Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig

Abstract

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model. Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model. Induced nuclear reprogramming through induced pluripotent stem cell (iPS) 2The abbreviations used are: iPSinduced pluripotent stem cellESCembryonic stem cellPEFporcine embryonic fibroblastsAPalkaline phosphataseTRITCtetramethylrhodamine isothiocyanateRTreverse transcriptase. 2The abbreviations used are: iPSinduced pluripotent stem cellESCembryonic stem cellPEFporcine embryonic fibroblastsAPalkaline phosphataseTRITCtetramethylrhodamine isothiocyanateRTreverse transcriptase. technology is an amazing achievement full of challenge to the intellect and important practical implications (1Tweedell K.S. Curr. Stem Cell Res. Ther. 2008; 3: 151-162Crossref PubMed Scopus (24) Google Scholar, 2Pei D. Cell Res. 2008; 18: 221-223Crossref PubMed Scopus (16) Google Scholar). Overexpression of exogenous factors that are highly enriched in embryonic stem cell (ESC) can rearrange the genetic program of different cell types, including somatic and adult stem cells, and induce a long lasting ESC-like pluripotent state (3Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18482) Google Scholar, 4Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3498) Google Scholar, 5Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, 6Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar, 7Wernig M. Meissner A. Foreman R. Brambrink T. Ku M. Hochedlinger K. Bernstein B.E. Jaenisch R. Nature. 2007; 448: 318-324Crossref PubMed Scopus (2204) Google Scholar). The repercussions of iPS technology are vast: it provides a way to create patient-specific stem cells that bypasses ethical and technical issues surrounding human ESC derivation and somatic cell nuclear transfer (8Thomson J.A. Kalishman J. Golos T.G. Durning M. Harris C.P. Becker R.A. Hearn J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7844-7848Crossref PubMed Scopus (909) Google Scholar, 9Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H. Nature. 1997; 385: 810-813Crossref PubMed Scopus (4010) Google Scholar), a state of the art model for studying genetic diseases in vitro (10Park I.H. Arora N. Huo H. Maherali N. Ahfeldt T. Shimamura A. Lensch M.W. Cowan C. Hochedlinger K. Daley G.Q. Cell. 2008; 134: 877-886Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar, 11Ebert A.D. Yu J. Rose Jr., F.F. Mattis V.B. Lorson C.L. Thomson J.A. Svendsen C.N. Nature. 2009; 457: 277-280Crossref PubMed Scopus (1170) Google Scholar), and an incredible backwards route that can crystallize our current understanding of developmental and stem cell biology. Many questions, especially mechanistic, remain unanswered, but the current rhythm of research may bring iPS to clinical application sooner than expected. However, before jumping onto such extraordinary endeavor, safety must be scrupulously tested in an animal model close enough to humans. Nowadays that iPS technology is expanding, with improved delivery systems, chemical additions, new tissue culture conditions, and multiple cell sources being reported regularly, such animal model is essential to set up quality standards (12Kaji K. Norrby K. Paca A. Mileikovsky M. Mohseni P. Woltjen K. Nature. 2009; 458: 771-775Crossref PubMed Scopus (1035) Google Scholar, 13Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. Kaji K. Sung H.K. Nagy A. Nature. 2009; 458: 766-770Crossref PubMed Scopus (1432) Google Scholar, 14Okita K. Nakagawa M. Hyenjong H. Ichisaka T. Yamanaka S. Science. 2008; 322: 949-953Crossref PubMed Scopus (1588) Google Scholar, 15Stadtfeld M. Nagaya M. Utikal J. Weir G. Hochedlinger K. Science. 2008; 322: 945-949Crossref PubMed Scopus (1321) Google Scholar, 16Aoi T. Yae K. Nakagawa M. Ichisaka T. Okita K. Takahashi K. Chiba T. Yamanaka S. Science. 2008; 321: 699-702Crossref PubMed Scopus (859) Google Scholar, 17Hanna J. Markoulaki S. Schorderet P. Carey B.W. Beard C. Wernig M. Creyghton M.P. Steine E.J. Cassady J.P. Foreman R. Lengner C.J. Dausman J.A. Jaenisch R. Cell. 2008; 133: 250-264Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, 18Shi C. S. Cell Stem Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). and as the to reprogramming and but and are for safety in humans. are an but in ethical remain to and are to to a of have of and physiology is to up as the attractive model for from is used to pig and have and to human for V. Stem Cell 2008; PubMed Scopus Google Scholar). have also for of a in of more genetic D. M. S. J. J.L. Liu C.N. A. E.