SCD  Vol.2 No.2 , April 2012
Temporal epigenetic modifications differentially regulate ES cell-like colony formation and maturation
Human somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells by forced expression of the transcription factors Oct4, Sox2, and either Klf4 and cMyc or Nanog and Lin28, using virus-based systems. However, low reprogramming efficiency and the potential for deleterious virus-induced genomic modification limit the clinical potential of this technology. Recent reports indicate, however, that the generation of iPS cells can be enhanced by the addition of synthetic small molecules, including epigenetic modulators. In this report, we demonstrate that the epigenetic modifiers Valproic Acid (VPA) and 5-azacytidine activate the reciprocal transcriptional regulation of endogenous pluripotency transcription factor genes in human dermal fibroblasts and that VPA alone can directly activate endogenous Oct4 in the absence of transgenes. Moreover, using human adipose cells, we demonstrate that histone deacetylase inhibition, prior to reprogramming factor transfection, increases embryonic stem (ES) cell-like colony formation ~2 - 3 fold. In addition, DNA methyltransferase (DNMT) inhibition during human ES cell culture promotes maturation of reprogrammed somatic cells, increasing the yield ~4 fold. These data provide proof of principle that reprogramming efficiency can be improved by inhibiting specific repressive epigenetic regulatory components at the levels of ES cell-like colony formation and maturation. In addition, these studies raise the interesting possibility that a more efficient small molecule-based reprogramming system may provide a superior alternative to current virus-based approaches.

Cite this paper
S. Rim, J. , L. Strickler, K. , W. Barnes, C. , L. Harkins, L. , Staszkiewicz, J. , M. Gimble, J. , H. Leno, G. and J. Eilertsen, K. (2012) Temporal epigenetic modifications differentially regulate ES cell-like colony formation and maturation. Stem Cell Discovery, 2, 45-57. doi: 10.4236/scd.2012.22008.
[1]   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. doi:10.1016/j.cell.2006.07.024

[2]   Park, I.-H., Arora., N., Huo, H.G., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M.W., Cowan, C., Hochedlinger, K. and Daley, G. Q., (2008) Disease-Specific induced pluripotent stem cells. Cell, 134, 1-10. doi:10.1016/j.cell.2008.07.041

[3]   Yu, J.Y., Vodyanik, M.A., Smuga-Otto, K., AntosiewiczBourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I. and Thomson, J.A. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science, 318, 1917-1920. doi:10.1126/science.1151526

[4]   Meng, X.L., Shen, J.S., Kawagoe, S., Ohashi, T., Brady, R.O., et al. (2010) Induced pluripotent stem cells derived from mouse models of lysosomal storage disorders. Proceedings of the National Academy of Sciences of USA, 107, 7886-7891. doi:10.1073/pnas.1002758107

[5]   Ebert, A.D., Yu, J.Y., Rose, F.F. Jr., Mattis, V.B., Lorson, C.L., et al. (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature, 457, 277-280.

[6]   Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., et al. (2009) Modelling pathogenesis and treatment of familial dysautonomia using patientspecific iPSCs. Nature, 461, 402-406.

[7]   Rashid, S.T., Corbineau, S., Hannan, N., Marciniak, S.J., Miranda, E., et al. (2010) Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. The Journal of Clinical Investigation, 120, 3127-3136. doi:10.1172/JCI43122

[8]   Zhang, N., An, M.C., Montoro, D., Ellerby, L.M. (2010) Characterization of human huntington’s disease cell model from induced pluripotent stem cells. PLoS Currents, 2, RRN1193. doi:10.1371/currents.RRN1193

[9]   Zhao, T. and Xu, Y. (2010) p53 and stem cells: New developments and new concerns. Trends in Cell Biology, 20, 170-175. doi:10.1016/j.tcb.2009.12.004

[10]   Yu, J.Y., Hu, K.J., Smuga-Otto, K., Tian, S.L., Stewart, R., Slukvin, I.I. and Thomson, J.A., (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science, 10, 797-801. doi:10.1126/science.1172482

[11]   Sommer, C.A., Stadtfeld, M., Murphy, G.J., Hochedlinger, K., Kotton, D.N., et al. (2009) Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells, 27, 543-549. doi:10.1634/stemcells.2008-1075

[12]   Huangfu, D.W., Maehr, R., Guo, W.J., Eijkelenboom, A., Snitow, M., Chen, A.E. and Melton, D.A. (2008) Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature Biotechnology, 26, 795-797.

[13]   Okita, K., Nakagawa, M., H., Hong, Ichisaka, T. and Yamanaka, S., (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322, 949-953. doi:10.1126/science.1164270

[14]   Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.-H., Li, H., et al. (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 7, 618-630. doi:10.1016/j.stem.2010.08.012

[15]   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. doi:10.1016/j.bbrc.2010.02.150

[16]   Anokye-Danso, F., Trivedi, C.M., Juhr, D., Gupta, M., Cui, Z., et al. (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, 8, 376-388. doi:10.1016/j.stem.2011.03.001

[17]   Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., et al. (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 458, 766-770.

