Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells

Department of Biochemistry and Molecular Biology, Paul D Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA 30602, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2011; 108(48):19240-5. DOI: 10.1073/pnas.1113746108
Source: PubMed


Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.


Available from: Parker Antin, Apr 17, 2014
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    • "The critical role of WNT signaling in our NC-induction conditions is consistent with previous findings in avian systems, which demonstrated that WNT signaling is necessary and sufficient for NC induction (García-Castro et al., 2002). Similar requirements for WNT signaling to induce a p75, HNK-1 coexpressing human NC population have also been reported (Lee et al., 2007; Menendez et al., 2011); however, our study is unique in addressing the temporal requirements for WNT signaling during human NC specification. The very narrow window during which WNT signaling promoted Sox10::GFP induction was unexpected and offers a powerful tool to mechanistically define competency factors that act together with WNT in NC specification. "
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    Cell Reports 04/2013; 3(4). DOI:10.1016/j.celrep.2013.03.025 · 8.36 Impact Factor
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    • "However, we observed that there was a loss of neurogenic potential with sequential passages of neurospheres, resulting in more gliogenesis, which was previously reported [27]. More recently, small molecule inhibitors (SMIs) that inhibit SMAD signaling have been used for the differentiation of ES and iPS cells to rapidly generate specific CNS cell types [44]–[47]. This approach, which bypasses the use of stromal cell lines or the generation of neurospheres, directly yields neural progenitor cells from which specific CNS cell types can be derived. "
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    PLoS ONE 12/2012; 7(12):e53010. DOI:10.1371/journal.pone.0053010 · 3.23 Impact Factor
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    • "By this way cells can be manipulated utilizing the knowledge on the signaling pathways for chondrogenic differentiation of MSCs [166], skin aging [167], fracture healing of the bone [168], liver regeneration [169] or cellular apoptosis [170], in order to provide solutions for engineering complex tissues. For example, human pluripotent stem cells were reprogrammed into multipotent neural crest cells through activation of the canonical Wnt signaling and suppression of the Activin A/Nodal pathway simultaneously [171]. It was Fig. 6. (A) Schematics of the simultaneous protein immobilization using 2 photon laser scanning method. "
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