建立以重組蛋白生產安全之自體多能性幹細胞的方法

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研究生：	趙冠豪 Guan-Hao Zhao
論文名稱：	建立以重組蛋白生產安全之自體多能性幹細胞的方法 Formulating a protocol for generating safe pluripotent stem cells from somatic cells by recombinant proteins
指導教授：	陳盛良 Shen-Liang Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2014
畢業學年度：	102
語文別：	中文
論文頁數：	91
中文關鍵詞：	誘導性多功能幹細胞、蛋白轉導區塊、重新編成
外文關鍵詞：	induced pluripotent stem cells, protein transduction domain, Reprogramming
相關次數：	點閱：8 下載：0
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利用特定轉錄因子將體細胞轉變為誘導多能性幹細胞在再生醫學上有令人期待的前景。至今已有許多方法能產生誘導多能性幹細胞，例如使用反轉錄病毒、慢病毒、質體、轉位子、合成信使核醣核酸以及重組蛋白。然而，外送的基因物質可能造成不預期的基因組突變。相較之下，以送入重組蛋白產生多能性幹細胞的方式可以避免去氧核醣核酸隨機插入的可能性。在這篇研究中，我們利用聚精胺酸蛋白轉導區塊來送達重組的重新編成因子。五個重新編成因子：Oct4,Sox2,Nanog,Lin28和c-Myc的羧基端都黏合上聚精胺酸蛋白轉導區塊。我們能將這些因子順利的表達並純化；然而，Oct4和Nanog蛋白的表現量相對來說非常低。令我們驚訝的是，將MyoD轉活區塊黏合在Oct4和Nanog的氨基端顯著性的增加蛋白表現。這些蛋白表現在大腸桿菌的包涵體或是原生型態都可以被溶解、重新摺疊並以鎳螯合物凝膠純化。純化的蛋白能轉導通過細胞膜並轉移至細胞核。現今我們正以這些蛋白組合一些小分子化合物，如丙戊酸，來測定最適合重新編成體細胞轉變為誘導多能性幹細胞以及種系特異性幹細胞的條件。

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined factors has showed hopeful perspective in regenerative medicine. Currently, many approaches are employed to generate iPSCs, including retroviruses, lentiviruses, adenoviruses, plasmids, transposons, synthetic mRNAs and recombinant proteins; however, the introduction of ectopic genetic materials may cause unexpected genetic modifications. In contrast, delivery of recombinant proteins can avoid the possibility of random DNA integration. In this study, we utilized poly-arginine (poly-R) protein transduction domain (PTD) for delivery of recombinant reprogramming factors. The PTD was fused to the C-terminus of five reprogramming factors: Oct4, Sox2, Nanog, Lin28 and c-Myc. These chimera factors could be expressed and purified with normal function in E.coli; however, the expression of Oct4 and Nanog was relatively low. To our surprise, fusion of the powerful MyoD transactivation domain to the N-terminus of Oct4 and Nanog significantly enhanced the protein yield. These proteins expressed in E.coli as inclusion bodies or native forms were solubilized, refolded, and purified using Nickel-Chelating Sepharose. These purified proteins were able to transduce the plasma membrane and translocate into nucleus. Currently, we are combining these proteins and small compounds, such as valproic acid, to determine the optimal condition for reprogram somatic cells into iPSC and lineage-specific stem cells.

