br Results br Discussion We

Results
Discussion We have established a novel serum-free culture method for generating and expanding CD271+CD73+SOX9+ chondrogenic ectomesenchymal diacylglycerol kinase from hPSC-derived, CD271hiCD73− neural crest-like progeny. The differentiation pathways from hPSCs to chondrocytes via chondrogenic ectomesenchymal cells postulated from this study are summarized in the Graphical Abstract. Under optimal conditions, the generation of neural crest-like progeny from hPSCs in CDM was achieved more quickly than previously reported, and the resultant ectomesenchymal cells displayed long-term chondrogenic activity. Quantitative data on the efficiency of genesis of SOX9-expressing chondroprogenitors from hESCs were first shown by Oldershaw et al. (2010). Under their conditions in which hESC differentiation is directed initially to mesoderm then toward chondrocytes, approximately 8.5 chondroprogenitors of 75%–97% SOX9+ are produced per hESC by day 14 of differentiation. In contrast, while only about 0.6 CD271hiCD73− neural crest-like progeny were generated per hPSC by day 6 of differentiation in CDM plus SB431542 (data not shown), our method for neural crest-like cell culture in CDM in the presence of FGF2+SB431542 led to 19 to 38 CD271+CD73+ chondroprogenitors per hPSC by day 14 (Figure S3A). Approximately 76%–85% of these cells continued to express SOX9 protein on days 27–33 (p8–p10), a proportion that increased slightly to about 97% after 3 days of treatment with FGF2+TGFβ3. Further expansion yielded approximately 1 × 107 chondrogenic ectomesenchymal cells per hPSC by day 51 (p16, 24 population doublings). Thus, the method described here allows human chondrogenic cells to expand for an extended period without the loss of purity and chondrogenic activity, resulting in a much larger yield of chondroprogenitor cells (although not homogenous) from hPSCs than previously attained. The effect of SB431542 on expansion of endothelial progenitors derived from mouse and human ESCs has also been reported (James et al., 2010; Watabe et al., 2003). A lack of Nodal/Activin/TGFβ signaling may generally promote proliferation and prevent terminal differentiation of embryonic stem/progenitor cells. In this respect, the ways in which FGF2+SB431542 helps to maintain SOX9 and CD271 while suppressing N-cadherin expression during expansion of the chondrogenic CD271+CD73+ cells and in which CD271hiCD73− neural crest-like cells are directed to give rise to such cells are interesting topics for future study. In this respect, comparative transcriptome analysis has revealed that the CD271+CD73+ cells accumulated under FGF2+SB431542 conditions possess a similar mRNA profile to primitive neural crest/ectomesenchymal cells, although they lacked SOX10 expression, which is critical for neural and melanocytic lineage commitment. Thus, suppression of Nodal/Activin/TGFβ signaling does not seem to freeze the developmental stage of the hPSC-derived neural crest during expansion. Such suppression may instead simply support the high proliferative potential of the cells as well as the expression of SOX9 (and COL2A1), and thereby maintain chondrogenic activity. SOX9 expression initiated at the specification and premigratory stages is transient in trunk neural crest but persists in cranial neural crest (Cheung and Briscoe, 2003; Cheung et al., 2005; McKeown et al., 2005). The chondrogenic CD271+CD73+ ectomesenchymal cells that maintain SOX9 transcription and translation (SOX9-GFP+/lo) may therefore represent proliferating cranial neural crest, with a slight commitment to non-neural lineages. Genesis of MSC-like cells from hESCs directly or via neuroectoderm specification has been demonstrated (Chambers et al., 2009; Mahmood et al., 2010). As shown in Figure S4D, the colonogenic (CFU-F) activity, representing self-renewal activity of MSCs, emerged as early as p2 of the expansion culture under FGF2 alone (i.e., in CD271−CD73 cells) or FGF2+SB431542 (i.e., in CD271+CD73+ cells). However, we were unable to demonstrate significant adipogenic or osteogenic activity in either cell type (data not shown), although the GO analysis predicted osteogenic activity in cells generated under FGF2 (data not shown) and FGF2+SB431542→FGF2 conditions. This discrepancy may be due to some degree of heterogeneity in the cell types generated under these conditions or to the assay method employed, which is widely used for MSCs (Pittenger et al., 1999), but is less well validated for mesenchymal cells derived from human neural crest. Furthermore, the FGF2+SB431542 conditions applied to the expansion culture of mouse bone marrow MSCs resulted in inhibition of proliferation and promotion of adipocytic differentiation (data not shown). The CD271+CD73+ ectomesenchymal cells are therefore very likely not to be MSCs, although the possibility that they are derived from an MSC-like precursor has not been excluded.