Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

BackgroundNeural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).
Methodology/Principal FindingsWe performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184+/CD271−/CD44−/CD24+ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184−/CD44−/CD15LOW/CD24+ and a population of glia that was CD184+/CD44+ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.
Conclusions/SignificanceThese findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

研究背景：人类多能干细胞的神经诱导过程通常会产生异质性细胞群体，这会干扰定量与比较分析。当前亟需优化的分化与富集流程，以获取高纯度的神经干细胞（NSC）、胶质细胞与神经元群体。解决该问题的一种思路是筛选细胞表面标志物组合，从而通过荧光激活细胞分选术（FACS）从异质性细胞群体中分离目标细胞类型。
方法与研究结果：我们针对未成熟人类胚胎干细胞（hESC）以及神经分化培养体系中的细胞衍生物，使用190种细胞表面标志物抗体开展了无偏倚的基于FACS与成像的免疫表型分析。通过该分析，我们鉴定出可用于分离NSC、胶质细胞与神经元的潜在细胞表面标志物组合。我们从hESC与人类诱导多能干细胞（hiPSC）的神经诱导培养物中，分离得到了CD184+/CD271−/CD44−/CD24+的NSC群体。经分选的NSC可多次传代扩增，并能在体外与体内分化为神经元与胶质细胞的混合培养体系。随后，我们从分化中的NSC培养物中分别纯化得到CD184−/CD44−/CD15LOW/CD24+的神经元群体，以及CD184+/CD44+的胶质细胞群体。纯化后的神经元具有活性，表达成熟神经元及亚型特异性标志物，并可产生动作电位；纯化后的胶质细胞具有增殖活性，可在体外与体内分化为表达胶质纤维酸性蛋白（GFAP）的星形胶质细胞。
结论与意义：本研究证实了免疫表型筛选在鉴定人类多能干细胞来源神经细胞表面标志物组合中的应用价值。此类标志物组合可通过FACS分离得到高纯度的活性NSC、胶质细胞与神经元群体。本文所述方法可支持后续需要标准化、明确神经细胞群体的相关研究。