Table of Contents
中文摘要    i
Abstract    ii
Declaration    iii
Acknowledgement    iv
Abbreviations    v
Table of Contents    vi
I.    Introduction    1
II.    Materials and Methods    8
1.    Cloning    8
1-1. Plasmid    8
1-1.1. pET-LpRH, pET-M3-LpRH and pET-HA2-LpRH    8
1-1.2. pET-M3-Oct4-LpRH    8
1-1.3. pET-M3-Nanog-LpRH, pET-HA2-Nanog-LpRH    8
1-1.4. pET-Klf4-LpRH and pET-M3-Klf4-LpRH    8
1-1.5. pET-HA2-p53R248Q-LpRH and pET-HA2-p53R248W-LpRH    9
1-1.6. pGEX4T1-LIF    9
1-1.7. pStable-Fgf4-promoter-enhancer    9
1-2. Molecular cloning technique    9
1-2.1. Polymerase chain reaction    9
1-2.2. Overlap extension polymerase chain reaction (OE-PCR)    10
1-2.3. Enzymatic digestion    10
1-2.4. Blunt-end formation    11
1-2.5. Phosphorylation of insert DNA    11
1-2.6. De-phosphorylation of vector DNA    11
1-2.7. Ligation    11
1-2.8. Transformation    11
1-2.9. Alkaline lysis method for plasmid DNA isolation    12
2.    Protein expression and purification    12
2-1. Induction of recombinant protein    12
2-2. Column preparation    13
2-3. Purification of recombinant protein under native condition    14
2-4. Isolation and solubilization of inclusion bodies    14
2-5. Purification of recombinant protein under denature condition    15
2-6. Refolding and Storage    15
3.    Cell culture and transfection    16
3-1. Cell line    16
3-2. Stable cell line    16
3-3. Primary culture    17
4.    Generation of induced pluripotent stem cells    17
4-1. Feeder cell preparation    17
4-2. iPSCs generation    18
5.    Analytical technique    18
5-1. Luciferase assay    18
5-2. Reverse transcription polymerase chain reaction (RT-PCR) analysis    18
5-3. Western blot analysis    19
5-4. Immunofluorescence staining    21
5-5. Trypan blue staining    22
III.    Results    23
1. Construction of recombinant reprogramming factor-expressing plasmids    23
2. Expression and purification of recombinant reprogramming factors    25
3. Characterization of recombinant reprogramming factors    29
4. Generation of iPS cells from mouse somatic cells    31
IV.    Discussion    33
V.    Reference    38
VI.    Figures    45
Fig. 1. Cloning strategy of pET-Reprogramming factor-LpRH expressing plasmids.    46
Fig. 2. Cloning strategy of pET-M3-Reprogramming factor-LpRH expressing plasmids.    47
Fig. 3. Accuracy of pET-HA2-LpRH, pET-HA2-p53R248Q and p53R248W-LpRH and pGEX4T1-LIF    49
Fig. 4. Protein expression of RFs in Rosetta.    50
Fig. 5. Solubility of RFs overexpressed in Rosetta.    51
Fig. 6. Comparison of strategy 1 and strategy 2 in purifying M3O.    52
Fig. 7. Purification of six RFs.    54
Fig. 8. Avoiding aggregate formation during dialysis.    55
Fig. 9. Purification of p53R248Q, p53R248W and GST-LIF.    56
Fig. 10. Characterization of protein translocation of M3O in C3H10T1/2 by immunofluorescence.    57
Fig. 11. Characterization of 12R-mediated translocation of LpRH-containing proteins in C3H10T1/2.    59
Fig. 12. Live cell analysis of LpRH-mediated protein transduction.    60
Fig. 13. Reprogramming somatic cells using RFs.    62
VII.    Appendixes    63
Appendix 1：Supplementary figures    63
Fig. S1. Stem cell hierarchy.    63
Fig. S2. Factor delivery methods for iPSC derivation.    64
Fig. S3. Pathway for the transduction of R9 into cells.    64
Fig. S4. Using luciferase assay to monitor the transcriptional activity of RFs.    66
Fig. S5. Using electrophoresis mobility shift assay to analyze DNA binding activity of M3O and Sox2 on Fgf4 enhancer.    67
Appendix 2：Plasmid and primer list    68
Appendix 3：Length and molecular weight of the recombinant proteins    73
Appendix 4：Solutions    74


                                

Anokye-Danso, F., Trivedi, C.M., Juhr, D., Gupta, M., Cui, Z., Tian, Y., Zhang, Y., Yang, W., Gruber, P.J., Epstein, J.A., et al. (2011). Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell stem cell 8, 376-388.
Beerens, A.M., Al Hadithy, A.F., Rots, M.G., and Haisma, H.J. (2003). Protein transduction domains and their utility in gene therapy. Current gene therapy 3, 486-494.
Biswas, A., and Hutchins, R. (2007). Embryonic stem cells. Stem cells and development 16, 213-222.
Campbell, K.H., McWhir, J., Ritchie, W.A., and Wilmut, I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64-66.
Chen, B., Retzlaff, M., Roos, T., and Frydman, J. (2011). Cellular strategies of protein quality control. Cold Spring Harbor perspectives in biology 3, a004374.
Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B., Robson, P., et al. (2005). Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Molecular and cellular biology 25, 6031-6046.
Cowan, C.A., Atienza, J., Melton, D.A., and Eggan, K. (2005). Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369-1373.
Cowan, K.J., Diamond, M.I., and Welch, W.J. (2003). Polyglutamine protein aggregation and toxicity are linked to the cellular stress response. Human molecular genetics 12, 1377-1391.
Crombez, L., Charnet, A., Morris, M.C., Aldrian-Herrada, G., Heitz, F., and Divita, G. (2007). A non-covalent peptide-based strategy for siRNA delivery. Biochemical Society transactions 35, 44-46.
Deana, A., Ehrlich, R., and Reiss, C. (1998). Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo. Nucleic acids research 26, 4778-4782.
Derossi, D., Joliot, A.H., Chassaing, G., and Prochiantz, A. (1994). The third helix of the Antennapedia homeodomain translocates through biological membranes. The Journal of biological chemistry 269, 10444-10450.
Deshmukh, R.S., Kovacs, K.A., and Dinnyes, A. (2012). Drug discovery models and toxicity testing using embryonic and induced pluripotent stem-cell-derived cardiac and neuronal cells. Stem cells international 2012, 379569.
El-Andaloussi, S., Johansson, H.J., Holm, T., and Langel, U. (2007). A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids. Molecular therapy : the journal of the American Society of Gene Therapy 15, 1820-1826.
Elena, C., Ravasi, P., Castelli, M.E., Peiru, S., and Menzella, H.G. (2014). Expression of codon optimized genes in microbial systems: current industrial applications and perspectives. Frontiers in microbiology 5, 21.
Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156.
Frankel, A.D., and Pabo, C.O. (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189-1193.
Fribley, A., Zhang, K., and Kaufman, R.J. (2009). Regulation of apoptosis by the unfolded protein response. Methods in molecular biology 559, 191-204.
Fuchs, S.M., and Raines, R.T. (2004). Pathway for polyarginine entry into mammalian cells. Biochemistry 43, 2438-2444.
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. Proceedings of the Japan Academy Series B, Physical and biological sciences 85, 348-362.
Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001). Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. The Journal of biological chemistry 276, 5836-5840.
Gahrton, G., and Bjorkstrand, B. (2000). Progress in haematopoietic stem cell transplantation for multiple myeloma. Journal of internal medicine 248, 185-201.
Golovanov, A.P., Hautbergue, G.M., Wilson, S.A., and Lian, L.Y. (2004). A simple method for improving protein solubility and long-term stability. Journal of the American Chemical Society 126, 8933-8939.
Griswold, K.E., Mahmood, N.A., Iverson, B.L., and Georgiou, G. (2003). Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm. Protein expression and purification 27, 134-142.
Gurdon, J.B. (1962). The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. Journal of embryology and experimental morphology 10, 622-640.
Han, J., Sachdev, P.S., and Sidhu, K.S. (2010). A combined epigenetic and non-genetic approach for reprogramming human somatic cells. PloS one 5, e12297.
Han, X., Bushweller, J.H., Cafiso, D.S., and Tamm, L.K. (2001). Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin. Nature structural biology 8, 715-720.
Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P., Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., et al. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920-1923.
Hirai, H., Tani, T., Katoku-Kikyo, N., Kellner, S., Karian, P., Firpo, M., and Kikyo, N. (2011). Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD. Stem cells 29, 1349-1361.
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.
Hu, P.F., Guan, W.J., Li, X.C., and Ma, Y.H. (2012). Construction of recombinant proteins for reprogramming of endangered Luxi cattle fibroblast cells. Molecular biology reports 39, 7175-7182.
Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and Melton, D.A. (2008). Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature biotechnology 26, 1269-1275.
Johnson, R.M., Harrison, S.D., and Maclean, D. (2011). Therapeutic applications of cell-penetrating peptides. Methods in molecular biology 683, 535-551.
Jones, S.W., Christison, R., Bundell, K., Voyce, C.J., Brockbank, S.M., Newham, P., and Lindsay, M.A. (2005). Characterisation of cell-penetrating peptide-mediated peptide delivery. British journal of pharmacology 145, 1093-1102.
