Lmx1a Allows Context-Specific Isolation of Progenitors of GABAergic Or Dopaminergic Neurons During Neural Differentiation of Embryonic Stem Cells
Lmx1a Allows Context-Specific Isolation of Progenitors of GABAergic Or Dopaminergic Neurons During Neural Differentiation of Embryonic Stem Cells
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DOI：
10.1002/stem.1105
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发表时间：
2012-07-01
期刊：
影响因子：
5.2
通讯作者：
Pouton, Colin W.
中科院分区：
文献类型：
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作者：
Nefzger, Christian M.;Su, Colin T.;Pouton, Colin W.
LIM同源框转录因子1α(Lmx1a)是中脑多巴胺能神经元发育、顶板形成和皮质HEM发育所必需的。我们为Lmx1a建立了一个报告胚胎干细胞(ESC)系，并用它来跟踪分化并从分化的小鼠ESCs中提取神经前体细胞。Lmx1a(+)细胞可分化为功能性皮质上层GABA能神经元或多巴胺能神经元。在化学定义的神经基础条件下，ESC的分化导致Lmx1a的广泛和瞬时表达，而不添加外源性因素，如sonic hedgehog(Shh)、Wnts和/或骨形态发生蛋白(BMPs)。在中性条件下，Lmx1a(+)细胞表达已知位于Lmx1(+)和皮质HEM标记Wnt3a和p73下游的基因。其中大多数细胞不表达腹侧中脑多巴胺能标记Foxa2或背侧顶板标记BMP-2。Lmx1a(+)-Foxa2(-)细胞被诱导成为SatB2(+)GABA能神经元，并且似乎对多巴胺能模式提示具有抵抗力。PA6共培养产生大量表达Foxa2的Lmx1a(+)前体细胞，进一步分化可产生高频多巴胺能神经元。我们认为Lmx1a是提取GABA能或多巴胺能神经元祖细胞的有用标志物。我们告诫不要认为它预示着胚胎干细胞体外分化过程中的多巴胺能参与。事实上，在神经基础条件下的单层培养中，无论是否添加Shh和成纤维细胞生长因子8(FGF8)，Lmx1a(+)细胞都是前脑GABA能神经元的主要前体细胞。通过与PA6细胞共培养，我们获得了大量的多巴胺能细胞。干细胞2012；30：1349-1361
LIM homeobox transcription factor 1 alpha (Lmx1a) is required for the development of midbrain dopaminergic neurons, roof plate formation, and cortical hem development. We generated a reporter embryonic stem cell (ESC) line for Lmx1a and used it to track differentiation and extract neural progenitors from differentiating mouse ESCs. Lmx1a(+) cells gave rise to functional cortical upper layer GABAergic neurons or dopaminergic neurons depending on the culture conditions used for differentiation. Under chemically defined neurobasal conditions, ESC differentiation resulted in widespread and transient expression of Lmx1a, without the addition of exogenous factors such as sonic hedgehog (Shh), Wnts, and/or bone morphogenic proteins (BMPs). Under neutral conditions, Lmx1a(+) cells express genes known to be downstream of Lmx1(+) and cortical hem markers Wnt3a and p73. The majority of these cells did not express the ventral midbrain dopaminergic marker Foxa2 or dorsal roof plate marker BMP-2. Lmx1a(+)-Foxa2(-) cells were primed to become SatB2(+) GABAergic neurons and appeared to be resistant to dopaminergic patterning cues. PA6 coculture produced a substantial population of Lmx1a(+) progenitors that also expressed Foxa2 and on further differentiation gave rise to dopaminergic neurons at high frequency. We conclude that Lmx1a is a useful marker for the extraction of progenitors of GABAergic or dopaminergic neurons. We caution against the assumption that it indicates dopaminergic commitment during in vitro differentiation of ESCs. Indeed, in monolayer culture under neurobasal conditions, with or without the addition of Shh and fibroblast growth factor 8 (FGF8), Lmx1a(+) cells were predominantly progenitors of forebrain GABAergic neurons. We obtained dopaminergic cells in large numbers only by coculture with PA6 cells. STEM CELLS 2012;30:1349-1361