Article

Isolation and characterization of neural crest cells derived from in vitro differentiated human embryonic stem cells

Divisions of Hematology-Oncology, The Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California 90027, USA.
Stem cells and development (Impact Factor: 4.2). 12/2008; 18(7):1059-70. DOI: 10.1089/scd.2008.0362
Source: PubMed

ABSTRACT The neural crest is a transient structure of vertebrate embryos that initially generates neural crest stem cells (NCSCs) which then migrate throughout the body to produce a diverse array of mature tissue types. Due to the rarity of adult NCSCs as well as ethical and technical issues surrounding isolation of early embryonic tissues, biologic studies of human NCSCs are extremely challenging. Thus, much of what is known about human neural crest development has been inferred from model organisms. In this study, we report that functional NCSCs can be rapidly generated and isolated from in vitro-differentiated human embryonic stem cells (hESCs). Using the stromal-derived inducing activity (SDIA) of PA6 fibroblast co-culture we have induced hESCs to differentiate into neural crest. Within 1 week, migrating cells that express the early neural crest markers p75 and HNK1 as well as numerous other genes associated with neural crest induction such as SNAIL, SLUG, and SOX10 are detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the p75-positive population enriches for cells with genetic, phenotypic, and functional characteristics of NCSCs. These p75-enriched cells readily form neurospheres in suspension culture, self-renew to form secondary spheres, and give rise under differentiation conditions to multiple neural crest lineages including peripheral nerves, glial, and myofibroblastic cells. Importantly, these cells differentiate into neural crest derivatives when transplanted into developing chick embryos in vivo. Thus, this SDIA protocol can be used to successfully and efficiently isolate early human NCSCs from hESCs in vitro. This renewable source of NCSCs provides an invaluable source of cells for studies of both normal and disordered human neural crest development.

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    • "Cells were collected, washed and then prepared into single cell suspension. Neural crest stem cells were screened using flow-cytometric cell sorting as described previously (Jiang et al., 2009; Yang and Xu, 2013). Briefly, cells were diluted with PBS to a final concentration of 10–20 × 10 6 cells/mL. "
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    ABSTRACT: Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3'-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3'-UTR of SOX2 mRNA.
    Neural Regeneration Research 04/2014; 9(8):828-36. DOI:10.4103/1673-5374.131599 · 0.23 Impact Factor
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    • "Xenopus, Chick, Mouse, Lamprey, Human Luo et al., 2003; Mitchell et al., 1991; Nikitina et al., 2008; Sauka-Spengler et al., 2007; Shen et al., 1997; Thomas et al., 2008 Brn3a (POU4F1) + + Curchoe et al., 2010; Goldstein et al., 2010; Jiang et al., 2009; Lee et al., 2007, 2010; Pomp et al., 2005, 2008 Mouse, Chick Fedtsova and Turner, 1995; Lindeberg et al., 1997 Cadherin-11 + Zhou and Snead, 2008 Xenopus, Chick, Mouse Chalpe et al., 2010; Kimura et al., 1995; Vallin et al., 1998 Frizzled-3 + Zhou and Snead, 2008 Xenopus Deardorff et al., 2001 HNK-1 epitope + + Chambers et al., 2009; Curchoe et al., 2010; Goldstein et al., 2010; Jiang et al., 2009; Lee et al., 2007, 2010 Chick, Dog, Pig, Human Tucker et al., 1984, 1988 NCAM + Goldstein et al., 2010; Jiang et al., 2009; Pomp et al., 2005, 2008 Xenopus, Chick, Mouse, Human Balak et al., 1987; Moase and Trasler, 1991; Thiery et al., 1982; Thomas et al., 2008 p75 NTR (NGFR) + + Chambers et al., 2009; Colleoni et al., 2010; Curchoe et al., 2010; Goldstein et al., 2010; Jiang et al., 2009; Lee et al., 2007, 2010; Pomp et al., 2005 Chick, Mouse, Human Heuer et al., 1990; Thomas et al., 2008; Wilson et al., 2004 PAX7 + Chambers et al., 2009; Colleoni et al., 2010 Xenopus, Chick, Mouse, Lamprey, Human Basch et al., 2006; Jostes et al., 1990; Maczkowiak et al., 2010; Mansouri et al., 1996; Nikitina et al., 2008; Sauka-Spengler et al., 2007; Thomas et al., 2008 Sox10 + + Colleoni et al., 2010; Curchoe et al., 2010; Jiang et al., 2009 Xenopus, Chick, Mouse, Lamprey, Human Aoki et al., 2003; Betters et al., 2010; Cheng et al., 2000; Honore et al., 2003; Kuhlbrodt et al., 1998; Sauka- Spengler et al., 2007 dHAND + Pomp et al., 2005 Xenopus, Zebrafish, Chick, Mouse Angelo et al., 2000; Srivastava et al., 1995 FoxD3 + Colleoni et al., 2010; Goldstein et al., 2010; Pomp et al., 2005, 2008 Xenopus, Chick, Mouse, Lamprey, Human Dottori et al., 2001; Kos et al., 2001; Sasai et al., 2001; Sauka-Spengler et al., 2007; Thomas et al., 2008 Msx-1 + Goldstein et al., 2010; Jiang et al., 2009; Pomp et al., 2005, 2008 Xenopus, Chick, Mouse, Lamprey, Human Hill et al., 1989; Nikitina et al., 2008; Sauka-Spengler et al., 2007; Suzuki et al., 1991, 1997; Thomas et al., 2008 Pax3 + Goldstein et al., 2010; Jiang et al., 2009; Lee et al., 2007; Pomp et al., 2008 Xenopus, Chick, Mouse, Lamprey, Human Bang et al., 1999; Betters et al., 2010; Goulding et al., 1991; Matsunaga et al., 2001; Nikitina et al., 2008; Sauka-Spengler et al., 2007; Thomas et al., 2008 Snail1 + Goldstein et al., 2010; Jiang et al., 2009; Lee et al., 2007; Pomp et al., 2005, 2008 Xenopus, Chick, Mouse, Lamprey Essex et al., 1993; Marin and Nieto, 2004; Sauka- Spengler et al., 2007; Smith et al., 1992 Sox9 + Goldstein et al., 2010; Jiang et al., 2009; Pomp et al., 2005, 2008 Xenopus, Chick, Mouse, Lamprey, Human Barrionuevo et al., 2008; Betters et al., 2010; Cheung and Briscoe, 2003; Saint-Germain et al., 2004; Sauka- Spengler et al., 2007; Thomas et al., 2008 Trkc + Jiang et al., 2009; Pomp et al., 2005 Chick, Mouse Donovan et al., 1996; Kahane and Kalcheim, 1994 premigratory neural crest markers fail to be expressed in the depleted cells and there were effects on migration in vitro (Bajpai et al., 2010). CHD7 role in neural crest development was further confirmed in vivo, using Xenopus embryos, showing the evolutionary conserved function of this gene. "
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    Developmental Biology 01/2012; 366(1):96-9. DOI:10.1016/j.ydbio.2012.01.016 · 3.64 Impact Factor
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    • "Recent studies showed that NCSCs could be isolated from ESCs and purified by sorting for p75 + cells with or without coculturing with stromal cells [Lee et al., 2007; Jiang et al., 2009], and SM22 ␣ -positive cells were obtained by sorting for CD73 and NCAM. In this study, we developed a protocol to induce the differentiation of human ESCs and iPSCs into NCSCs in a high yield without coculture . "
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    Cells Tissues Organs 01/2012; 195(1-2):5-14. DOI:10.1159/000331412 · 2.14 Impact Factor
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