Judson, R.L., Babiarz, J.E., Venere, M., and Blelloch, R. (2009). Embryonic stem cell-specific microRNAs promote induced pluripotency. Nature biotechnology 27, 459-461.
Kane, J.F. (1995). Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Current opinion in biotechnology 6, 494-500.
Karanes, C., Nelson, G.O., Chitphakdithai, P., Agura, E., Ballen, K.K., Bolan, C.D., Porter, D.L., Uberti, J.P., King, R.J., and Confer, D.L. (2008). Twenty years of unrelated donor hematopoietic cell transplantation for adult recipients facilitated by the National Marrow Donor Program. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 14, 8-15.
Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R., et al. (2009). Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell stem cell 4, 472-476.
Knoblich, J.A. (2008). Mechanisms of asymmetric stem cell division. Cell 132, 583-597.
Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S.Y., Suemori, H., Nakatsuji, N., and Tada, T. (2005). Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Molecular and cellular biology 25, 2475-2485.
Lee, B.J., Cansizoglu, A.E., Suel, K.E., Louis, T.H., Zhang, Z., and Chook, Y.M. (2006). Rules for nuclear localization sequence recognition by karyopherin beta 2. Cell 126, 543-558.
Lo, B., and Parham, L. (2009). Ethical issues in stem cell research. Endocrine reviews 30, 204-213.
Lundberg, M., and Johansson, M. (2001). Is VP22 nuclear homing an artifact? Nature biotechnology 19, 713-714.
Lundberg, P., and Langel, U. (2003). A brief introduction to cell-penetrating peptides. Journal of molecular recognition : JMR 16, 227-233.
Magico, A.C., and Bell, J.B. (2011). Identification of a classical bipartite nuclear localization signal in the Drosophila TEA/ATTS protein scalloped. PloS one 6, e21431.
Makkerh, J.P., Dingwall, C., and Laskey, R.A. (1996). Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Current biology : CB 6, 1025-1027.
Martin, G.R. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of the National Academy of Sciences of the United States of America 78, 7634-7638.
Michiue, H., Tomizawa, K., Wei, F.Y., Matsushita, M., Lu, Y.F., Ichikawa, T., Tamiya, T., Date, I., and Matsui, H. (2005). The NH2 terminus of influenza virus hemagglutinin-2 subunit peptides enhances the antitumor potency of polyarginine-mediated p53 protein transduction. The Journal of biological chemistry 280, 8285-8289.
Miyoshi, N., Ishii, H., Nagano, H., Haraguchi, N., Dewi, D.L., Kano, Y., Nishikawa, S., Tanemura, M., Mimori, K., Tanaka, F., et al. (2011). Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell stem cell 8, 633-638.
Morgani, S.M., Canham, M.A., Nichols, J., Sharov, A.A., Migueles, R.P., Ko, M.S., and Brickman, J.M. (2013). Totipotent embryonic stem cells arise in ground-state culture conditions. Cell reports 3, 1945-1957.
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. Nature biotechnology 26, 101-106.
Nemes, C., Varga, E., Polgar, Z., Klincumhom, N., Pirity, M.K., and Dinnyes, A. (2014). Generation of mouse induced pluripotent stem cells by protein transduction. Tissue engineering Part C, Methods 20, 383-392.
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.
Pan, C., Lu, B., Chen, H., and Bishop, C.E. (2010). Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC. Molecular biology reports 37, 2117-2124.
Pooga, M., Kut, C., Kihlmark, M., Hallbrink, M., Fernaeus, S., Raid, R., Land, T., Hallberg, E., Bartfai, T., and Langel, U. (2001). Cellular translocation of proteins by transportan. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 15, 1451-1453.
Richard, J.P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M.J., Chernomordik, L.V., and Lebleu, B. (2003). Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. The Journal of biological chemistry 278, 585-590.
Rinas, U., and Bailey, J.E. (1992). Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli. Applied microbiology and biotechnology 37, 609-614.
Robinton, D.A., and Daley, G.Q. (2012). The promise of induced pluripotent stem cells in research and therapy. Nature 481, 295-305.
Rodda, D.J., Chew, J.L., Lim, L.H., Loh, Y.H., Wang, B., Ng, H.H., and Robson, P. (2005). Transcriptional regulation of nanog by OCT4 and SOX2. The Journal of biological chemistry 280, 24731-24737.