[18]   Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P., et al. (2009) Virus-Free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458, 771-775.

[19]   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. and Kim, K.-S. (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell, 4, 472-476. doi:10.1016/j.stem.2009.05.005

[20]   Zhou, H.Y., Wu, S.L., Joo, J.Y., Zhu, S.Y., Han, D.W., Lin, T.X., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Scholer, H.R., Duan, L.X. and Ding, S. (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4, 381-384.

[21]   Zhu, S.Y., Li, W.L., Zhou, H.Y., Wei, W.G., Ambasudhan, R., et al. (2010) Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell, 7, 651-655. doi:10.1016/j.stem.2010.11.015

[22]   Li, Y., Zhang, Q., Yin, X., Yang, W., Du, Y., et al. (2011) Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Research, 21, 196-204. doi:10.1038/cr.2010.142

[23]   Chen, J.K., Liu, J., Yang, J.Q., Chen, Y., Chen, J., et al. (2011) BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone. Cell Research, 21, 205-212. doi:10.1038/cr.2010.172

[24]   Yuan, X., Wan, H., Zhao, X., Zhu, S., Zhou, Q., et al. (2011) Combined chemical treatment enables Oct4-induced reprogramming from mouse embryonic fibroblasts. Stem Cells, 29, 549-553. doi:10.1002/stem.594

[25]   Zhu, S., Wei, W. and Ding, S. (2011) Chemical Strategies for stem cell biology and regenerative medicine. Annual Review of Biomedical Engineering, 13, 73-90.

[26]   Jaenisch, R. and Young, R. (2008) Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell, 132, 567-582. doi:10.1016/j.cell.2008.01.015

[27]   Kim, J., Chu, J.L., Shen, X.H., Wang, J.L. and Orkin, S.H. (2008) An extended transcriptional network for pluripotency of embryonic stem cells. Cell, 132, 1049-1061. doi:10.1016/j.cell.2008.02.039

[28]   Sun, N., Panetta, N.J., Gupta, D.M., Wilson, K.D., Lee, A., et al. (2009) Feeder-Free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proceedings of the National Academy of Sciences of USA, 106, 15720-15725. doi:10.1073/pnas.0908450106

[29]   Chan, E.M., Ratanasirintrawoot, S., Park, I.-H., Manos, P.D., Loh, Y.-H., et al. (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nature Biotechnology, 27, 1033-1037.

[30]   Stadtfeld, M., Apostolou, E., Akutsu, H., Fukuda, A., Follett, P., et al. (2010) Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature, 465, 175-181.

[31]   Sul, H.S., Smas, C., Mei, B. and Zhou, L. (2000) Function of pref-1 as an inhibitor of adipocyte differentiation. International Journal of Obesity and Related Metabolic Disorders, 24, 15-19.

[32]   Wylie, A.A., Murphy, S.K., Orton, T.C. and Jirtle, R.L. (2000) Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation. Genome Research, 10, 1711-1718. doi:10.1101/gr.161600

[33]   Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D.A., Huang J.I., et al. (2002) Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell, 13, 4279-4295. doi:10.1091/mbc.E02-02-0105

[34]   Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., et al. (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering, 7, 211-228. doi:10.1089/107632701300062859

[35]   Izadpanah, R., Trygg, C., Patel, B., Kriedt, C., Dufour, J., Gimble, J.M. and Bunnell, B.A. (2006) Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. Journal of Cellular Biochemistry, 99, 1285-1297. doi:10.1002/jcb.20904

[36]   Lin, T.X., Ambasudhan, R., Yuan, X., Li, W.L., Hilcove, S., et al. (2009) A chemical platform for improved induction of human iPSCs. Nature Methods, 6, 805-808.

[37]   Li, W.L., Zhou, H.Y., Abujarour, R., Zhu, S.Y., Joo, Y.J., et al. (2009) Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2. Stem Cells, 27, 2992-3000. doi:10.1002/stem.240

[38]   Ichida, J.K., Blanchard, J., Lam, K., Son, E.Y., Chung, J.E., et al. (2009) A small-molecule inhibitor of tgf-β signaling replaces Sox2 in reprogramming by inducing Nanog. Cell Stem Cell, 5, 491-503. doi:10.1016/j.stem.2009.09.012

[39]   Esteban, M.A., Wang, T., Qin, B.M., Yang, J.Y., Qin, D.J., et al. (2010) Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 6, 71-79. doi:10.1016/j.stem.2009.12.001

[40]   Shi, Y., Desponts, C., Do, J.T., Hahm, H.S., Scholer, H.R., et al. (2008) Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with smallmolecule compounds. Cell Stem Cell, 3, 568-574. doi:10.1016/j.stem.2008.10.004

[41]   Huangfu, D.W., Osafune, K., Maehr, R., Guo, W.J., Eijkelenboom, A., Chen, S.B., 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.