Nanofibrous Electrospun Polymers for Reprogramming Human Cells
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Forced expression of transcription factors epigenetically reprograms somatic cells harvested from routine skin biopsies into induced pluripotent stem cells (iPSCs). Human iPSCs are key resources for drug discovery, regenerative medicine and tissue engineering. Here we developed a materials approach to explore how culture substrates could impact factor-mediated reprogramming of human fibroblasts. A materials library consisting of nanofibrous substrates with randomly oriented and aligned structures was prepared by electrospinning four polymers [polylactic acid (PLA), polycaprolactone (PCL), thermoplastic polyurethane (TPU) and polypropylene carbonate (PPC)] into nanofiber orientations. Adsorbing protein to each substrate permitted robust attachment of fibroblasts to all substrates. Fibroblasts on aligned substrates had elongated nuclei, but after reprogramming factor expression, nuclei became more circular. Reprogramming factors could override the nuclear shape constraints imposed by nanofibrous substrates, and the majority of substrates supported full reprogramming. Early culture on PCL and TPU substrates promoted reprogramming, and TGF-β repressed substrate effects. Partial least squares modeling of the biochemical and biophysical cues within our reprogramming system identified TGF-β and polymer identity as important cues governing cellular reprogramming responses. We believe that our approach of using a nanofibrous materials library can be used to dissect molecular mechanisms of reprogramming and generate novel substrates that enhance epigenetic reprogramming.
KeywordsPluripotent stem cells Reprogramming Biomaterials Electrospinning Nuclear shape
We acknowledge generous financial support from the Wisconsin Institute for Discovery (T.C. and K.S.), a Grainger Fellowship (J.C-S.) and the Society in Science Foundation (K.S.). We also would like to thank all members of the Saha lab and BIONATES theme for advice and support throughout this project. We acknowledge Dr. Rob McClain and the University of Wisconsin-Madison Biochemistry department for the use and expertise in collecting surface area data via the BET Micromeritics Gemini VII instrument.
Conflict of interest
Travis Cordie, Ty Harkness, Xin Jing, Jared Carlson-Stevermer, Hao-Yang Mi, Lih-Sheng Turng and Krishanu Saha declare that they have no conflicts of interest. Dr. Saha reports grants from Society in Science Foundation during the conduct of the study.
No human and animal studies were carried out by the authors for this article. All work with human embryonic stem cell lines was carried out in accordance with institutional, national, and international guidelines and approved by the Stem Cell Research Oversight Committee at the University of Wisconsin-Madison.
- 2.Armond, J. W., K. Saha, A. A. Rana, C. J. Oates, R. Jaenisch, M. Nicodemi, and S. Mukherjee. A stochastic model dissects cell states in biological transition processes. Sci. Rep. 4:3692, 2014.Google Scholar
- 4.Beers, J., D. R. Gulbranson, N. George, L. I. Siniscalchi, J. Jones, J. A. Thomson, and G. Chen. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7:2029–2040, 2012.Google Scholar
- 6.Chen, G., D. R. Gulbranson, Z. Hou, J. M. Bolin, V. Ruotti, M. D. Probasco, K. Smuga-Otto, S. E. Howden, N. R. Diol, N. E. Propson, R. Wagner, G. O. Lee, J. Antosiewicz-Bourget, J. M. Teng, and J. A. Thomson. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8:424–429, 2011.Google Scholar
- 10.Downing, T. L., J. Soto, C. Morez, T. Houssin, A. Fritz, F. Yuan, J. Chu, S. Patel, D. V. Schaffer, and S. Li. Biophysical regulation of epigenetic state and cell reprogramming. Nat. Mater. 12:1154–1162, 2013.Google Scholar
- 11.Eriksson, L. Multi- and Megavariate Data Analysis, MKS Umetrics AB, 2006.Google Scholar
- 15.Grskovic, M., A. Javaherian, B. Strulovici, and G. Q. Daley. Induced pluripotent stem cells—opportunities for disease modelling and drug discovery. Nat. Rev. Drug Discov. 10:915–929, 2011.Google Scholar
- 17.Hanna, J., K. Saha, B. Pando, J. van Zon, C. J. Lengner, M. P. Creyghton, A. van Oudenaarden, and R. Jaenisch. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462:595–601, 2009.Google Scholar
