Customized stem cells for clinical application in blood disorders

描述(申请人提供)：这项建议融合了发育生物学和化学生物学、计算分析和生物工程，以解决血液学研究中最重要的挑战之一--从多能干细胞体外获得长期可植入的造血干细胞。到目前为止，从胚胎干细胞(ESCs)和诱导多能干细胞(IPSCs)定向分化为造血干细胞(HSCs)的相当多的努力被证明只在小鼠身上部分成功，在人类身上大部分不成功。未能产生真正的ESC/IPSC来源的HSC仍然是利用患者来源的IPSC来模拟血液疾病的障碍，并排除了利用这一潜在的变革性治疗技术的可能性。这次续签申请将建立在前两年赠款期间取得的进展的基础上，以解决核心问题：在胚胎中驱动造血干细胞形成的发育途径是什么，以及我们如何利用这一知识来指导将多能干细胞分化为临床相关的造血祖细胞？我们将把计算算法应用于由鱼类、小鼠和人类的造血干细胞和祖细胞群体的单细胞RNA-SEQ生成的增强表达数据集。我们将探索指导造血和淋巴发育的基因调控网络，并测试候选转录因子、非编码RNA和形态原，假设它们通过得失功能策略单独和联合管理这些网络。在第二个推力中，我们将在斑马鱼胚球和人类多能干细胞中进行筛选，以发现HSC的新的化学和生物调节因子，并将化学物质整合到细胞分化方案中，以推动HSC的承诺和增强HSC的功能。最后，利用生物工程平台模拟作用于胚胎血液内皮细胞的生物力学力量，我们将配置“芯片上的血液内皮细胞”来询问促进造血干细胞出现和造血干/祖细胞扩增的效应器和下游信号通路。我们在定义造血发育中的遗传、化学和生物力学机制方面的努力代表着互补和协同的策略，以解决我们在体外获得造血干细胞的关键挑战。

DESCRIPTION (provided by applicant): This proposal merges developmental and chemical biology, computational analysis, and bioengineering to tackle one of the most significant challenges in hematology research-the in vitro derivation of long-term engraftable hematopoietic stem cells from pluripotent stem cells. To date, considerable efforts at directed differentiation o hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have proven only partially successful in mice and largely unsuccessful in humans. The failure to generate true ESC/iPSC-derived HSCs remains a barrier for exploiting patient-derived iPSCs for modeling blood diseases, and precludes harnessing this potentially transformative technology for therapy. This renewal application will build upon progress made during the first two years of the prior grant period to address the central question: What are the developmental pathways that drive the formation of HSCs in embryos, and how can we exploit this knowledge to direct the differentiation of pluripotent stem cells into clinically relevant hematopoietic lineages? We will apply computational algorithms to enhanced expression datasets generated by single cell RNA-seq of hematopoietic stem and progenitor populations from fish, mouse, and human. We will probe gene regulatory networks that direct hematopoietic and lymphoid development, and test candidate transcription factors, non-coding RNAs, and morphogens hypothesized to govern these networks, alone and in combination, through gain and loss-of-function strategies. In a second thrust, we will perform screens in zebrafish blastomeres and human pluripotent stem cells to discover novel chemical and biological regulators of HSCs, and will incorporate chemicals into cell differentiation protocols to drive HSC commitment and enhance HSC function. Lastly, using bioengineered platforms which mimic the biomechanical forces that act on the embryonic hemogenic endothelium, we will configure "hemogenic endothelium-on-a-chip" to interrogate the effectors and downstream signaling pathways that promote emergence of HSCs and expansion of hematopoietic stem and progenitor cells. Our efforts at defining genetic, chemical and biomechanical mechanisms in hematopoietic development represent complementary as well as synergistic strategies to solve our key challenge of deriving HSCs in vitro.