Induction and isolation of hNCSCs from hESCs.

<p><b>(A):</b> A colony of H9 hESCs double stained for p75 (green) and HNK-1 (red) after one week of culture on PA6. Note that the expression of p75 and HNK-1 is outside or at the edge of the hESC colony, scale bar = 200μm; <b>(B):</b> Reverse transcription-polymerase chain reaction analysis (RT-PCR) of <i>p75</i> expression, note that the expression of <i>p75</i> is gradually increased up to day 10; <b>(C):</b> Analysis of H9-derived p75⁺ and HNK1⁺ cells by FACS analysis. The dot plots are representative of three independent experiments; <b>(D):</b> Different gene expression profiles are displayed in p75⁺ and p75^- populations as assessed by RT-PCR. Note that p75⁺ cells express markers of NCSC and sympathetic neurons. The image is the representative of three independent experiments.</p

<i>MycN</i> Is Critical for the Maintenance of Human Embryonic Stem Cell-Derived Neural Crest Stem Cells

<div><p>The biologic studies of human neural crest stem cells (hNCSCs) are extremely challenging due to the limited source of hNCSCs as well as ethical and technical issues surrounding isolation of early human embryonic tissues. On the other hand, vast majority of studies on <i>MycN</i> have been conducted in human tumor cells, thus, the role of <i>MycN</i> in normal human neural crest development is completely unknown. In the present study, we determined the role of <i>MycN</i> in hNCSCs isolated from <i>in vitro</i>-differentiating human embryonic stem cells (hESCs). For the first time, we show that suppression of <i>MycN</i> in hNCSCs inhibits cell growth and cell cycle progression. Knockdown of <i>MycN</i> in hNCSCs increases the expression of <i>Cdkn1a</i>, <i>Cdkn2a</i> and <i>Cdkn2b</i>, which encodes the cyclin-dependent kinases p21^CIP1, p16 ^INK4a and p15^INK4b. In addition, <i>MycN</i> is involved in the regulation of human sympathetic neurogenesis, as knockdown of <i>MycN</i> enhances the expression of key transcription factors involved in sympathetic neuron differentiation, including <i>Phox2a</i>, <i>Phox2b</i>, <i>Mash1</i>, <i>Hand2</i> and <i>Gata3</i>. We propose that unlimited source of hNCSCs provides an invaluable platform for the studies of human neural crest development and diseases.</p></div

<i>MycN</i> knockdown suppresses cell cycle progression in hNCSCs.

<p>FACS-sorted p75⁺ hNCSCs were transduced with concentrated pGLVH1/GFP-MYCN shRNA virus and selected in puromycin for two weeks.<b>(A):</b> Cell growth of control and <i>MycN</i>kd hNCSCs was detected by MTT assay, cell growth is presented as percentage increase: Absorbance _dn–absorbance _d0 / absorbance _d0and error bars representing 95% confidence intervals are shown. Data are from three independent experiments, ***p<0.001; <b>(B):</b> Cell cycle was assessed in adherent control and <i>MycN</i>kd hNCSCs 2weeks after lentiviral transduction; <b>(C)</b>: Flow cytometric of DNA content showed <i>MycN</i> kd hNCSCs was arrested in G0/G1, *p<0.05, **p<0.01.</p

Induction and isolation of hNCSCs from hESCs.

<p><b>(A):</b> A colony of H9 hESCs double stained for p75 (green) and HNK-1 (red) after one week of culture on PA6. Note that the expression of p75 and HNK-1 is outside or at the edge of the hESC colony, scale bar = 200μm; <b>(B):</b> Reverse transcription-polymerase chain reaction analysis (RT-PCR) of <i>p75</i> expression, note that the expression of <i>p75</i> is gradually increased up to day 10; <b>(C):</b> Analysis of H9-derived p75⁺ and HNK1⁺ cells by FACS analysis. The dot plots are representative of three independent experiments; <b>(D):</b> Different gene expression profiles are displayed in p75⁺ and p75^- populations as assessed by RT-PCR. Note that p75⁺ cells express markers of NCSC and sympathetic neurons. The image is the representative of three independent experiments.</p

<i>MycN</i> knockdown affects the expression of cell cycleand autonomic neuron development-related genes.

<p>FACS-sorted p75⁺ hNCSCs were transduced with concentrated pGLVH1/GFP-MYCN shRNA virus and selected in puromycin for two weeks.<b>(A):</b> Real-time PCR analysis showing the expression of cell cycle-related genes was altered by <i>MycN</i> suppression, *p<0.05, ***p<0.001; ****p<0.0001; quantitative data was acquired from three independent experiments;<b>(B):</b> Western blot shows knockdown of <i>MycN</i> increases the expression of p15, whereas decreases the expression of Cyclin D1; <b>(C)</b> Real-time PCR analysis showing the expression of polycomb genes and autonomic neuronal markers was altered by <i>MycN</i> suppression, *p<0.05, **p<0.01;***p<0.001; ****p<0.0001, quantitative data were acquired from three independent experiments.</p

Induction of neural crest cells from hESCs.

<p>For neural crest induction, H9 cells were plated at 5-10x10²/cm² on a confluent layer of PA6 cells in 6 well plates or chamber slides in induction medium as described in <i>Materials and Methods</i>. <b>(A):</b>Phase contrast photographs of neural crest differentiation of H9 induced by PA6, scale bar = 200μm;<b>(B):</b>Dynamic expression of sympathetic neuronal markers, <i>c-Myc</i> and <i>MycN</i> in NC differentiating cells from hESCs as determined by PCR. The image is the representative of three independent experiments.</p

<i>MycN</i> knockdown inhibits cell growth in hNCSCs.

<p>FACS-sorted p75⁺ hNCSCs were plated at a cell density of 4×10³ cells/cm² in self-renewal media on 6-well plates that were pre-coated with 15 μg/ml polyornithine, 1 μg/ml laminin and 10 μg/ml fibronectin for 24 hours. Freshly isolated hNCSCs were transduced with concentrated pGLVH1/GFP-<i>MycN</i>shRNA virus and selected in puromycin (2 μg/ml) for two weeks, as described in Materials and Methods.<b>(A):</b> The GFP expression in control and <i>MycN</i>-transduced hNCSCs; <b>(B):</b> FACS analysis shows the similar transduction efficiency in control and <i>MycN</i>-transduced hNCSCs; <b>(C):</b> RT-PCR analysis showed the expression of <i>MycN</i> in control and <i>MycN</i>kd hNCSCs after 2 weeks of antibiotics selection. Image shown is the representative of three independent experiments; <b>(D):</b> Freshly sorted cells were plated onto 15 μg/ml polyornithine, 1 μg/ml laminin and 10 μg/ml fibronectin coated plates at a concentration of 10x10³ cells/well and grown in NC media. Within two days to adherent plate, <i>MycN</i> knocked down cells initiated a change from small, NCSC-like to a larger shape.</p