- 19.Hockemeyer, D., F. Soldner, E. G. Cook, Q. Gao, M. Mitalipova, and R. Jaenisch. A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell 3:346–353, 2008.Google Scholar
- 21.Jiao, J., Y. Dang, Y. Yang, R. Gao, Y. Zhang, Z. Kou, X. F. Sun, and S. Gao. Promoting reprogramming by FGF2 reveals that the extracellular matrix is a barrier for reprogramming fibroblasts to pluripotency. Stem Cells 31:729–740, 2012.Google Scholar
- 24.Li, R., J. Liang, S. Ni, T. Zhou, X. Qing, H. Li, W. He, J. Chen, F. Li, Q. Zhuang, B. Qin, J. Xu, W. Li, J. Yang, Y. Gan, D. Qin, S. Feng, H. Song, D. Yang, B. Zhang, L. Zeng, L. Lai, M. A. Esteban, and D. Pei. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 7:51–63, 2010.Google Scholar
- 25.Liu, X., H. Sun, J. Qi, L. Wang, S. He, J. Liu, C. Feng, C. Chen, W. Li, Y. Guo, D. Qin, G. Pan, J. Chen, D. Pei, and H. Zheng. Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT–MET mechanism for optimal reprogramming. Nat. Cell Biol. 15:829–838, 2013.Google Scholar
- 26.Mali, P., L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville, and G. M. Church. RNA-guided human genome engineering via Cas9. Science 339:823–826, 2013.Google Scholar
- 28.McNulty, J. D., T. Klann, J. Sha, M. Salick, G. T. Knight, L. S. Turng, and R. S. Ashton. High-precision robotic microcontact printing (R-μCP) utilizing a vision guided selectively compliant articulated robotic arm. Lab Chip 14:1923–1930, 2014.Google Scholar
- 30.Ohgushi, M., M. Matsumura, M. Eiraku, K. Murakami, T. Aramaki, A. Nishiyama, K. Muguruma, T. Nakano, H. Suga, M. Ueno, T. Ishizaki, H. Suemori, S. Narumiya, H. Niwa, and Y. Sasai. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7:225–239, 2010.Google Scholar
- 31.Quintanilla, R. H., Jr., J. S. T. Asprer, C. Vaz, V. Tanavde, and U. Lakshmipathy. CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming. PLoS ONE 9:e85419, 2014.Google Scholar
- 32.Rais, Y., A. Zviran, S. Geula, O. Gafni, E. Chomsky, S. Viukov, A. A. Mansour, I. Caspi, V. Krupalnik, M. Zerbib, I. Maza, N. Mor, D. Baran, L. Weinberger, D. A. Jaitin, D. Lara-Astiaso, R. Blecher-Gonen, Z. Shipony, Z. Mukamel, T. Hagai, S. Gilad, D. Amann-Zalcenstein, A. Tanay, I. Amit, N. Novershtern, and J. H. Hanna. Deterministic direct reprogramming of somatic cells to pluripotency. Nature 502:65–70, 2013.Google Scholar
- 33.Rnjak-Kovacina, J., S. G. Wise, Z. Li, P. K. Maitz, C. J. Young, Y. Wang, and A. S. Weiss. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering. Biomaterials 32:6729–6736, 2011.Google Scholar
- 35.Saha, K., Y. Mei, C. M. Reisterer, N. K. Pyzocha, J. Yang, J. Muffat, M. C. Davies, M. R. Alexander, R. Langer, D. G. Anderson, and R. Jaenisch. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions. PNAS 108:18714–18719, 2011.Google Scholar
- 36.Samavarchi-Tehrani, P., A. Golipour, L. David, H. K. Sung, T. A. Beyer, A. Datti, K. Woltjen, A. Nagy, J. L. Wrana. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7:64–77, 2010.Google Scholar
- 37.Schindelin, J., I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9:676–682, 2012.Google Scholar
- 39.Shinagawa, T., T. Takagi, D. Tsukamoto, C. Tomaru, L. M. Huynh, P. Sivaraman, T. Kumarevel, K. Inoue, R. Nakato, Y. Katou, T. Sado, S. Takahashi, A. Ogura, K. Shirahige, and S. Ishii. Histone variants enriched in oocytes enhance reprogramming to induced pluripotent stem cells. Cell Stem Cell 14:217–227, 2014.Google Scholar
- 43.Wang, X., M. R. Salick, X. Wang, T. Cordie, W. Han, Y. Peng, Q. Li, and L. S. Turng. Poly (ε-caprolactone) nanofibers with a self-induced nanohybrid Shish–Kebab structure mimicking collagen fibrils. Biomacromolecules 14:3557–3569, 2013.Google Scholar
- 44.Watanabe, K., M. Ueno, D. Kamiya, A. Nishiyama, M. Matsumura, T. Wataya, J. B. Takahashi, S. Nishikawa, S. Nishikawa, K. Muguruma, and Y. Sasai. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25:681–686, 2007.Google Scholar
- 45.Yu, J., M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J. L. Frane, S. Tian, J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, I. I. Slukvin, and J. A. Thomson. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920, 2007.Google Scholar