The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting. 1. Introduction The feasibility of reprogramming somatic cells to induced pluripotent stem cells (iPS) [1–4] has led to the possibility of developing disease-specific iPS cells for improved disease modeling in vitro [5–7] and potential use in clinical applications [8, 9]. Since the initial generation of iPS cells from mouse embryonic fibroblast (MEF) cells [1], there have been numerous refinements of the method. The potential therapeutic application of initial iPS cell lines was hampered by the fact that applied methods of iPS cell derivation modified the host genome through the integration of DNA sequences [3, 10–15]. Kim and colleagues [16] showed that it is possible to reprogram human foreskin fibroblasts through exposure to membrane-permeable recombinant proteins of the pluripotency factors Oct-4, Sox-2, Klf-4, and c-Myc. The factors were fused to a 9-arginine sequence to establish the ability of cell penetration. HEK 293 cells were transfected with plasmids for producing the described proteins. The whole HEK 293 cell extract was used for reprogramming. The method was refined by Zhou et al. [17] for MEF cells using recombinant cell-penetrating proteins. It has been shown that the modified mRNA-mediated delivery of reprogramming factors based on nucleofection is an efficient and nontoxic alternative approach to cell modification [18] which has recently facilitated the derivation of iPS cell lines [19–21]. Here, we investigate in vitro transcribed mRNA transfection as a method for producing iPS cells that does not bear any risk with respect
References
[1]
K. Takahashi and S. Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors,” Cell, vol. 126, no. 4, pp. 663–676, 2006.
[2]
K. Takahashi, K. Tanabe, M. Ohnuki et al., “Induction of pluripotent stem cells from adult human fibroblasts by defined factors,” Cell, vol. 131, no. 5, pp. 861–872, 2007.
[3]
J. Yu, M. A. Vodyanik, K. Smuga-Otto et al., “Induced pluripotent stem cell lines derived from human somatic cells,” Science, vol. 318, no. 5858, pp. 1917–1920, 2007.
[4]
W. E. Lowry, L. Richter, R. Yachechko et al., “Generation of human induced pluripotent stem cells from dermal fibroblasts,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 8, pp. 2883–2888, 2008.
[5]
J. T. Dimos, K. T. Rodolfa, K. K. Niakan et al., “Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons,” Science, vol. 321, no. 5893, pp. 1218–1221, 2008.
[6]
I. H. Park, N. Arora, H. Huo et al., “Disease-specific induced pluripotent stem cells,” Cell, vol. 134, no. 5, pp. 877–886, 2008.
[7]
A. D. Ebert, J. Yu, F. F. Rose et al., “Induced pluripotent stem cells from a spinal muscular atrophy patient,” Nature, vol. 457, no. 7227, pp. 277–280, 2009.
[8]
E. Kiskinis and K. Eggan, “Progress toward the clinical application of patient-specific pluripotent stem cells,” Journal of Clinical Investigation, vol. 120, no. 1, pp. 51–59, 2010.
[9]
C. Freund and C. L. Mummery, “Prospects for pluripotent stem cell-derived cardiomyocytes in cardiac cell therapy and as disease models,” Journal of Cellular Biochemistry, vol. 107, no. 4, pp. 592–599, 2009.
[10]
M. Stadtfeld, M. Nagaya, J. Utikal, G. Weir, and K. Hochedlinger, “Induced pluripotent stem cells generated without viral integration,” Science, vol. 322, no. 5903, pp. 945–949, 2008.
[11]
K. Okita, M. Nakagawa, H. Hyenjong, T. Ichisaka, and S. Yamanaka, “Generation of mouse induced pluripotent stem cells without viral vectors,” Science, vol. 322, no. 5903, pp. 949–953, 2008.
[12]
F. Soldner, D. Hockemeyer, C. Beard et al., “Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors,” Cell, vol. 136, no. 5, pp. 964–977, 2009.
[13]
K. Kaji, K. Norrby, A. Paca, M. Mileikovsky, P. Mohseni, and K. Woltjen, “Virus-free induction of pluripotency and subsequent excision of reprogramming factors,” Nature, vol. 458, no. 7239, pp. 771–775, 2009.
[14]
K. Woltjen, I. P. Michael, P. Mohseni et al., “PiggyBac transposition reprograms fibroblasts to induced pluripotent stem cells,” Nature, vol. 458, no. 7239, pp. 766–770, 2009.
[15]
D. Huangfu, K. Osafune, R. Maehr et al., “Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2,” Nature Biotechnology, vol. 26, no. 11, pp. 1269–1275, 2008.
[16]
D. Kim, C. H. Kim, J. I. Moon et al., “Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins,” Cell Stem Cell, vol. 4, no. 6, pp. 472–476, 2009.
[17]
H. Zhou, S. Wu, J. Y. Joo et al., “Generation of induced pluripotent stem cells using recombinant proteins,” Cell Stem Cell, vol. 4, no. 5, pp. 381–384, 2009.
[18]
J. M. Wiehe, P. Ponsaerts, M. T. Rojewski et al., “mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression,” Journal of Cellular and Molecular Medicine, vol. 11, no. 3, pp. 521–530, 2007.
[19]
L. Warren, P. D. Manos, T. Ahfeldt et al., “Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA,” Cell Stem Cell, vol. 7, no. 5, pp. 618–630, 2010.
