Direct reprogramming of mouse fibroblasts to neural progenitors

Janghwan Kim

Jem Efe

Saiyong Zhu

Maria Talantova

Supplementary Material

Supporting Information:

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^{Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;}

^{Development and Differentiation Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, 305-806, Republic of Korea;}

^{Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;}

^{Institute for Genomic Medicine and Shiley Eye Center, University of California at San Diego, La Jolla, CA 92093;}

^{Molecular Medicine Research Center and Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610065, China; and}

^{Gladstone Institute of Cardiovascular Disease, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158}

^{To whom correspondence should be addressed. E-mail:}ude.fscu.enotsdalg@gnid.gnehs.

Edited^* by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved March 29, 2011 (received for review February 24, 2011)

Author contributions: J.K., K.Z., and S.D. designed research; J.K., J.A.E., S.Z., M.T., X.Y., S.W., and S.A.L. performed research; J.K., J.A.E., S.Z., M.T., X.Y., S.A.L., and S.D. analyzed data; and J.K., K.Z., and S.D. wrote the paper.

Edited^* by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved March 29, 2011 (received for review February 24, 2011)

Abstract

The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor–based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.

Abstract

Although successful transdifferentiation from one cell type to another by overexpressing lineage-specific genes in vivo (1, 2) and in vitro (3, 4) has been reported, until recently these methods were only effective for fate switching within the major lineages, i.e., ectoderm, mesoderm, and endoderm. However, the generation of iN cells (5) using neural-specific transcription factors has established that interlineage transdifferentiation is also possible in vitro. These transdifferentiation schemes entail overexpression of different sets of lineage-specific transcription factors. A more recent example reported single-factor transdifferentiation of fibroblasts into blood precursors using long-term ectopic expression of OCT4 (6); through extensive binding to the regulatory regions of key hematopoietic genes, OCT4 also appears to be participating in regulating hematopoietic programs acting as a lineage-specific transcription factor in this context. An important aspect of this study is the ability to generate a mitotically active progenitor population that can be further differentiated into a variety of blood cells—a critical feat that has yet to be accomplished in transdifferentiation to neural and endoderm lineages.

In an effort to devise a more general transdifferentiation strategy that might give rise to a broad array of unrelated cell types—including lineage-specific precursors—we attempted to direct conventional four iPSC-factor–based reprogramming (7, 8) toward alternative outcomes. Specifically, studies indicating that iPSCs are generated in a sequential and stochastic manner (9 –11) led us to hypothesize that we might be able to manipulate cells at an early and epigenetically highly unstable state induced by the reprogramming factors. Different conditions could potentially give rise to a multitude of cell types (12) with more stable epigenetic profiles. In this context, induced pluripotency is only one—and perhaps among the less likely—of many possible outcomes. Indeed, studies have found partially or incompletely reprogrammed cells expressing multiple lineage-specific markers (7, 13 –17), although these cells did not appear to represent physiologically relevant cell types. Accordingly, we hypothesized that it might be possible to deliberately bias the early reprogramming process toward a defined cell type by using inductive and/or permissive signaling conditions, after which the desired cells could be selected and/or expanded. On the basis of this same hypothesis and using a similar methodology, our group has recently shown that direct reprogramming into cardiomyocytes can be achieved (18).

In the present study, we have directly reprogrammed fibroblasts to functional neural stem/progenitor cells (NPCs) over an abbreviated period of four-factor induction. This direct reprogramming process is clearly distinct from conventional reprogramming to iPSCs or forward differentiation of pluripotent cells. Our findings not only represent a unique successful transdifferentiation of somatic cells into proliferating NPCs, but also form the basis of a methodology for interlineage transdifferentiation into multi- or oligopotent cells.

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Acknowledgments

We thank Wenlin Li, Per Stehmeier, and Tongxiang Lin for critical reading of the manuscript. We appreciate the assistance of Ella Kothari and Jun Zhao at the University of California at San Diego, La Jolla, CA Stem Cell Core in blastocyst injections and Chao Zhao in caring for mice. We thank Debbie Watry for flow cytometry and general assistance. The pHAGE2-TetOminiCMV-STEMCCA plasmid was a generous gift from Dr. Gustavo Mostoslavsky. S.D. and K.Z. are supported by the National Institutes of Health (NIH) Director's Transformative R01 Program (R01 EY021374). S.D. is supported by funding from the National Institute of Child Health and Human Development, National Heart, Lung, and Blood Institute; the National Institute of Mental Health/NIH; the California Institute for Regenerative Medicine; the Prostate Cancer Foundation, Fate Therapeutics; the Esther B. O'Keeffe Foundation; and The Scripps Research Institute. K.Z. is supported by grants from the Chinese National 985 Project to Sichuan University and West China Hospital, the National Eye Institute/NIH, Veterans Administration Merit Award, the Macula Vision Research Foundation, Research to Prevent Blindness, a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, and the Dick and Carol Hertzberg Fund. S.A.L. and M.T. were supported in part by NIH Grants P01 HD29587, P01 ES016738, P30 NS057096, and R01 EY05477 and by the California Institute for Regenerative Medicine.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103113108/-/DCSupplemental.

Footnotes

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