Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), NIH, DHHS, Baltimore, MD, USA.
PLoS One. 2009 Aug 12;4(8):e6606. doi: 10.1371/journal.pone.0006606.
Stromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed "SDIA" is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC).
METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT) in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections.
CONCLUSIONS/SIGNIFICANCE: The combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products.
基质衍生诱导活性（SDIA）是从胚胎干细胞（ESC）中产生多巴胺能（DA）神经元的最有效方法之一。通过将 ESC 与小鼠基质细胞系 PA6 或 MS5 共培养，可以实现 DA 神经元的诱导。这种效应被称为“SDIA”，但其分子性质目前尚不清楚。最近，我们发现 PA6 细胞分泌的因子为诱导人 ESC（hESC）向 DA 分化提供了谱系特异性指令。
方法/主要发现：在本研究中，我们将 PA6 细胞与缺乏 SDIA 效应的各种细胞系进行了比较，并采用基因组表达分析来鉴定差异表达的信号分子。在 PA6 细胞高表达的因子中，有基质细胞衍生因子 1（SDF-1/CXCL12）、多效蛋白（PTN）、胰岛素样生长因子 2（IGF2）和 Ephrin B1（EFNB1）与 CNS 发育有关。当将这四种因子（称为 SPIE）应用于 hESC 时，它们在体外诱导 TH 阳性神经元的分化。RT-PCR 和 Western blot 分析证实了这些四种分子作用下培养物中中脑特异性标记物的表达，包括 engrailed 1、Nurr1、Pitx3 和多巴胺转运体（DAT）。电生理记录显示，用 SPIE 处理 hESC 可诱导产生动作电位和形成功能突触连接的神经元分化。
结论/意义：SDF-1、PTN、IGF2 和 EFNB1 的组合模拟了 SDIA 诱导 DA 表型的特性，足以促进 hESC 分化为功能性中脑 DA 神经元。这些发现为 hESC 分化形成 DA 神经元提供了一种方法，无需使用动物源性细胞系或产品。
