Characterization of neural crest-derived ASCs.

<p><b><i>A</i></b>. Quantification of surface marker expression in both GFP+ and GFP− ASCs of either P0-Cre/FR or Wnt1-Cre/FR mice with flow cytometric analysis. The majority of both GFP+ and GFP− cells expressed MSC markers including CD29, CD44, CD90, CD105, and Sca-1. <b><i>B</i></b>. The proportion of both CD24- and CD34-positive cells in the GFP+ population was also higher, and the proportion of double-positive cells in the GFP+ population was more than five-fold higher compared to those in GFP− cells. Representative flow cytometry profiles of CD24/CD34 double-positive cells from P0-Cre/FR mice are shown. <b><i>C</i></b>. The GFP+ population had a significantly higher proportion of p75NTR-positive cells. Data (<i>A–C</i>) are shown as the mean + SEM of 4 independent experiments for each condition. *<i>p</i><0.05 versus GFP− cells, <i>t</i> test. <b><i>D</i></b>. Immunofluorescent staining for p75NTR, S100, Nestin, GFAP, and fibronectin (<i>red</i>) of either P0-Cre/FR ASCs (<i>upper five panels</i>) or Wnt1-Cre/FR (<i>lower two panels</i>) shows that the majority of GFP+ cells (<i>green</i>) co-expressed p75NTR, S100, and Nestin (<i>arrow</i>) but were negative for GFAP and fibronectin. A few GFP-negative but p75NTR- S100-, or Nestin-positive cells were also observed (<i>arrowhead</i>). Note that the majority of GFP− cells were positive for fibronectin. Scale bar = 50 µm. <i>E</i>. RT-qPCR analysis for pluripotent markers <i>Sox2, Nanog</i>, and <i>Oct3/4</i> on FACS-purified GFP− and GFP+ cells from P0-Cre/FR mice. Results are normalized based on <i>GAPDH</i> expression and shown as relative changes to GFP− cells. Data are shown as the mean + SEM of 3–4 independent experiments for each condition.