Neural Specification of Embryonic Stem Cells

描述（由申请人提供）：人类胚胎干细胞（hESCs）可以定向到多种细胞谱系，包括神经谱系。因此，他们提供了一个独特的模型来揭示发生在实验上难以接近的早期人类胚胎中早期神经规范的分子相互作用。在我们之前的R01项目中，我们意外地发现转录因子Pax6在hESCs分化的早期或原始神经上皮（NE）细胞中均匀且特异性地表达。这让人想起脊椎动物中最早的NE转录因子Sox1。我们现在发现，强迫表达Pax6导致人而非小鼠ESCs的神经上皮分化，而抑制Pax6表达抑制人而非小鼠ESCs的神经上皮分化。因此，我们假设Pax6是hESCs NE命运的关键介质，并且可以通过调节Pax6的表达来实现对人类NE规范的控制。我们将构建有条件表达Pax6或Sox1的hESC细胞系，并通过RNA干扰（RNAi）抑制Pax6或Sox1的表达。使用我们定义的系统将这些转基因hESCs分化为NE将解决Pax6对于hESCs的NE规格是否必要和/或充分的问题。这将揭示保守蛋白Pax6在人类NE规范中的新作用。然后，我们将确定调节NE规范的细胞外因子，并通过使用染色质免疫沉淀和DNA结合试验直接调节Pax6表达来确定这些因子是否起作用。这将首次将细胞外因子与神经外胚层转录因子直接联系起来。最后，我们将评估是否hESC的NE规范，特别是NE的原始状态的维持以及重新模式的可塑性可以通过使用我们的转基因Pax6 hESC系调节Pax6表达来控制。如果是这样，我们将发现一种维持体细胞（神经）干细胞的方法。总之，从这一建议中获得的信息将为我们提供一种控制人类干细胞命运选择的手段。它也将有助于扩大我们大脑中存在的干细胞的修复潜力。

DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs) can be directed to multiple cell lineages including the neural lineage. They thus offer a unique model to reveal molecular interactions underlying early neural specification that occurs in an experimentally inaccessible early human embryo. In our previous R01 project, we discovered, unexpectedly, that the transcription factor Pax6 is expressed uniformly and specifically in early or primitive neuroepithelial (NE) cells that are differentiated from hESCs. This is reminiscent of that of Sox1, the earliest and definitive NE transcription factor in vertebrate animals. We now show that forced expression of Pax6 results in neuroepithelial differentiation in human but not mouse ESCs whereas inhibition of Pax6 expression suppresses neuroepithelial differentiation from human but not mouse ESCs. We therefore hypothesize that Pax6 acts as a key mediator of the NE fate of hESCs and that control of human NE specification may be achieved through regulation of Pax6 expression. We will build hESC lines that conditionally express Pax6 or Sox1 and inhibit Pax6 or Sox1 expression via RNA interference (RNAi). Differentiation of these transgenic hESCs to NE using our defined system will address whether Pax6 is necessary and/or sufficient for NE specification from hESCs. This will reveal a novel role of the conserved protein Pax6 in human NE specification. We will then identify extracellular factors that regulate NE specification and determine if these factors do so by directly regulating Pax6 expression using chromatin immunoprecipitation and DNA binding assays. This will directly link extracellular factors to neuroectoderm transcription factors for the first time. Finally, we will assess whether NE specification from hESCs, especially the maintenance of the primitive state of NE and hence the plasticity to be re-patterned may be controlled by regulating Pax6 expression using our transgenic Pax6 hESC lines. If so, we will uncover a way to maintain somatic (neural) stem cells. Together, information gained from this proposal will provide us with a means of controlling the fate choice of human stem cells. It will also be instrumental to expanding the repair potential of the stem cells that are present in our brain. PUBLIC HEALTH RELEVANCE: The proposed study will reveal how the transition from human embryonic stem cells to neural stem cells is regulated at the genetic and epigenetic levels. Understanding the molecular regulators in directing the fate of neural stem cells will significantly expand our ability to instigate regeneration of our aging and/or diseased brain.