Rodda, S.J., Kavanagh, S.J., Rathjen, J., and Rathjen, P.D. (2002). Embryonic stem cell differentiation and the analysis of mammalian development. The International journal of developmental biology 46, 449-458.
Sarig, R., Rivlin, N., Brosh, R., Bornstein, C., Kamer, I., Ezra, O., Molchadsky, A., Goldfinger, N., Brenner, O., and Rotter, V. (2010). Mutant p53 facilitates somatic cell reprogramming and augments the malignant potential of reprogrammed cells. The Journal of experimental medicine 207, 2127-2140.
Shekkeris, A.S., Jaiswal, P.K., and Khan, W.S. (2012). Clinical applications of mesenchymal stem cells in the treatment of fracture non-union and bone defects. Current stem cell research & therapy 7, 127-133.
Stadtfeld, M., and Hochedlinger, K. (2010). Induced pluripotency: history, mechanisms, and applications. Genes & development 24, 2239-2263.
Stadtfeld, M., Maherali, N., Borkent, M., and Hochedlinger, K. (2010). A reprogrammable mouse strain from gene-targeted embryonic stem cells. Nature methods 7, 53-55.
Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., and Hochedlinger, K. (2008). Induced pluripotent stem cells generated without viral integration. Science 322, 945-949.
Stefani, M., and Dobson, C.M. (2003). Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. Journal of molecular medicine 81, 678-699.
Sumer, H., Liu, J., Tat, P., Heffernan, C., Jones, K.L., and Verma, P.J. (2009). Somatic cell nuclear transfer: pros and cons. Journal of stem cells 4, 85-93.
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.
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.
Tapscott, S.J. (2005). The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development 132, 2685-2695.
Tavernier, G., Wolfrum, K., Demeester, J., De Smedt, S.C., Adjaye, J., and Rejman, J. (2012). Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc. Biomaterials 33, 412-417.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147.
Toma, J.G., Akhavan, M., Fernandes, K.J., Barnabe-Heider, F., Sadikot, A., Kaplan, D.R., and Miller, F.D. (2001). Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nature cell biology 3, 778-784.
Tuch, B.E. (2006). Stem cells--a clinical update. Australian family physician 35, 719-721.
Vives, E., Brodin, P., and Lebleu, B. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. The Journal of biological chemistry 272, 16010-16017.
Wang, S., and Kaufman, R.J. (2012). The impact of the unfolded protein response on human disease. The Journal of cell biology 197, 857-867.
Wang, Y., Chen, J., Hu, J.L., Wei, X.X., Qin, D., Gao, J., Zhang, L., Jiang, J., Li, J.S., Liu, J., et al. (2011). Reprogramming of mouse and human somatic cells by high-performance engineered factors. EMBO reports 12, 373-378.
Wobus, A.M., and Boheler, K.R. (2005). Embryonic stem cells: prospects for developmental biology and cell therapy. Physiological reviews 85, 635-678.
Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., et al. (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766-770.
Xu, J., Wang, F., Tang, Z., Zhan, Y., Zhang, J., Yan, Q., Xiong, Y., Xie, X., Wu, J., and Zhang, S. (2010). Role of leukaemia inhibitory factor in the induction of pluripotent stem cells in mice. Cell biology international 34, 791-797.
Yakubov, E., Rechavi, G., Rozenblatt, S., and Givol, D. (2010). Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochemical and biophysical research communications 394, 189-193.
Ying, Q.L., Nichols, J., Evans, E.P., and Smith, A.G. (2002). Changing potency by spontaneous fusion. Nature 416, 545-548.
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., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.
Yuan, H., Corbi, N., Basilico, C., and Dailey, L. (1995). Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes & development 9, 2635-2645.
Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J., Wong, J., and Jin, Y. (2012). Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors. Biomaterials 33, 5047-5055.
Zhao, Y., Yin, X., Qin, H., Zhu, F., Liu, H., Yang, W., Zhang, Q., Xiang, C., Hou, P., Song, Z., et al. (2008). Two supporting factors greatly improve the efficiency of human iPSC generation. Cell stem cell 3, 475-479.
Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., et al. (2009). Generation of induced pluripotent stem cells using recombinant proteins. Cell stem cell 4, 381-384.

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