Reprogramming mouse ear mesenchymal stem cells (EMSC) expressing the Dlk1-Dio3 imprinted gene cluster


The identification of a single, early marker for full developmental potential of induced pluripotent stem (iPS) cells has proven elusive. Recently, however, activation of the imprinted gene cluster, Dlk1-Dio3 has emerged as a viable candidate in the mouse. To explore the relationship between Dlk1-Dio3 expression and developmental potential more fully, we used murine ear mesenchymal stem cells (mEMSC) for iPS cell induction. Mouse EMSC are easily obtained and share functional characteristics with embryonic stem (ES) cells and therefore, may be a reliable non-embryonic source for iPS cell production. We report that mEMSC express high levels of Gtl2, a maternally expressed gene within the Dlk1-Dio3 imprinted cluster. Moreover, mEMSC produce Gtl2 expressing (Gtl2on) iPSC clones that share functional characteristics with ES cell clones. The production of Gtl2on iPS cell clones from mEMSC provides a new model with which to investigate the regulation of Dlk1-Dio3 cluster activity during direct cell reprogramming.

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Gao, R. , S. Rim, J. , L. Strickler, K. , W. Barnes, C. , L. Harkins, L. , Staszkiewicz, J. , M. Gimble, J. , Gawronska-Kozak, B. , H. Leno, G. and J. Eilertsen, K. (2013) Reprogramming mouse ear mesenchymal stem cells (EMSC) expressing the Dlk1-Dio3 imprinted gene cluster. Stem Cell Discovery, 3, 64-71. doi: 10.4236/scd.2013.31010.

Conflicts of Interest

The authors declare no conflicts of interest.


