Using MicroRNAs to Enhance the Generation of Induced Pluripotent Stem Cells
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
Abstract
Somatic cells reprogrammed to acquire an ES?like state are termed iPS cells. In this unit, a protocol to use microRNAs as enhancers to increase the reprogramming efficiency is described. Mouse embryonic fibroblasts (MEFs) are isolated from E13.5 mouse embryos and seeded for reprogramming by defined factors. microRNA mimics are transfected into MEFs at two time points during this process to enhance the overall reprogramming efficiency. Two standard protocols for characterization of these miR?iPSCs, embryoid body formation and teratoma formation, are also provided. By using this method, the investigators can obtain a significantly higher number of bona?fide iPSC colonies and miR?iPSCs can be derived at a faster rate than with non?treated cells. Curr. Protoc. Stem Cell Biol. 20:4A.4.1?4A.4.14. © 2012 by John Wiley & Sons, Inc.
Keywords: reprogramming; microRNA; iPSC; embryonic body; teratoma
Table of Contents
- Introduction
- Basic Protocol 1: Preparation of Mouse Embryonic Fibroblasts (MEFs)
- Basic Protocol 2: Use microRNAs as an Enhancer to Reprogram Mouse Embryonic Fibroblasts
- Basic Protocol 3: Derivation and Characterization of miR‐iPSC Colonies
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol 1: Preparation of Mouse Embryonic Fibroblasts (MEFs)
Materials
Basic Protocol 2: Use microRNAs as an Enhancer to Reprogram Mouse Embryonic Fibroblasts
Materials
Basic Protocol 3: Derivation and Characterization of miR‐iPSC Colonies
Materials
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Figures
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Figure 4.A0.1 Scheme for iPSC generatioin with microRNAs as enhancers. (A ) microRNAs were transfected both on day 0 and day 5 at a final concentration of 50 nM. Infected MEFs were first cultured in MEF medium until day 3 post‐4F transduction and then switched to mES culture medium containing LIF supplement. Typically, for 4F‐transduced cells, Oct4‐GFP‐positive colonies could be readily picked around day 11 to establish iPSC lines, and for OSK‐transduced cells, GFP‐positive colonies could be picked around day 15. (B ) Derived miR‐iPSC clone. miR‐93 iPSC no. 5 clone was derived as mentioned in A and the cells have acquired typical mouse embryonic stem cell morphology. They have also turned on the endogenous locus for Oct4 and thus become GFP‐positive. View Image -
Figure 4.A0.2 iPSC characterization by embryoid body formation assay. iPS cells were first trypsinized to become a single cell suspension and diluted to a final concentration of 1 × 105 ∼1.5 × 105 cells/ml. Cells were then seeded in 20‐µl drops onto the cover of petri dishes to reach 2000∼3000 cells/drop. Embryoid bodies (mEBs) were formed by inverted culture of the cells in petri dishes with ∼15 ml PBS, which could prevent the drops from drying. mEBs would be harvested at day 2 to 3 and they typically have universal morphology by this method. mEBs were then seeded onto tissue culture‐treated 6‐well plates to allow them attach the bottom. Attached mEBs were further maintained in mEB differentiation medium until around day 13 to 14 for immunostaining of different lineage markers. View Image -
Figure 4.A0.3 iPSC characterization by teratoma formation. (A ) 4‐ to 6‐week‐old athymus nude mice were anesthetized with avertin and injected with ∼1 × 106 iPS cells at the dorsal neck region. After 3 to 4 weeks, the tumors were then harvested and fixed in zinc formalin solution before further paraffin embedding and H&E staining. (B ) Teratomas contain tissues from different lineages. Cartilage, neural tissue, skeleton muscle, and epidermis tissue were shown. View Image
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Literature Cited
Literature Cited | |
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. | |
Giorgetti, A., Montserrat, N., Aasen, T., Gonzalez, F., Rodriguez‐Piza, I., Vassena, R., Raya, A., Boue, S., Barrero, M.J., Corbella, B.A., Torrabadella, M., Veiga, A., and Izpisua Belmonte, J.C. 2009. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell 5:353‐357. | |
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., Huangfu, D., Akutsu, H., Liu, D.R., Rubin, L.L., and Eggan, K. 2009. A small‐molecule inhibitor of Tgf‐beta signaling replaces Sox2 in reprogramming by inducing nanog. Cell Stem Cell 5:491‐503. | |
Itzhaki, I., Maizels, L., Huber, I., Zwi‐Dantsis, L., Caspi, O., Winterstern, A., Feldman, O., Gepstein, A., Arbel, G., Hammerman, H., Boulos, M., and Gepstein, L. 2011. Modeling the long QT syndrome with induced pluripotent stem cells. Nature 471:225‐229. | |
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. | |
Latronico, M.V. and Condorelli, G. 2009. MicroRNAs and cardiac pathology. Nat. Rev. Cardiol. 6:419‐429. | |
Li, Z., Yang, C.S., Nakashima, K., and Rana, T.M. 2011. Small RNA‐mediated regulation of iPS cell generation. EMBO J. 30:823‐834. | |
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., Bollong, M., Kunick, C., Brinker, A., Cho, C.Y., Schultz, P.G., and Jaenisch, R. 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. | |
Maherali, N. and Hochedlinger, K. 2009. Tgf‐beta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr. Biol. 19:1718‐1723. | |
Marchetto, M.C., Carromeu, C., Acab, A., Yu, D., Yeo, G.W., Mu, Y., Chen, G., Gage, F.H., and Muotri, A.R. 2010. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143:527‐539. | |
Meissner, A., Wernig, M., and Jaenisch, R. 2007. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25:1177‐1181. | |
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. | |
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. | |
Shi, Y., Zhao, Y., and Deng, H. 2010. Powering reprogramming with vitamin C. Cell Stem Cell 6:1‐2. | |
Subramanyam, D., Lamouille, S., Judson, R.L., Liu, J.Y., Bucay, N., Derynck, R., and Blelloch, R. 2011. Multiple targets of miR‐302 and miR‐372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat. Biotechnol. 29:443‐448. | |
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. | |
Wang, Y., Baskerville, S., Shenoy, A., Babiarz, J.E., Baehner, L., and Blelloch, R. 2008. Embryonic stem cell‐specific microRNAs regulate the G1‐S transition and promote rapid proliferation. Nat. Genet. 40:1478‐1483. | |
Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J.A., and Kosik, K.S. 2009. MicroRNA‐145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137:647‐658. | |
Yazawa, M., Hsueh, B., Jia, X., Pasca, A.M., Bernstein, J.A., Hallmayer, J., and Dolmetsch, R.E. 2011. Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471:230‐234. | |
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., Slukvin, I.I., and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917‐1920. | |
Zhang, J., Lian, Q., Zhu, G., Zhou, F., Sui, L., Tan, C., Mutalif, R.A., Navasankari, R., Zhang, Y., Tse, H.F., Stewart, C.L., and Colman, A. 2011. A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects. Cell Stem Cell 8:31‐45. |