailed analysis of the most divergent genes between the different hiPSC cell types revealed that embryo-derived hESCs and factor-free hiPSCs were more closely related to each other than to the provirus-containing parental hiPSCs. The remaining minor differences in gene expression between vector-free hiPSCs and hESCs may be due to the expression of transactivators that were not excised in our experiments. These results presented in this example provide clear evidence that provirus-based expression carried in conventional iPSCs can affect the molecular characteristics of the cells. The system described in this example provides a basis for further elucidating the effect of residual transgene expression, for example in the context of in vitro and in vivo differentiation. Moreover, these results demonstrate that the derivation of reprogramming factor-free hiPSCs is of great benefit not only for potential therapeutic applications, but also for biomedical research to develop reliable and reproducible in vitro models of disease. To this end, we propose that the generation of transgene-free hiPSCs by Cre-mediated excision offers significant advantages, such as its high efficiency and experimental simplicity. The system described in this example has the potential to become a routine technique for the derivation of hiPSCs, allowing the generation of standardized hiPSCs from various sources using various combinations of reprogramming factors.

[0204] [Table 4] N / A Not available aFurther information regarding these fibroblast cell lines is available from the Coriell Institute. b PDB 3F -12d was isolated in an experiment to determine the temporal requirements for transgene expression. PDB 3F -12d was isolated from a culture exposed to doxycycline for 12 days. c These cells were induced in experiments to determine the temporal requirements for transgene expression. PDB 4F -l to -3 were isolated from cultures exposed to doxycycline for 8 days, whereas PDB 4F -4 and -5 were isolated from cultures exposed to doxycycline for 10 and 12 days, respectively. d These hiPSC cells have been previously characterized in Hockemeyer et al., 2008.

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Claims

[Claim 1] A nucleic acid construct as described in the specification.

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