Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

Abstract

Differentiated cells can be reprogrammed into pluripotent stem cells, known as “induced pluripotent stem cells” (iPSCs), through the overexpression of defined transcription factors. The creation of iPSC lines has opened new avenues for patient-specific cell replacement therapies for regenerative medicine. However, the clinical utilization of iPSCs is largely impeded by two limitations. The first limitation is the low efficiency of iPSCs generation from differentiated cells. The second limitation is that many iPSC lines are not authentically pluripotent, as many cell lines inefficiently differentiate into differentiated cell types when they are tested for their ability to complement embryonic development. Thus, the “quality” of iPSCs must be increased if they are to be differentiated into specialized cell types for cell replacement therapies. Overcoming these two limitations is paramount to facilitate the widespread employment of iPSCs for therapeutic purposes. Here, we summarize recent progress made in strategies enabling the efficient production of high-quality iPSCs, including choice of reprogramming factors, choice of target cell type, and strategies to improve iPSC quality.

Keywords

iPSCs / reprogramming / embryonic stem cells / efficiency and quality

References

[1] Aasen, T., Raya, A., Barrero, M.J., Garreta, E., Consiglio, A., Gonzalez, F., Vassena, R., Bilic, J., Pekarik, V., Tiscornia, G., . (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26, 1276-1284 .10.1038/nbt.1503
[2] Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T., and Yamanaka, S. (2008). Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 321, 699-702 .10.1126/science.1154884
[3] Blelloch, R., Venere, M., Yen, J., and Ramalho-Santos, M. (2007). Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell 1, 245-247 .10.1016/j.stem.2007.08.008
[4] Bosnali, M., and Edenhofer, F. (2008). Generation of transducible versions of transcription factors Oct4 and Sox2. Biol Chem 389, 851-861 .10.1515/BC.2008.106
[5] Brambrink, T., Foreman, R., Welstead, G.G., Lengner, C.J., Wernig, M., Suh, H., and Jaenisch, R. (2008). Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151-159 .10.1016/j.stem.2008.01.004
[6] Carlson, H., Ota, S., Song, Y., Chen, Y., and Hurlin, P.J. (2002). Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic differentiation. Oncogene 21, 3827-3835 .10.1038/sj.onc.1205476
[7] Chang, C.W., Lai, Y.S., Pawlik, K.M., Liu, K., Sun, C.W., Li, C., Schoeb, T.R., and Townes, T.M. (2009). Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27, 1042-1049 .10.1002/stem.39
[8] Eminli, S., Foudi, A., Stadtfeld, M., Maherali, N., Ahfeldt, T., Mostoslavsky, G., Hock, H., and Hochedlinger, K. (2009). Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nat Genet 41, 968-976 .10.1038/ng.428
[9] Eminli, S., Utikal, J., Arnold, K., Jaenisch, R., and Hochedlinger, K. (2008). Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression. Stem Cells 26, 2467-2474 .10.1634/stemcells.2008-0317
[10] Esteban, M.A., and Pei, D. (2012). Vitamin C improves the quality of somatic cell reprogramming. Nat Genet 44, 366-367 .10.1038/ng.2222
[11] Esteban, M.A., Wang, T., Qin, B., Yang, J., Qin, D., Cai, J., Li, W., Weng, Z., Chen, J., Ni, S., . (2010). Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6, 71-79 .10.1016/j.stem.2009.12.001
[12] Esteban, M.A., Xu, J., Yang, J., Peng, M., Qin, D., Li, W., Jiang, Z., Chen, J., Deng, K., Zhong, M., . (2009). Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem 284, 17634-17640 .10.1074/jbc.M109.008938
[13] Ezashi, T., Telugu, B.P., Alexenko, A.P., Sachdev, S., Sinha, S., and Roberts, R.M. (2009). Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci U S A 106, 10993-10998 .10.1073/pnas.0905284106
[14] Feng, B., Jiang, J., Kraus, P., Ng, J.H., Heng, J.C., Chan, Y.S., Yaw, L.P., Zhang, W., Loh, Y.H., Han, J., . (2009a). Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat Cell Biol 11, 197-203 .10.1038/ncb1827
[15] Feng, B., Ng, J.H., Heng, J.C., and Ng, H.H. (2009b). Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells. Cell Stem Cell 4, 301-312 .10.1016/j.stem.2009.03.005
[16] Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., and Hasegawa, M. (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85, 348-362 .10.2183/pjab.85.348