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of ESC from or animal species from mouse, human, and more the rat, of A. Stem Cell 2008; PubMed Scopus Google Scholar, I. A. J. 2007; PubMed Scopus Google Scholar). porcine genetic can be through and somatic cell nuclear transfer D. J.L. M. A. A. C.N. Science. PubMed Scopus Google Scholar). porcine embryonic fibroblasts the cell for somatic cell nuclear have a that D. C. A. M. P. J. P. Stem 2009; PubMed Scopus (24) Google Scholar). of pig iPS cell lines provide an of for a different from porcine iPS may as the of the miniature pig as a for of the pig is and and Liu Yu M. K. 2006; PubMed Scopus Google Scholar). of and the standard to induce and been used in mouse, human, and cells K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3498) Google Scholar, 5Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, H. J. P. P. H. W. J. Liu M. K. T. D. G. W. M. H. Cell Stem Cell. 2008; 3: Full Text Full Text PDF PubMed Scopus Google Scholar, W. W. S. J. T. A. H. S. Cell Stem Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). factors can human cells P. Yu J. G. J. Stem 2008; PubMed Scopus Google Scholar). and human factors by means of and used as a with close to culture for standard ESC and modified high with of ESC as and but with and a and of the for the cells in a set as is the in and we it the with or human iPS generation in cells onto with human ESC-like and with more the in and of the factors cell also and to more standard with human ESC-like in the and of in the and of in the human, the and positive for These cell lines be without but or without induced and the a high with of human and H. J. P. P. H. W. J. Liu M. K. T. D. G. W. M. H. Cell Stem Cell. 2008; 3: Full Text Full Text PDF PubMed Scopus Google Scholar), and positive of the into the of tested iPS cell lines an of high or in and high in iPS of of pluripotency porcine iPS positive for the human and and factors and in or iPS cell lines resulting from or human or in high of with that the and also of and for the also been by in human and iPS cell lines J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar, J. C. S. J. J. H. N. H. Cell Stem Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). can the of iPS cell lines into full iPS J. Ku M. Wernig M. Schorderet P. Bernstein B.E. Jaenisch R. Meissner A. Nature. 2008; PubMed Scopus Google Scholar, J. J. J. A. 2008; PubMed Scopus Google Scholar). of pig iPS cell lines with a of any in cell or ESC markers of and and but levels of the tested ESC markers pig iPS cell lines into the of in teratomas from the three germ and and of pig iPS cell lines that pluripotent with of and have been of isolated porcine cells into V. Stem Cell 2008; PubMed Scopus Google Scholar). the of our study we pig iPS cell lines into from and into The of such is our of using pig ESC that been for to of pig iPS and the of from pig ESC been reported V. Stem Cell 2008; PubMed Scopus Google of porcine iPS cell with ESC-like with a are pig iPS is the and positive for is in with of exogenous or human factors into the of pig iPS to and for the high in iPS with with iPS and M. H. of porcine iPS cell of the ESC are the and for iPS that and serve as an are of iPS cell the and are also and used as used as of human factors and iPS with of ESC markers iPS and with that the of the in iPS cell lines with from cells used as for the M. H. iPS cell lines are in into the three germ layers. from the are a with are the and is for and are in and a in and tissue is in an of in different human iPS cell lines with that the iPS cell lines to different H. we a for reprogramming into iPS cells, and provide for The of of for the in our cell lines are may have an the of our cell lines to differentiate into different and may the long for The of different cell other than fibroblasts a iPS generation D. K. Wang H. W. Wang T. W. J. G. M.A. S. D. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, V. G. P. K. D. M. D. J. K. C. C. M. H. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Utikal J. K. Jaenisch R. Hochedlinger K. Stem 2008; PubMed Scopus Google Scholar). more of to iPS different porcine be important and full by the of the from an cell that the animal be for of exogenous factors also of the exogenous and we are up for porcine iPS D. Beard C. G. A. M. Jaenisch R. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). of as to be in pig may from established and human and in in are the may be The can be or other for the Tibetan in of have for of in a and have that to iPS of cells from other pig be important as are using in iPS cell lines be the long of iPS generation is an it is in The the in of can be more than the and in the of an model for porcine iPS research is and Induced nuclear reprogramming through induced pluripotent stem cell (iPS) 2The abbreviations used are: iPSinduced pluripotent stem cellESCembryonic stem cellPEFporcine embryonic fibroblastsAPalkaline phosphataseTRITCtetramethylrhodamine isothiocyanateRTreverse transcriptase. 2The abbreviations used are: iPSinduced pluripotent stem cellESCembryonic stem cellPEFporcine embryonic fibroblastsAPalkaline phosphataseTRITCtetramethylrhodamine isothiocyanateRTreverse transcriptase. technology is an amazing achievement full of challenge to the intellect and important practical implications (1Tweedell K.S. Curr. Stem Cell Res. Ther. 2008; 3: 151-162Crossref PubMed Scopus (24) Google Scholar, 2Pei D. Cell Res. 2008; 18: 221-223Crossref PubMed Scopus (16) Google Scholar). Overexpression of exogenous factors that are highly enriched in embryonic stem cell (ESC) can rearrange the genetic program of different cell types, including somatic and adult stem cells, and induce a long lasting ESC-like pluripotent state (3Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18482) Google Scholar, 4Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3498) Google Scholar, 5Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, 6Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar, 7Wernig M. Meissner A. Foreman R. Brambrink T. Ku M. Hochedlinger K. Bernstein B.E. Jaenisch R. Nature. 2007; 448: 318-324Crossref PubMed Scopus (2204) Google Scholar). The repercussions of iPS technology are vast: it provides a way to create patient-specific stem cells that bypasses ethical and technical issues surrounding human ESC derivation and somatic cell nuclear transfer (8Thomson J.A. Kalishman J. Golos T.G. Durning M. Harris C.P. Becker R.A. Hearn J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7844-7848Crossref PubMed Scopus (909) Google Scholar, 9Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H. Nature. 1997; 385: 810-813Crossref PubMed Scopus (4010) Google Scholar), a state of the art model for studying genetic diseases in vitro (10Park I.H. Arora N. Huo H. Maherali N. Ahfeldt T. Shimamura A. Lensch M.W. Cowan C. Hochedlinger K. Daley G.Q. Cell. 2008; 134: 877-886Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar, 11Ebert A.D. Yu J. Rose Jr., F.F. Mattis V.B. Lorson C.L. Thomson J.A. Svendsen C.N. Nature. 2009; 457: 277-280Crossref PubMed Scopus (1170) Google Scholar), and an incredible backwards route that can crystallize our current understanding of developmental and stem cell biology. Many questions, especially mechanistic, remain unanswered, but the current rhythm of research may bring iPS to clinical application sooner than expected. However, before jumping onto such extraordinary endeavor, safety must be scrupulously tested in an animal model close enough to humans. Nowadays that iPS technology is expanding, with improved delivery systems, chemical additions, new tissue culture conditions, and multiple cell sources being reported regularly, such animal model is essential to set up quality standards (12Kaji K. Norrby K. Paca A. Mileikovsky M. Mohseni P. Woltjen K. Nature. 2009; 458: 771-775Crossref PubMed Scopus (1035) Google Scholar, 13Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. Kaji K. Sung H.K. Nagy A. Nature. 2009; 458: 766-770Crossref PubMed Scopus (1432) Google Scholar, 14Okita K. Nakagawa M. Hyenjong H. Ichisaka T. Yamanaka S. Science. 2008; 322: 949-953Crossref PubMed Scopus (1588) Google Scholar, 15Stadtfeld M. Nagaya M. Utikal J. Weir G. Hochedlinger K. Science. 2008; 322: 945-949Crossref PubMed Scopus (1321) Google Scholar, 16Aoi T. Yae K. Nakagawa M. Ichisaka T. Okita K. Takahashi K. Chiba T. Yamanaka S. Science. 