[20]
M. Angel and M. F. Yanik, “Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins,” PLoS ONE, vol. 5, no. 7, Article ID e11756, 2010.
[21]
E. Yakubov, G. Rechavi, S. Rozenblatt, and D. Givol, “Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors,” Biochemical and Biophysical Research Communications, vol. 394, no. 1, pp. 189–193, 2010.
[22]
J. A. Thomson, “Embryonic stem cell lines derived from human blastocysts,” Science, vol. 282, no. 5391, pp. 1145–1147, 1998.
[23]
C. T. Freberg, J. A. Dahl, S. Timoskainen, and P. Collas, “Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract,” Molecular Biology of the Cell, vol. 18, no. 5, pp. 1543–1553, 2007.
[24]
T. Schnell, P. Foley, M. Wirth, J. Münch, and K. überla, “Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus,” Human Gene Therapy, vol. 11, no. 3, pp. 439–447, 2000.
[25]
J. K. Yee, T. Friedmann, and J. C. Burns, “Generation of high-titer pseudotyped retroviral vectors with very broad host range,” Methods in Cell Biology, vol. 43, part A, pp. 99–112, 1994.
[26]
S. Schuck, A. Manninen, M. Honsho, J. Füllekrug, and K. Simons, “Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 14, pp. 4912–4917, 2004.
[27]
C. M. Counter, W. C. Hahn, W. Wei et al., “Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 25, pp. 14723–14728, 1998.
[28]
C. Dai, L. Whitesell, A. B. Rogers, and S. Lindquist, “Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis,” Cell, vol. 130, no. 6, pp. 1005–1018, 2007.
[29]
H. Binder and S. Preibisch, “‘Hook’-calibration of GeneChip-microarrays: theory and algorithm,” Algorithms for Molecular Biology, vol. 3, no. 1, article 12, 2008.
[30]
B. M. Bolstad, R. A. Irizarry, M. ？strand, and T. P. Speed, “A comparison of normalization methods for high density oligonucleotide array data based on variance and bias,” Bioinformatics, vol. 19, no. 2, pp. 185–193, 2003.
[31]
K. Kadota, Y. Nakai, and K. Shimizu, “A weighted average difference method for detecting differentially expressed genes from microarray data,” Algorithms for Molecular Biology, vol. 3, no. 1, article 8, 2008.
[32]
N. Jain, J. Thatte, T. Braciale, K. Ley, M. O'Connell, and J. K. Lee, “Local-pooled-error test for identifying differentially expressed genes with a small number of replicated microarrays,” Bioinformatics, vol. 19, no. 15, pp. 1945–1951, 2003.
[33]
Y. Xie, W. Pan, and A. B. Khodursky, “A note on using permutation-based false discovery rate estimates to compare different analysis methods for microarray data,” Bioinformatics, vol. 21, no. 23, pp. 4280–4288, 2005.
[34]
L. Tian, S. A. Greenberg, S. W. Kong, J. Altschuler, I. S. Kohane, and P. J. Park, “Discovering statistically significant pathways in expression profiling studies,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 38, pp. 13544–13549, 2005.
[35]
B. Bhattacharya, T. Miura, R. Brandenberger et al., “Gene expression in human embryonic stem cell lines: unique molecular signature,” Blood, vol. 103, no. 8, pp. 2956–2964, 2004.
[36]
B. Efron and R. Tibshirani, “On testing the significance of sets of genes,” Annals of Applied Statistics, vol. 1, pp. 107–129, 2007.
[37]
V. F. I. Van Tendeloo, P. Ponsaerts, F. Lardon et al., “Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells,” Blood, vol. 98, no. 1, pp. 49–56, 2001.
[38]
O. Adewumi, B. Aflatoonian, L. Ahrlund-Richter et al., “Characterization of human embryonic stem cell lines by the International Stem Cell Initiative,” Nature Biotechnology, vol. 25, no. 7, pp. 803–816, 2007.
[39]
E. M. Chan, S. Ratanasirintrawoot, I. H. Park et al., “Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells,” Nature Biotechnology, vol. 27, no. 11, pp. 1033–1037, 2009.
[40]
P. Mali, Z. Ye, H. H. Hommond et al., “Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts,” Stem Cells, vol. 26, no. 8, pp. 1998–2005, 2008.
[41]
E. P. Papapetrou, M. J. Tomishima, S. M. Chambers et al., “Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 31, pp. 12759–12764, 2009.
[42]
C. E. Forristal, K. L. Wright, N. A. Hanley, R. O. C. Oreffo, and F. D. Houghton, “Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions,” Reproduction, vol. 139, no. 1, pp. 85–97, 2010.
[43]
S. M. Fleming, P.-O. Fernagut, and M.-F. Chesselet, “Genetic mouse models of Parkinsonism: strengths and limitations,” NeuroRx, vol. 2, no. 3, pp. 495–503, 2005.
[44]
M. Newman, F. I. Musgrave, and M. Lardelli, “Alzheimer disease: amyloidogenesis, the presenilins and animal models,” Biochimica et Biophysica Acta, vol. 1772, no. 3, pp. 285–297, 2007.
[45]
J. A. Potashkin, S. R. Blume, and N. K. Runkle, “Limitations of animal models of Parkinson's disease,” Parkinson's Disease, vol. 2011, Article ID 658083, 7 pages, 2011.
