Isolation and characterization of neural crest stem cells derived from in vitro-differentiated human embryonic stem cells.
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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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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ABSTRACT: Controling embryonic stem cell fate in vitro has been a major challenge in the past decade. Several protocols have been developed to obtain neural crest derivatives in culture, using more or less defined conditions. Here, we present various strategies used to date to obtain neural crest specification and the markers that can be used to identify human neural crest cells.Developmental Biology 01/2012; 366(1):96-9. DOI:10.1016/j.ydbio.2012.01.016 · 3.64 Impact Factor
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ABSTRACT: The heterogeneity of vascular smooth muscle cells (SMCs) is related to their different developmental origins such as the neural crest and mesoderm. Derivation of SMCs from different origins will provide valuable in vitro models for the investigation of vascular development and diseases. From the perspective of regenerative medicine and tissue engineering, an expandable cell source of SMCs is required for the construction of tissue-engineered blood vessels. In this study, we developed a robust protocol to derive neural crest stem cells (NCSCs) from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). NCSCs derived from ESCs and iPSCs were expandable with similar cell doubling times. NCSCs were capable of differentiating into neural and mesenchymal lineages. TGF-β1 induced the expression of SMC markers calponin-1, SM22α, and smooth muscle myosin heavy chain and resulted in the assembly of smooth muscle α-actin, calponin-1, and SM22α into stress fibers. This work provides a basis for using iPSCs to study SMC biology and deriving vascular cells for tissue engineering.Cells Tissues Organs 01/2012; 195(1-2):5-14. DOI:10.1159/000331412 · 2.14 Impact Factor