Isolation and Characterization of Neural Crest Stem Cells Derived From In Vitro-Differentiated Human Embryonic Stem Cells
Isolation and Characterization of Neural Crest Stem Cells Derived From In Vitro-Differentiated Human Embryonic Stem Cells
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DOI：
10.1089/scd.2008.0362
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发表时间：
2009-09-01
影响因子：
4
通讯作者：
Lawlor, Elizabeth R.
中科院分区：
文献类型：
--
作者：
Jiang, Xiaohua;Gwye, Ynnez;Lawlor, Elizabeth R.
神经嵴是脊椎动物胚胎的一种瞬时结构，最初产生神经嵴干细胞（NCSCs），然后在全身迁移，产生各种成熟组织类型。由于成人NCSCs的罕见性以及围绕早期胚胎组织分离的伦理和技术问题，人类NCSCs的生物学研究极具挑战性。因此，关于人类神经嵴发育的许多已知信息都是从模式生物中推断出来的。在这项研究中，我们报道了功能性NCSCs可以从体外分化的人胚胎干细胞（hESCs）中快速生成和分离。利用PA6成纤维细胞共培养的基质诱导活性（SDIA）诱导hESCs向神经嵴分化。在1周内，可以检测到表达早期神经嵴标志物p75和HNK1以及许多其他与神经嵴诱导相关的基因（如SNAIL、SLUG和SOX10）的迁移细胞。基于荧光激活细胞分选（FACS）的p75阳性群体的分离丰富了具有NCSCs遗传、表型和功能特征的细胞。这些富含p75的细胞在悬浮培养中容易形成神经球，自我更新形成次级球，并在分化条件下产生多种神经嵴谱系，包括周围神经、胶质细胞和肌成纤维细胞。重要的是，这些细胞在体内移植到发育中的鸡胚胎后分化为神经嵴衍生物。因此，该SDIA协议可用于体外成功有效地分离早期人类NCSCs和hESCs。这种可再生的神经干细胞为研究正常和紊乱的人类神经嵴发育提供了宝贵的细胞来源。
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.