Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany.
Institute of Human Genetics, Life & Brain Center, University of Bonn Medical Faculty, 53127 Bonn, Germany.
Methods. 2018 Jan 15;133:65-80. doi: 10.1016/j.ymeth.2017.09.012. Epub 2017 Oct 14.
The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC). Typically, these protocols comprise several cell culture steps to enrich for NCCs in the neural derivatives of the differentiating hPSCs. Here we report on a remarkable and hitherto unnoticed in vitro segregation phenomenon that enables direct extraction of virtually pure NCCs during the earliest stages of hPSC differentiation. Upon aggregation to embryoid bodies (EB) and replating, differentiating hPSCs give rise to a population of NCCs, which spontaneously segregate from the EB outgrowth to form conspicuous, macroscopically visible atoll-shaped clusters in the periphery of the EB outgrowth. Isolation of these NC clusters yields p75NTR(+)/SOXE(+) NCCs, which differentiate to peripheral neurons and glia as well as mesenchymal derivatives. Our data indicate that differentiating hPSC cultures recapitulate, in a simplified manner, the physical segregation of central nervous system (CNS) tissue and NCCs. This phenomenon may be exploited for NCC purification and for studying segregation and differentiation processes observed during early human NC development in vitro.
神经嵴（NC）是一种具有显著特征的短暂胚胎细胞群体。在从神经管分离后，NC 细胞（NCC）广泛迁移，遍布身体的几乎所有组织，并分化为高度多样化的细胞类型，如外周神经元和神经胶质细胞，但也包括间充质细胞，如软骨细胞、成骨细胞和脂肪细胞。虽然 NC 已在几种动物模型中得到广泛研究，但对人类 NC 发育知之甚少。已经建立了许多方法来从人类多能干细胞（hPSC）体外衍生 NCC。通常，这些方案包括几个细胞培养步骤，以在分化的 hPSC 的神经衍生物中富集 NCC。在这里，我们报告了一个显著的、迄今为止未被注意到的体外分离现象，该现象能够在 hPSC 分化的最早阶段直接提取几乎纯的 NCC。在聚集形成类胚体（EB）并重新铺板后，分化的 hPSC 会产生一群 NCC，这些 NCC 会自发地从 EB 外生体中分离出来，形成在 EB 外生体边缘明显的、肉眼可见的环形簇。分离这些 NC 簇可获得 p75NTR(+) / SOXE(+) NCC，其分化为外周神经元和神经胶质细胞以及间充质衍生物。我们的数据表明，分化的 hPSC 培养物以简化的方式再现了中枢神经系统（CNS）组织和 NCC 的物理分离。这种现象可用于 NCC 的纯化以及研究体外早期人类 NC 发育过程中观察到的分离和分化过程。