[17] Gonzalez, F., Barragan Monasterio, M., Tiscornia, G., Montserrat Pulido, N., Vassena, R., Batlle Morera, L., Rodriguez Piza, I., and Izpisua Belmonte, J.C. (2009). Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector. Proc Natl Acad Sci U S A 106, 8918-8922 .10.1073/pnas.0901471106
[18] Gore, A., Li, Z., Fung, H.L., Young, J.E., Agarwal, S., Antosiewicz-Bourget, J., Canto, I., Giorgetti, A., Israel, M.A., Kiskinis, E., . (2011). Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63-67 .10.1038/nature09805
[19] Graf, T., and Enver, T. (2009). Forcing cells to change lineages. Nature 462, 587-594 .10.1038/nature08533
[20] Han, J., Yuan, P., Yang, H., Zhang, J., Soh, B.S., Li, P., Lim, S.L., Cao, S., Tay, J., Orlov, Y.L., . (2010). Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature 463, 1096-1100 .10.1038/nature08735
[21] Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., . (2008). Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250-264 .10.1016/j.cell.2008.03.028
[22] Heng, J.C., Feng, B., Han, J., Jiang, J., Kraus, P., Ng, J.H., Orlov, Y.L., Huss, M., Yang, L., Lufkin, T., . (2010). The nuclear receptor Nr5a2 can replace Oct4 in the reprogramming of murine somatic cells to pluripotent cells. Cell Stem Cell 6, 167-174 .10.1016/j.stem.2009.12.009
[23] Hochedlinger, K., and Jaenisch, R. (2006). Nuclear reprogramming and pluripotency. Nature 441, 1061-1067 .10.1038/nature04955
[24] Hong, H., Takahashi, K., Ichisaka, T., Aoi, T., Kanagawa, O., Nakagawa, M., Okita, K., and Yamanaka, S. (2009). Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460, 1132-1135 .10.1038/nature08235
[25] Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A.E., and Melton, D.A. (2008a). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 26, 795-797 .10.1038/nbt1418
[26] Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and Melton, D.A. (2008b). Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol 26, 1269-1275 .10.1038/nbt.1502
[27] Hussein, S.M., Batada, N.N., Vuoristo, S., Ching, R.W., Autio, R., Narva, E., Ng, S., Sourour, M., Hamalainen, R., Olsson, C., . (2011). Copy number variation and selection during reprogramming to pluripotency. Nature 471, 58-62 .10.1038/nature09871
[28] Ichida, J.K., Blanchard, J., Lam, K., Son, E.Y., Chung, J.E., Egli, D., Loh, K.M., Carter, A.C., Di Giorgio, F.P., Koszka, K., . (2009). A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5, 491-503 .10.1016/j.stem.2009.09.012
[29] Ivanova, N., Dobrin, R., Lu, R., Kotenko, I., Levorse, J., DeCoste, C., Schafer, X., Lun, Y., and Lemischka, I.R. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533-538 .10.1038/nature04915
[30] Judson, R.L., Babiarz, J.E., Venere, M., and Blelloch, R. (2009). Embryonic stem cell-specific microRNAs promote induced pluripotency. Nat Biotechnol 27, 459-461 .10.1038/nbt.1535
[31] Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P., and Woltjen, K. (2009). Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458, 771-775 .10.1038/nature07864
[32] Kawamura, T., Suzuki, J., Wang, Y.V., Menendez, S., Morera, L.B., Raya, A., Wahl, G.M., and Belmonte, J.C. (2009). Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460, 1140-1144 .10.1038/nature08311
[33] Keefer, C.L., Pant, D., Blomberg, L., and Talbot, N.C. (2007). Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod Sci 98, 147-168 .10.1016/j.anireprosci.2006.10.009
[34] Kim, J.B., Greber, B., Arauzo-Bravo, M.J., Meyer, J., Park, K.I., Zaehres, H., and Scholer, H.R. (2009a). Direct reprogramming of human neural stem cells by OCT4. Nature 461, 649-643 .10.1038/nature08436
[35] Kim, J.B., Sebastiano, V., Wu, G., Arauzo-Bravo, M.J., Sasse, P., Gentile, L., Ko, K., Ruau, D., Ehrich, M., van den Boom, D., . (2009b). Oct4-induced pluripotency in adult neural stem cells. Cell 136, 411-419 .10.1016/j.cell.2009.01.023
[36] Kim, J.B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-Bravo, M.J., Ruau, D., Han, D.W., Zenke, M., . (2008). Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454, 646-650 .10.1038/nature07061
[37] Kishigami, S., Mizutani, E., Ohta, H., Hikichi, T., Thuan, N.V., Wakayama, S., Bui, H.T., and Wakayama, T. (2006). Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem Biophys Res Commun 340, 183-189 .10.1016/j.bbrc.2005.11.164
[38] Krizhanovsky, V., and Lowe, S.W. (2009). Stem cells: The promises and perils of p53. Nature 460, 1085-1086 .10.1038/4601085a
