Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.
Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.
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DOI：
10.1371/journal.pone.0017540
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发表时间：
2011-03-02
期刊：
影响因子：
3.7
通讯作者：
Carson CT
中科院分区：
文献类型：
--
作者：
Yuan SH;Martin J;Elia J;Flippin J;Paramban RI;Hefferan MP;Vidal JG;Mu Y;Killian RL;Israel MA;Emre N;Marsala S;Marsala M;Gage FH;Goldstein LS;Carson CT
人多能干细胞的神经诱导通常产生异质细胞群，这可能妨碍定量和比较分析。需要改进的分化和富集程序，以产生高纯度的神经干细胞（NSC）、神经胶质和神经元群体。解决这个问题的一种方法是鉴定细胞表面特征，其使得能够通过荧光激活细胞分选（FACS）从异质细胞群体中分离这些细胞类型。我们使用190种抗体对幼稚人胚胎干细胞（hESC）和神经分化培养物的细胞衍生物进行了无偏倚的FACS和基于图像的免疫表型分析。从该分析中，我们确定了用于分离NSC、神经胶质细胞和神经元的预期细胞表面特征。我们从hESC和人诱导多能干细胞（hiPSC）的神经诱导培养物中分离出一群CD 184 +/CD 271 −/CD 44 −/CD 24+的NSC。分选后的神经干细胞在体外和体内均可传代，并可分化为神经元和胶质细胞的混合培养物。随后从分化的NSC培养物中纯化了一群CD 184 −/CD 44 −/CD 15 LOW/CD 24+的神经元和一群CD 184 +/CD 44+的神经胶质。纯化的神经元是可行的，表达成熟和亚型特异性神经元标记物，并能发射动作电位。纯化的胶质细胞在体外和体内均能成熟为表达GFAP的星形胶质细胞。这些发现说明了免疫表型筛选用于鉴定源自人多能干细胞的神经细胞的细胞表面特征的效用。这些特征可用于通过FACS分离高纯度的活NSC、神经胶质和神经元群体。这里描述的方法将使下游研究，需要一致的和明确的神经细胞群。
Neural 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). We 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. These 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.
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