[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] 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. doi:10.1038/nature05944
[3] Hanna, 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. and Jaenisch, R. (2008) Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell, 133, 250-264. doi:10.1016/j.cell.2008.03.028
[4] Maherali, N., Ahfeldt, T., Rigamonti, A., Utikal, J., Cowan, C. and Hochedlinger, K. (2008) A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell, 3, 340-345. doi:10.1016/j.stem.2008.08.003
[5] Mikkelsen, T.S., Hanna, J., Zhang, X., Ku, M., Wernig, M., Schorderet, P., Bernstein, B.E., Jaenisch, R., Lander, E.S. and Meissner, A. (2008) Dissecting direct reprogramming through integrative genomic analysis. Nature, 454, 49-55. doi:10.1038/nature07056
[6] Okita, K., Ichisaka, T. and Yamanaka, S. (2007) Generation of germline-competent induced plu-ripotent stem cells. Nature, 448, 313-317. doi:10.1038/nature05934
[7] Boland, M.J., Hazen, J.L., Nazor, K.L., Rodriguez, A.R., Gifford, W., Martin, G., Kupriyanov, S. and Baldwin, K.K. (2009) Adult mice generated from induced plu-ripotent stem cells. Nature, 461, 91-94. doi:10.1038/nature08310
[8] Zhao, X.-Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., Hao, J., Guo, C.-L., Ma, Q.-W., Wang, L., Zeng, F. and Zhou, Q. (2009) iPS cells produce viable mice through tetraploid complementation. Nature, 461, 86-90. doi:10.1038/nature08267
[9] Kang, L., Wang, J., Zhang, Y., Kou, Z. and Gao, S. (2009) iPS cells can support full-term development of tetraploid blastocyst-complemented embryos. Cell Stem Cell, 5, 135-138. doi:10.1016/j.stem.2009.07.001
[10] Rubin, L.L. and Haston, K.M. (2011) Stem cell biology and drug discovery. BMC Biology, 7, 9-42.
[11] Chin, M.H., Mason, M.J., Xie, W., Volinia, S., Singer, M., Peterson, C., Ambartsumyan, G., Aimiuwu, O., Richter, L., Zhang, J., Khvorostov, I., Ott, V., Grunstein, M., La-von, N., Benvenisty, N., Croce, C.M., Clark, A.T., Baxter, T., Pyle, A.D., Teitell, M.A., Pelegrini, M., Plath, K. and Lowry, W.E. (2009) Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell, 5, 111-123. doi:10.1016/j.stem.2009.06.008
[12] Marchetto, M.C., Yeo, G.W., Kainohana, O., Marsala, M., Gage, F.H. and Muotri, A.R. (2009) Transcriptional signature and memory retention of human induced pluripotent stem cells. PLoS One, 4, e7076. doi:10.1371/journal.pone.0007076
[13] Wilson, K.D., Venkatasubrahmanyam, S., Jia, F., Sun, N., Butte, A.J. and Wu, J.C. (2009) MicroRNA profiling of human-induced pluripotent stem cells. Stem Cells and Development, 18, 749-758. doi:10.1089/scd.2008.0247
[14] Kim, J.B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Araúzo-Bravo, M.J., Ruau, D., Han, D.W., Zenke, M. and Sch?ler, H.R. (2008) Pluripotent stem cells induced from adult neural stem cells by re-programming with two factors. Nature, 454, 646-650. doi:10.1038/nature07061
[15] Meissner, A., Wernig, M. and Jaenisch, R. (2007) Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nature Biotechnology, 25, 1177-1181. doi:10.1038/nbt1335
[16] Zho, T., Zhang, Z.N., Rong, Z. and Xu, Y. (2011) Immuno-genicity of induced pluripotent stem cells. Nature, 474, 212-215.
[17] Pera, M.F. (2011) Stem cells: The dark side of induced pluripotency. Nature, 471, 46-47. doi:10.1038/471046a
[18] Gawronska-Kozak, B. (2004) Regeneration in the ears of immunodeficient mice: Identification and lineage analysis of mesenchymal stem cells. Tissue Engineering, 10, 1251-1265.
[19] Gawronska-Kozak, B., Manuel, J.A. and Prpic, V. (2007) Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells. Journal of Cellular Biochemistry, 102, 122-135. doi:10.1002/jcb.21286
[20] Rim, J.S., Mynatt, R.L. and Gawronska-Kozak, B. (2005) Mesenchymal stem cells from the outer ear: a novel adult stem cell model system for the study of adipogenesis. The FASEB Journal, 19, 1205-1207.
[21] Staszkiewicz, J., Frazier, T.P., Rowan, B.G., Bunnell, B.A., Chiu, E.S., Gimble, J.M. and Gawronska-Kozak, B. (2010) Cell growth characteristics, differentiation frequency, and immunophenotype of adult ear mesenchymal stem cells. Stem Cells and Development, 19, 83-92. doi:10.1089/scd.2009.0051
[22] Abdallah, B.M., Jensen, C.H., Gutierrez, G., Leslie, R.G. Q., Jensen, T.G. and Kassem, M. (2004) Regulation of human skeletal stem cells differentiation by Dlk1/Pref1. Journal of Bone and Mineral Research, 19, 841-852. doi:10.1359/jbmr.040118
[23] Smas, C.M., Green, D. and Sul, H.S. (1993) Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1. Biochemistry, 33, 9257-9265.
[24] Smas, C.M. and Sul, H.S. (1993) Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell, 73, 725-734. doi:10.1016/0092-8674(93)90252-L
[25] 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, 24, S15.
[26] Moon, Y.S., Smas, C.M., Lee, K., Villena, J.A. and Kim, K.H. (2002) Mice lacking paternally expressed Pref-1/ Dlk1 display growth retardation and accelerated adiposity. Mol. The Journal of Cell Biology, 22, 5585-5592. doi:10.1128/MCB.22.15.5585-5592.2002
[27] Lee, K., Villena, J.A., Moon, Y.S., Kim, K.H., Lee, S., Kang, C. and Sul, H.S. (2003) Inhibition of adipogenesis and development of glucose intolerance by soluble preadipocyte factor-1 (Pref-1). Journal of Clinical Investigation, 111, 453-461.
[28] Wang, Y., Kim, K.-A., Kim, J.-H. and Sul, H.S. (2006) Pref-1, a preadipocyte secreted factor that inhibits adipogenesis. Journal of Nutrition, 136, 2953-2956.
[29] Liu, L., Luo, G.-Z., Yang, W., Zhao, X., Zheng, Q., Lv, Z., Li, W., Wu, H.-J., Wang, L., Wang, X.-J. and Zhou, Q. (2010) Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells. JBC, 285, 19483-19490. doi:10.1074/jbc.M110.131995
[30] 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, 175-183. doi:10.1038/nature09017
[31] Niibe, K., Kawamura, Y., Araki, D., Morikawa, S., Miura, K., Suzuki, S., Shimmura, S., Sunabori, T., Mabuchi, Y., Nagai, Y., Nakagawa, T., Okano, H. and Matsuzaki, Y. (2011) Purified mesenchymal stem cells are an efficient source for ips cell induction. PLoS One, 6, 1-9. doi:10.1371/journal.pone.0017610
[32] Staszkiewicz, J., Gimble, J.M., Manuel, J.A. and Gawronska-Kozak, B. (2008) IFATS collection: Stem cell antigen-1-positive ear mesenchymal stem cells display enhanced adipogenic potential. Stem Cells, 26, 2666-2673. doi:10.1634/stemcells.2008-0270
[33] Hotta, A. and Ellis, J. (2008) Retroviral vector silencing during iPS cell induction: An epigenetic beacon that signals distinct pluripotent states. Journal of Cellular Biochemistry, 105, 940-948. doi:10.1002/jcb.21912
[34] Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J. and Thorold, W. (2008) Theunissen, Austin Smith. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biology, 60, 2237-2247.
[35] Chan, E.M., Ratanasirintrawoot, S., Park, I.-H., Manos, P.D., Loh, Y.-H., Huo, H., Miller, J.D., Hartung, O., Rho, J., Ince, T.A., Daley, G.Q. and Schlaeger, T.M. (2009) Live cell imaging distinguishes bona fide human iPScells from partially reprogrammed cells. Nature Biotechnology, 27, 1033-1037. doi:10.1038/nbt.1580

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