[39] Kustikova, O., Fehse, B., Modlich, U., Yang, M., Dullmann, J., Kamino, K., von Neuhoff, N., Schlegelberger, B., Li, Z., and Baum, C. (2005). Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 308, 1171-1174 .10.1126/science.1105063
[40] Li, H., Collado, M., Villasante, A., Strati, K., Ortega, S., Canamero, M., Blasco, M.A., and Serrano, M. (2009a). The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460, 1136-1139 .10.1038/nature08290
[41] Li, W., Wei, W., Zhu, S., Zhu, J., Shi, Y., Lin, T., Hao, E., Hayek, A., Deng, H., and Ding, S. (2009b). Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4, 16-19 .10.1016/j.stem.2008.11.014
[42] Liao, J., Cui, C., Chen, S., Ren, J., Chen, J., Gao, Y., Li, H., Jia, N., Cheng, L., Xiao, H., . (2009). Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4, 11-15 .10.1016/j.stem.2008.11.013
[43] Lin, S.L., Chang, D.C., Chang-Lin, S., Lin, C.H., Wu, D.T., Chen, D.T., and Ying, S.Y. (2008). Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115-2124 .10.1261/rna.1162708
[44] Liu, H., Zhu, F., Yong, J., Zhang, P., Hou, P., Li, H., Jiang, W., Cai, J., Liu, M., Cui, K., . (2008). Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3, 587-590 .10.1016/j.stem.2008.10.014
[45] Lluis, F., Pedone, E., Pepe, S., and Cosma, M.P. (2008). Periodic activation of Wnt/beta-catenin signaling enhances somatic cell reprogramming mediated by cell fusion. Cell Stem Cell 3, 493-507 .10.1016/j.stem.2008.08.017
[46] Loh, Y.H., Agarwal, S., Park, I.H., Urbach, A., Huo, H., Heffner, G.C., Kim, K., Miller, J.D., Ng, K., and Daley, G.Q. (2009). Generation of induced pluripotent stem cells from human blood. Blood , 5476-5479 .10.1182/blood-2009-02-204800
[47] Lowry, W.E., Richter, L., Yachechko, R., Pyle, A.D., Tchieu, J., Sridharan, R., Clark, A.T., and Plath, K. (2008). Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci U S A 105, 2883-2888 .10.1073/pnas.0711983105
[48] Lyssiotis, C.A., Foreman, R.K., Staerk, J., Garcia, M., Mathur, D., Markoulaki, S., Hanna, J., Lairson, L.L., Charette, B.D., Bouchez, L.C., . (2009). Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4. Proc Natl Acad Sci U S A 106, 8912-8917 .10.1073/pnas.0903860106
[49] Maekawa, M., Yamaguchi, K., Nakamura, T., Shibukawa, R., Kodanaka, I., Ichisaka, T., Kawamura, Y., Mochizuki, H., Goshima, N., and Yamanaka, S. (2011). Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature 474, 225-229 .10.1038/nature10106
[50] Maherali, N., and Hochedlinger, K. (2008). Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell 3, 595-605 .10.1016/j.stem.2008.11.008
[51] Marion, R.M., Strati, K., Li, H., Murga, M., Blanco, R., Ortega, S., Fernandez-Capetillo, O., Serrano, M., and Blasco, M.A. (2009). A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460, 1149-1153 .10.1038/nature08287
[52] Markoulaki, S., Hanna, J., Beard, C., Carey, B.W., Cheng, A.W., Lengner, C.J., Dausman, J.A., Fu, D., Gao, Q., Wu, S., . (2009). Transgenic mice with defined combinations of drug-inducible reprogramming factors. Nat Biotechnol 27, 169-171 .10.1038/nbt.1520
[53] Marson, A., Foreman, R., Chevalier, B., Bilodeau, S., Kahn, M., Young, R.A., and Jaenisch, R. (2008). Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3, 132-135 .10.1016/j.stem.2008.06.019
[54] Matsui, T., Leung, D., Miyashita, H., Maksakova, I.A., Miyachi, H., Kimura, H., Tachibana, M., Lorincz, M.C., and Shinkai, Y. (2010). Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927-931 .10.1038/nature08858
[55] Melton, C., Judson, R.L., and Blelloch, R. (2010). Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature 463, 621-626 .10.1038/nature08725
[56] Miyabayashi, T., Teo, J.L., Yamamoto, M., McMillan, M., Nguyen, C., and Kahn, M. (2007). Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl Acad Sci U S A 104, 5668-5673 .10.1073/pnas.0701331104
[57] Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., and Yamanaka, S. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26, 101-106 .10.1038/nbt1374
[58] Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., and Yamanaka, S. (2008). Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949-953 .10.1126/science.1164270
[59] Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., and Daley, G.Q. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141-146 .10.1038/nature06534