2008; 321: 699-702Crossref PubMed Scopus (859) Google Scholar, 17Hanna J. Markoulaki S. Schorderet P. Carey B.W. Beard C. Wernig M. Creyghton M.P. Steine E.J. Cassady J.P. Foreman R. Lengner C.J. Dausman J.A. Jaenisch R. Cell. 2008; 133: 250-264Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, 18Shi C. S. Cell Stem Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). and as the to reprogramming and but and are for safety in humans. are an but in ethical remain to and are to to a of have of and physiology is to up as the attractive model for from is used to pig and have and to human for V. Stem Cell 2008; PubMed Scopus Google Scholar). have also for of a in of more genetic D. M. S. J. J.L. Liu C.N. A. E.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of ESC from or animal species from mouse, human, and more the rat, of A. Stem Cell 2008; PubMed Scopus Google Scholar, I. A. J. 2007; PubMed Scopus Google Scholar). porcine genetic can be through and somatic cell nuclear transfer D. J.L. M. A. A. C.N. Science. PubMed Scopus Google Scholar). porcine embryonic fibroblasts the cell for somatic cell nuclear have a that D. C. A. M. P. J. P. Stem 2009; PubMed Scopus (24) Google Scholar). of pig iPS cell lines provide an of for a different from porcine iPS may as the of induced pluripotent stem cell embryonic stem cell porcine embryonic fibroblasts alkaline transcriptase. induced pluripotent stem cell embryonic stem cell porcine embryonic fibroblasts alkaline transcriptase. the miniature pig as a for of the pig is and and Liu Yu M. K. 2006; PubMed Scopus Google Scholar). of and the standard to induce and been used in mouse, human, and cells K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3498) Google Scholar, 5Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, H. J. P. P. H. W. J. Liu M. K. T. D. G. W. M. H. Cell Stem Cell. 2008; 3: Full Text Full Text PDF PubMed Scopus Google Scholar, W. W. S. J. T. A. H. S. Cell Stem Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). factors can human cells P. Yu J. G. J. Stem 2008; PubMed Scopus Google Scholar). and human factors by means of and used as a with close to culture for standard ESC and modified high with of ESC as and but with and a and of the for the cells in a set as is the in and we it the with or human iPS generation in cells onto with human ESC-like and with more the in and of the factors cell also and to more standard with human ESC-like in the and of in the and of in the human, the and positive for These cell lines be without but or without induced and the a high with of human and H. J. P. P. H. W. J. Liu M. K. T. D. G. W. M. H. Cell Stem Cell. 2008; 3: Full Text Full Text PDF PubMed Scopus Google Scholar), and positive of the into the of tested iPS cell lines an of high or in and high in iPS of of pluripotency porcine iPS positive for the human and and factors and in or iPS cell lines resulting from or human or in high of with that the and also of and for the also been by in human and iPS cell lines J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar, J. C. S. J. J. H. N. H. Cell Stem Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). can the of iPS cell lines into full iPS J. Ku M. Wernig M. Schorderet P. Bernstein B.E. Jaenisch R. Meissner A. Nature. 2008; PubMed Scopus Google Scholar, J. J. J. A. 2008; PubMed Scopus Google Scholar). of pig iPS cell lines with a of any in cell or ESC markers of and and but levels of the tested ESC markers pig iPS cell lines into the of in teratomas from the three germ and and of pig iPS cell lines that pluripotent with of and have been of isolated porcine cells into V. Stem Cell 2008; PubMed Scopus Google Scholar). the of our study we pig iPS cell lines into from and into The of such is our of using pig ESC that been for to of pig iPS and the of from pig ESC been reported V. Stem Cell 2008; PubMed Scopus Google of porcine iPS cell of the ESC are the and for iPS that and serve as an are of iPS cell the and are also and used as used as of human factors and iPS with of ESC markers iPS and with that the of the in iPS cell lines with from cells used as for the M. H. iPS cell lines are in into the three germ layers. from the are a with are the and is for and are in and a in and tissue is in an of in different human iPS cell lines with that the iPS cell lines to different H. we a for reprogramming into iPS cells, and provide for The of of for the in our cell lines are may have an the of our cell lines to differentiate into different and may the long for The of different cell other than fibroblasts a iPS generation D. K. Wang H. W. Wang T. W. J. G. M.A. S. D. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, V. G. P. K. D. M. D. J. K. C. C. M. H. