Parts of a bony particle made in (C) and (D) were stained with H&E (G) or stained with hMit antibody (pink, G and H) and anti-type I collagen (COL1) antibody (green, H), followed by counterstaining with DAPI (blue). CD271hiCD73? fraction. The PAX3 protein (Physique?1E) and transcript (Physique?S1G) were also detected in the CD271hiCD73? fraction. Furthermore, in sharp contrast to the results obtained from the hPSC differentiation to paraxial mesoderm (Umeda et?al., 2012), when (an early mesendoderm gene)-green fluorescence protein (GFP) knockin hESCs (MIXL1-GFP) (Davis et?al., 2008) were differentiated under comparable conditions, no MIXL1-GFP+ progeny developed (Physique?1A). There was also negligible induction of a second mesendoderm transcript, (Physique?S1B) (Umeda et?al., 2012). Therefore, neither CD271hi(PDGFRlo)CD73? nor CD271lo(PDGFR?)CD73? cells were likely to be mesendodermal derivatives. BMP and WNT are implicated in the neural crest specification (Milet and Monsoro-Burq, 2012). As expected, the BMP inhibitor Noggin suppressed the SB431542-induced development of the CD271hiPDGFRlo(CD73?) neural crest-like progeny from H9 hESCs (Physique?S1E). The WNT inhibitor FZD also showed an inhibitory effect, consistent with the findings of Menendez et?al. (2011) (Physique?S1D). Interestingly, BMP4 at 10?ng/ml, a concentration sufficient to induce mesoderm (Wang and Nakayama, 2009), was as inhibitory as Noggin, and the GSK3 inhibitor that mimics canonical WNT signaling showed weakly inhibitory effects (Physique?S1E). However, when SOX9-GFP iPSCs were used, the GSK3 inhibitor was found to enhance the genesis of CD271hiCD73? cells (Physique?S1I). Thus, inhibition of Nodal/Activin/TGF signaling with appropriate levels of BMP and WNT signaling is required for the effective development of CD271hiPDGFRloCD73?CD13? neural D-(+)-Xylose crest-like progeny from hPSCs (hereafter called CD271hiCD73? progeny) more quickly than previously attained (Lee et?al., 2010; Menendez et?al., 2011), potentially reflecting the specification of cranial instead of trunk neural crest cells. Mesenchymal Cells Derived from the Nonmesendodermal hESC Progeny by Conventional Methods Show Weak, Transient Chondrogenic Activity The neural crest-like progeny were Rabbit Polyclonal to EPHA3/4/5 (phospho-Tyr779/833) then directed to commit to chondrogenic ectomesenchyme. First, using a conventional EB-outgrowth method (Hwang et?al., 2006) (Physique?S2A), we generated mesenchymal cells from the SB431542-treated H9 and MIXL1-GFP hESCs. In knockout serum replacement-based SR medium or serum-containing D10 medium, expansion of the outgrowth cells led to enhanced expression of CD73 and later CD13, but loss of the expression of CD271 (Figures S2D and S2E). As we reported previously (Umeda et?al., 2012), MIXL1-GFP+ mesendodermal progeny were never detected during such studies (data not shown). In 3D-pellet culture, the generated mesenchymal cells gave rise to a particle made up of an area that weakly stained metachromatically (pink to purple) with Toluidine Blue and immunostained with anti-type II collagen (COL2) antibody at passage 1 (p1) (Physique?S2F) and p2, but not from p3 to p5. The lack of chondrogenic activity in the primary outgrowth cells (p0), suggests that a short-term expansion of the outgrowth cells is required for its development and/or accumulation. However, as reported by others (Nakayama and Umeda, 2011), we did not observe robust chondrogenic activity leading to a full-cartilage particle, as found for paraxial mesoderm derived from mPSCs and hPSCs (Nakayama et?al., D-(+)-Xylose 2003; Umeda et?al., 2012). Thus, conventional culture methods failed to generate and maintain strong chondrogenic activity from hPSC-derived neural crest-like progeny. Generation and Selective Expansion of CD271+PDGFR+CD73+ Mesenchymal Cells in CDM in the Presence of FGF2 and SB431542 Either in a FACS-purified form or in an unpurified mixture with other nonmesendodermal (i.e., MIXL1?) cells, the CD271hiCD73? neural crest-like progeny failed to adhere to the culture dish in the absence of fibronectin and grew poorly in the medium in which they were specified, i.e., CDM plus D-(+)-Xylose SB431542 (SB; Figures 2B and S3A). Therefore, we tested the effects of growth factors, such as FGF2 that have been used for maintaining neural crest cells (Stemple and Anderson, 1992) and generating chondrogenic activity (Abzhanov et?al., 2003) in culture, and of other.