[60] Qin, D., Gan, Y., Shao, K., Wang, H., Li, W., Wang, T., He, W., Xu, J., Zhang, Y., Kou, Z., . (2008). Mouse meningiocytes express Sox2 and yield high efficiency of chimeras after nuclear reprogramming with exogenous factors. J Biol Chem 283, 33730-33735 .10.1074/jbc.M806788200
[61] Shi, Y., Desponts, C., Do, J.T., Hahm, H.S., Scholer, H.R., and Ding, S. (2008a). Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3, 568-574 .10.1016/j.stem.2008.10.004
[62] Shi, Y., Do, J.T., Desponts, C., Hahm, H.S., Scholer, H.R., and Ding, S. (2008b). A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2, 525-528 .10.1016/j.stem.2008.05.011
[63] Shi, Y., Zhao, Y., and Deng, H. (2010). Powering reprogramming with vitamin C. Cell Stem Cell 6, 1-2 .10.1016/j.stem.2009.12.012
[64] Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol 6, e253.10.1371/journal.pbio.0060253
[65] Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G.W., Cook, E.G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M., . (2009). Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-977 .10.1016/j.cell.2009.02.013
[66] Stadtfeld, M., Apostolou, E., Akutsu, H., Fukuda, A., Follett, P., Natesan, S., Kono, T., Shioda, T., and Hochedlinger, K. (2010). Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465, 175-181 .10.1038/nature09017
[67] Stadtfeld, M., Brennand, K., and Hochedlinger, K. (2008a). Reprogramming of pancreatic beta cells into induced pluripotent stem cells. Curr Biol 18, 890-894 .10.1016/j.cub.2008.05.010
[68] Stadtfeld, M., Maherali, N., Breault, D.T., and Hochedlinger, K. (2008b). Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230-240 .10.1016/j.stem.2008.02.001
[69] Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., and Hochedlinger, K. (2008c). Induced pluripotent stem cells generated without viral integration. Science 322, 945-949 .10.1126/science.1162494
[70] Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 .10.1016/j.cell.2007.11.019
[71] Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 .10.1016/j.cell.2006.07.024
[72] Trounson, A. (2009). Rats, cats, and elephants, but still no unicorn: induced pluripotent stem cells from new species. Cell Stem Cell 4, 3-4 .10.1016/j.stem.2008.12.002
[73] Utikal, J., Polo, J.M., Stadtfeld, M., Maherali, N., Kulalert, W., Walsh, R.M., Khalil, A., Rheinwald, J.G., and Hochedlinger, K. (2009). Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460, 1145-1148 .10.1038/nature08285
[74] Varas, F., Stadtfeld, M., de Andres-Aguayo, L., Maherali, N., di Tullio, A., Pantano, L., Notredame, C., Hochedlinger, K., and Graf, T. (2009). Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions. Stem Cells 27, 300-306 .10.1634/stemcells.2008-0696
[75] Wernig, M., Lengner, C.J., Hanna, J., Lodato, M.A., Steine, E., Foreman, R., Staerk, J., Markoulaki, S., and Jaenisch, R. (2008). A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 26, 916-924 .10.1038/nbt1483
[76] Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324 .10.1038/nature05944
[77] West, F.D., Terlouw, S.L., Kwon, D.J., Mumaw, J.L., Dhara, S.K., Hasneen, K., Dobrinsky, J.R., and Stice, S.L. (2010). Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev 19, 1211-1220 .10.1089/scd.2009.0458
[78] Wobus, A.M., and Boheler, K.R. (2005). Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 85, 635-678 .10.1152/physrev.00054.2003
[79] Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., . (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766-770 .10.1038/nature07863
[80] Wu, Z., Chen, J., Ren, J., Bao, L., Liao, J., Cui, C., Rao, L., Li, H., Gu, Y., Dai, H., . (2009). Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol 1, 46-54 .10.1093/jmcb/mjp003
[81] Yamanaka, S. (2007). Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1, 39-49 .10.1016/j.stem.2007.05.012
[82] Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, II, and Thomson, J.A. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801 .10.1126/science.1172482
[83] Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., . (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 .10.1126/science.1151526
[84] Zhao, Y., Yin, X., Qin, H., Zhu, F., Liu, H., Yang, W., Zhang, Q., Xiang, C., Hou, P., Song, Z., . (2008). Two supporting factors greatly improve the efficiency of human iPSC generation. CellS tem Cell 3, 475-479 .10.1016/j.stem.2008.10.002