Contribution of neural crest-derived cells to adult ASCs.

<p><b><i>A</i></b>. Schematic diagram of the Cre-loxP-mediated fate mapping of NC-derived cells. Either P0-Cre or Wnt1-Cre transgenic mice are crossed with CAG-CAT-EGFP mice to generate NC-specific Cre/Foxed-Reporter (FR) mice in which NC-derived cells can be traced by the expression of the reporter protein GFP. <b><i>B</i></b>. Flow cytometry profiles of ASCs from adult Cre/FR mice. The x axis is GFP fluorescent intensity, and the y axis is the side scatter channel (SSC). ASCs from non-transgenic controls served as a gating control. The proportions of GFP+ cells per total cells were 0.98±0.24% and 1.48±0.35% in P0-Cre/FR and in Wnt1-Cre/FR mice, respectively. <b><i>C</i></b>. Phase contrast and fluorescent micrographs of second passage ASC cultures isolated from adult P0-Cre/FR (<i>left</i>) and Wnt1-Cre/FR mice (<i>right</i>). The majority of GFP+ cells possess small soma with long slender processes (<i>arrow in bottom</i>), whereas a few cells show a flat/polygonal shape similar to GFP- ASCs (<i>arrowhead</i>). Scale bar  = 50 µm. <b><i>D</i></b>. The proportions of GFP+ cells in ASC cultures from P0-Cre/FR mice of different ages (1 week-, 3 week-, and 16 week-old) at different passages (from passage 0 to 6). Data are shown as the mean + SEM of 6 independent cultures. <b><i>E</i></b>. Growth curves of GFP+ and GFP− cells sorted from second passage ASCs of P0-Cre/FR mice. Results are shown as relative changes to the cell number on day 0 (after sorting). There was no significant difference in the population doubling time between GFP+ and GFP− cells. Data are shown as the mean ± SEM of 3 independent experiments for each condition.</p

RT-PCR primer sequences.

<p>RT-PCR primer sequences.</p

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.</p

Adipogenic potential of neural crest-derived ASCs.

<p><b><i>A</i></b>. Phase contrast and fluorescent images show that both GFP+ (<i>arrow</i>) and GFP− ASCs (<i>arrowhead</i>) from P0-Cre/FR mice contain lipid droplets after adipogenic differentiation. Scale bar  = 50 µm. <b><i>B</i></b>. FACS-purified GFP+ cells are positive for both perilipin immunostaining (<i>top left, top right, and bottom left</i>) and Oil red O staining (<i>bottom right</i>) after adipogenic differentiation. Nuclei were stained with DAPI (<i>blue</i>). Scale bar  = 50 µm. <b><i>C</i></b>. Representative images and quantification of adipogenic differentiation of FACS-purified GFP+ and GFP− cells. The percentage of perilipin-positive cells per total cells was more than 1.5-fold higher in the GFP+ cells than that of the GFP-cells. Data 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>. RT-qPCR analysis for preadipocyte markers <i>Pref-1, C/EBPδ</i>, and <i>GATA2</i>, as well as for mature adipocyte markers <i>PPARγ</i> and <i>aP2</i> on purified GFP− and GFP+ cells before or after (<i>adipo</i>) adipogenic induction. Results are normalized based on <i>GAPDH</i> expression and shown as relative changes to undifferentiated GFP− cells. Data are shown as the mean + SEM of 3–4 independent experiments for each condition.</p

Osteogenic and chondrogenic potential of neural crest-derived ASCs.

<p><b><i>A</i></b>. Bright-field images of alizarin staining (<i>top</i>) and fluorescent images of osteopontin immunostaning (<i>red in bottom</i>) in FACS-purified GFP- and GFP+ cells after osteogenic induction. Nuclei were stained with DAPI (<i>blue</i>). Scale bar  = 50 µm. <b><i>B</i></b>. The osteopontin-positive area was measured and expressed as a percentage of the total area. Data are shown as mean +SEM, n = 3. **<i>p</i><0.01 versus GFP− cells, <i>t</i> test. <b><i>C</i></b>. Bright-field images of Alcian blue staining in FACS-purified GFP+ and GFP− cells after chondrogenic induction. Scale bar = 100 µm. <b><i>D</i></b>. RT-qPCR analysis for chondrogenic markers <i>Aggrecan, COL2a1</i>, and <i>Sox9</i> in the GFP− and the GFP+ cells after chondrogenic differentiation. 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.</p

<i>In vivo</i> localization of neural crest-derived cells in the subcutaneous adipose tissue.

<p>Confocal micrographs of the reporter protein GFP (<i>green</i>) combined with immunofluorescence of various markers (<i>red</i>) in the adult subcutaneous tissue of either P0-Cre/FR (<i>A, B, D–I</i>) or Wnt1-Cre/FR (<i>C, J</i>) mice. <b><i>A–A″</i></b>. A small portion of perilipin-positive adipocytes expressed GFP in the cephalic region. <b><i>B</i></b>. Peripheral nerve tissue was GFP+. <b><i>C</i></b>. GFP+ cells with slender processes were identified in the stroma of both cephalic and trunk regions. <b><i>D–J</i></b>. These cells (<i>arrow</i>) were localized along the vessels or occasionally in the vicinity to GFP+ adipocytes (<i>arrowhead in E</i>). They were negative for an endothelial marker CD31 (<i>D</i>), mural markers PDGFRβ and αSMA (<i>E, F</i>), and a glial marker GFAP (<i>G</i>), but positive for p75NTR and S100 (<i>H–J</i>). Nuclei were stained with DAPI (<i>blue, D–F, H–J</i>). Scale bar = 20 µm (<i>A, B</i>), 10 µm (<i>C–J</i>).</p