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Utikal J. K. Jaenisch R. Hochedlinger K. Stem 2008; PubMed Scopus Google Scholar). more of to iPS different porcine be important and full by the of the from an cell that the animal be for of exogenous factors also of the exogenous and we are up for porcine iPS D. Beard C. G. A. M. Jaenisch R. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). of as to be in pig may from established and human and in in are the may be The can be or other for the Tibetan in of have for of in a and have that to iPS of cells from other pig be important as are using in iPS cell lines be the long of iPS generation is an it is in The the in of can be more than the and in the of an model for porcine iPS research is and the miniature pig as a for of the pig is and and Liu Yu M. K. 2006; PubMed Scopus Google Scholar). of and the standard to induce and been used in mouse, human, and cells K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3498) Google Scholar, 5Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14662) Google Scholar, H. J. P. P. H. W. J. Liu M. K. T. D. G. W. M. H. Cell Stem Cell. 2008; 3: Full Text Full Text PDF PubMed Scopus Google Scholar, W. W. S. J. T. A. H. S. Cell Stem Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). factors can human cells P. Yu J. G. J. Stem 2008; PubMed Scopus Google Scholar). and human factors by means of and used as a with close to culture for standard ESC and modified high with of ESC as and but with and a and of the for the cells in a set as is the in and we it the with or human iPS generation in cells onto with human ESC-like and with more the in and of the factors cell also and to more standard with human ESC-like in the and of in the and of in the human, the and positive for These cell lines be without but or without induced and the a high with of human and H. J. P. P. H. W. J. Liu M. K. T. D. G. W. M. H. Cell Stem Cell. 2008; 3: Full Text Full Text PDF PubMed Scopus Google Scholar), and positive of the into the of tested iPS cell lines an of high or in and high in iPS of of pluripotency porcine iPS positive for the human and and factors and in or iPS cell lines resulting from or human or in high of with that the and also of and for the also been by in human and iPS cell lines J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin I.I. Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8046) Google Scholar, J. C. S. J. J. H. N. H. Cell Stem Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). can the of iPS cell lines into full iPS J. Ku M. Wernig M. Schorderet P. Bernstein B.E. Jaenisch R. Meissner A. Nature. 2008; PubMed Scopus Google Scholar, J. J. J. A. 2008; PubMed Scopus Google Scholar). of pig iPS cell lines with a of any in cell or ESC markers of and and but levels of the tested ESC markers pig iPS cell lines into the of in teratomas from the three germ and and of pig iPS cell lines that pluripotent with of and have been of isolated porcine cells into V. Stem Cell 2008; PubMed Scopus Google Scholar). the of our study we pig iPS cell lines into from and into The of such is our of using pig ESC that been for to of pig iPS and the of from pig ESC been reported V. Stem Cell 2008; PubMed Scopus Google Scholar). we a for reprogramming into iPS cells, and provide for The of of for the in our cell lines are may have an the of our cell lines to differentiate into different and may the long for The of different cell other than fibroblasts a iPS generation D. K. Wang H. W. Wang T. W. J. G. M.A. S. D. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, V. G. P. K. D. M. D. J. K. C. C. M. H. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Utikal J. K. Jaenisch R. Hochedlinger K. Stem 2008; PubMed Scopus Google Scholar). more of to iPS different porcine be important and full by the of the from an cell that the animal be for of exogenous factors also of the exogenous and we are up for porcine iPS D. Beard C. G. A. M. Jaenisch R. Cell. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). of as to be in pig may from established and human and in in are the may be The can be or other for the Tibetan in of have for of in a and have that to iPS of cells from other pig be important as are using in iPS cell lines be the long of iPS generation is an it is in The the in of can be more than the and in the of an model for porcine iPS research is and of the for for and the of