Department of Neuroscience, University Medical Center Groningen, University of Groningen, A. Deusinglaan 1, 9713AV Groningen, The Netherlands.
Neuroscience. 2010 Oct 13;170(2):417-28. doi: 10.1016/j.neuroscience.2010.07.023. Epub 2010 Jul 17.
Neural stem cells (NSCs), either isolated from fetal or adult human brain or derived from induced pluripotent stem cells, are now considered major candidates for in vitro generation of transplantable dopaminergic (DA) neurons and modeling of Parkinson's disease. It is generally thought that in vitro differentiation of neural stem cells into meso-diencephalic dopaminergic neurons, requires recapitulation of dopaminergic differentiation pathway normally occurring in the ventral mesencephalon during embryogenesis. This dopaminergic pathway is partially activated by a combination of the extracellular induction factors Sonic Hedgehog (Shh), Fibroblast Growth Factor 8 (FGF8) and Wnt1 that trigger specific intracellular transcription cascades. In vitro mimicking of these embryonic ventral mesencephalic conditions has been successful for dopaminergic differentiation of embryonic stem cells and ventral mesencephalic NSCs. Dopaminergic differentiation of non-mesencephalic NSCs (nmNSCs), however, is considered arduous. Here we examine whether Shh, FGF8 and Wnt1 can activate typical dopaminergic transcription factors, such as Lmx1a, Msx1 and Otx2 in nmNSCs. We found that Shh, FGF8 and Wnt1 induced the expression of Lmx1a and Otx2 in nmNSCs resulting in the differentiation of up to 39% of the nmNSCs into neurons expressing Pitx3. However, only a low number ( approximately 13%) of these cells became more DA-like neurons also expressing tyrosine hydroxylase (TH). The histone deacetylase (HDAC)-inhibitor trichostatin A combined with Shh, FGF8 and Wnt1 caused orchestrated induction of Lmx1a, Otx2, Msx1 plus the early DA transcription factor En1. Now significantly increased numbers of TH ( approximately 22%) and Pitx3 ( approximately 33%) neurons were observed. Most of these cells coexpressed the DA markers DAT and Vmat2. Taken together, we demonstrate that nmNSCs indeed can be differentiated towards DA-like neurons, but this differentiation is far from complete in comparison to ventral mesencephalic NSCs and embryonic stem cells; most likely, the nmNSCs lack the proper "primed" epigenetic state of these cells for DA differentiation facilitating the induction of DA specific transcription factors.
神经干细胞（NSCs），无论是从胎儿或成人脑中分离出来的，还是从诱导多能干细胞中衍生出来的，现在都被认为是体外生成可移植的多巴胺能（DA）神经元和模拟帕金森病的主要候选者。一般认为，体外将神经干细胞分化为中脑多巴胺能神经元，需要重现胚胎发育过程中中脑腹侧多巴胺能分化途径。这个多巴胺能途径部分被 Sonic Hedgehog（Shh）、Fibroblast Growth Factor 8（FGF8）和 Wnt1 等细胞外诱导因子的组合激活，这些因子触发特定的细胞内转录级联反应。体外模拟这些胚胎腹侧中脑条件已经成功地用于胚胎干细胞和腹侧中脑 NSCs 的多巴胺能分化。然而，非中脑 NSCs（nmNSCs）的多巴胺能分化被认为是困难的。在这里，我们研究了 Shh、FGF8 和 Wnt1 是否可以在 nmNSCs 中激活典型的多巴胺能转录因子，如 Lmx1a、Msx1 和 Otx2。我们发现，Shh、FGF8 和 Wnt1 诱导 nmNSCs 表达 Lmx1a 和 Otx2，导致高达 39%的 nmNSCs 分化为表达 Pitx3 的神经元。然而，只有少数（约 13%）这些细胞成为更多的多巴胺能神经元，也表达酪氨酸羟化酶（TH）。组蛋白去乙酰化酶（HDAC）抑制剂 Trichostatin A 与 Shh、FGF8 和 Wnt1 联合使用，导致 Lmx1a、Otx2、Msx1 和早期 DA 转录因子 En1 的协调诱导。现在观察到显著增加数量的 TH（约 22%）和 Pitx3（约 33%）神经元。这些细胞中的大多数共同表达 DA 标志物 DAT 和 Vmat2。总之，我们证明了 nmNSCs 确实可以分化为多巴胺能样神经元，但与腹侧中脑 NSCs 和胚胎干细胞相比，这种分化还远远不够；很可能，nmNSCs 缺乏这些细胞进行 DA 分化的适当“启动”表观遗传状态，从而促进 DA 特异性转录因子的诱导。
