Cell extrinsic factors' roles on direct conversion to human induced neural crest

 描述（由申请人提供）：周围神经系统（PNS）的普遍遗传缺陷和获得性损伤对我们的医疗保健系统造成了重大的社会经济问题。然而，由于难以获得患者样本，对人PNS组织的详细研究相对较少。 我们已经证明，外周神经元和许旺细胞可以来源于人胚胎干细胞（hESC）和人诱导多能干细胞（hiPSC）;然而，这个过程是艰苦的长（至少超过5个月）。最近，我们开发了一种替代方法，一种新的方法，可以在两周内直接将人成纤维细胞转化为诱导神经嵴（iNC）。我们的iNC群体表现出神经嵴特异性的细胞和分子特征。 虽然iNC群体在单细胞水平上显示出多能性，但它们更倾向于感觉神经元的命运，而不是自主神经元谱系。在发育过程中，细胞命运由细胞外在因素如生长因子和形态发生素决定。如果这些细胞外在因素也在iNC生成过程中支配细胞命运，我们可能能够改变iNC的分化潜力。这一假说引发了一个问题，是否调制细胞外在因素（如腹和/或尾线索）可以影响细胞的命运在直接转换，以同样的方式，这种机制在发育细胞的决定。 目前，直接转化，例如我们的iNC，依赖于致癌病毒转导传递的转录因子。我们的第二个问题是“非遗传”小分子是否以及如何能够实现人成纤维细胞向诱导神经嵴的充分直接转化？我们目前的iNC诱导系统适合于研究这些关于细胞命运可塑性的基本问题，因为它仅采用单一转录因子，沿着高度定量检测方法（通过FACS检测SOX 10：：GFP）。 我们提出的实验预计：（i）扩大和加强我们目前的一般“遗传因子依赖性”直接转换的概念，和（ii）加速广泛的PNS疾病的研究，例如通过提供疾病特异性雪旺细胞或交感神经元直接从成纤维细胞的Charcot-Marie-Tooth 1A（CMT 1A）或家族性自主神经功能障碍患者。

 DESCRIPTION (provided by applicant): The prevalent genetic defects and acquired injuries of the peripheral nervous system (PNS) cause a significant socioeconomic issue on our healthcare system. However, there are relatively few detailed studies of human PNS tissues, due to the difficulty to obtaining patient samples. We have shown that peripheral neurons and Schwann cells can be derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs); however, the process is arduously long (at least over 5 months). Recently we developed an alternative, a new method that directly converts human fibroblasts into induced neural crest (iNC) in only two weeks. Our iNC population exhibits neural crest- specific cellular and molecular characteristics. Although the iNC population shows multipotency in a single cell level, they are more prone to a sensory neuron fate, rather than autonomic neuron lineages. During development, cell fates are determined by cell extrinsic factors, such as growth factors and morphogens. If such cell extrinsic factors also govern cell fates during generation of iNC, we may be able to change the differentiation potential of the iNC. This hypothesis incites a question, whether modulation of cell extrinsic factors (such as ventralization and/or caudalization cues) can influence the cellular fate during direct conversion, in the same way that this mechanism operates in developmental cell determination. Currently direct conversion, exemplified by our iNC, is dependent on transcription factors delivered by oncogenic viral transduction. Our second question is whether and how `non-genetic' small molecules can achieve sufficient direct conversion of human fibroblasts to induced neural crest? Our current iNC induction system is suitable for investigating these fundamental questions about cell fate plasticity, because it employs only a single transcription factor, along with highly quantitative detection method (SOX10::GFP detection by FACS). Our proposed experiments are expected to: (i) expand and strengthen our current conception of general `genetic factor-dependent' direct conversion, and (ii) accelerate a wide range of research on PNS disorders, e.g. by providing disease-specific Schwann cells or sympathetic neurons directly from fibroblasts of Charcot-Marie- Tooth1A (CMT1A) or familial dysautonomia patients.