DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction
DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction
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DOI：
10.1021/cn300029t
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发表时间：
2012-06-01
影响因子：
5
通讯作者：
Bowman, Aaron B.
中科院分区：
文献类型：
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作者：
Neely, M. Diana;Litt, Michael J.;Bowman, Aaron B.
最近在衍生人类诱导多能干细胞（hiPSC）方面的成功使得研究来自神经系统疾病患者的人类神经元成为可能。内源性拮抗剂头蛋白和小分子SB 431542分别同时抑制TGF-β信号传导途径的BMP和TGF-β 1分支，诱导hiPSC的有效神经化，这是一种称为双SMAD抑制的方法。使用小分子抑制剂代替其内源性对应物具有几个优点，包括较低的成本、一致的活性和维持无异种培养条件。我们测试了DMH 1（一种高选择性小分子BMP抑制剂）在hiPSC神经化中替代Noggin的潜力的功效。我们通过测量七天内多能性和神经前体标志物的蛋白质和mRNA水平来比较Noggin和DMH 1诱导的hiPSC神经化。所评估的六种标志物中的五种的调节在已显示有效抑制其他系统中的BMP信号传导的Noggin或DMH 1浓度的存在下是不可区分的。我们观察到，通过改变DMH 1或Noggin的浓度，我们可以选择性地调节表达SOX 1的细胞的数量，而PAX 6，另一种神经前体标志物，保持不变。SOX 1表达的水平和时间已经显示出影响神经诱导以及神经谱系。因此，我们的观察结果表明，BMP抑制剂的浓度需要仔细监测，以确保适当的表达水平的所有必要的转录因子诱导一个特定的神经元谱系。我们进一步证明，DMH 1诱导的神经祖细胞可以分化成β 3-微管蛋白表达神经元，其中一个子集也表达酪氨酸羟化酶。因此，DMH 1（一种高度特异性的BMP通路抑制剂）和SB 431542（一种TGF-β 1通路特异性抑制剂）的联合使用为我们提供了通过专门使用小分子抑制剂独立调节这两种通路的工具。
Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-beta 1 branches of the TGF-beta signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into beta 3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-beta 1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors.