Molecular Determinants of Hemogenic Endothelium

摘要 细胞重编程技术的进展已经为可规模生产细胞重编程蛋白质创造了替代平台。 通过在体细胞中诱导多能性用于输血、免疫治疗和移植的血细胞。 然而，即使有先进的造血分化方法， 主导多能干细胞（PSC）分化培养物和区分原始和 确定性淋巴骨髓样造血仍是未知的。因此，将hPSC进一步翻译为 血液学临床需要更好地理解指导确定性淋巴骨髓样瘤的分子程序 造血在发育过程中，淋巴祖细胞和造血干细胞（HSC）从 血管内皮细胞（HE），而不是血管。缺乏静脉对HSC的贡献沿着 动脉命运获取和HSC发育所需的共同信号传导途径，导致 假设动脉特化是HSC形成关键先决条件。但是，一个直接的祖先- 动脉内皮和永久性淋巴骨髓造血之间的后代联系从未被 演示。在本申请中，我们提出证明动脉特化是一个假设， 确定造血的必要先决条件，并证明促进HE的动脉模式 可以提供一种新的策略来帮助从hPSC产生用于免疫治疗的淋巴样细胞。在目标1中， 我们将确定动脉型HE（AHE），并证明AHE和 使用动脉特异性增强子-Cre追踪系统的确定性淋巴骨髓样造血。在目标2中，我们将 表明动脉程序激活对于建立明确的淋巴骨髓样病变是必要的， 造血计划我们将证明，增强来自hPSC的确定性造血程序可以 通过用动脉源性ETS和SOXF转录因子（TF）激活动脉程序来实现， 以及使用小分子调节动脉生成中涉及的分子途径。相反，我们将 表明抑制HE特化后的动脉化会消除明确的造血作用。使用 通过RNAseq和ChipSeq分析，我们将确定一个连接动脉和决定性的基因调控网络， 造血计划在目标3中，基于在理解动脉生成的作用中获得的知识， 在淋巴细胞生成中的因子，我们将开发用于从hPSC产生T细胞的正向编程系统 使用修饰的mRNA，并评估它们对体内CAR-T细胞疗法的适用性。总体而言，拟议 研究将首次建立动脉编程和确定性之间的分子联系。 造血，并提供证据表明，促进hPSC培养物中的动脉图案化可以有助于体外造血。 指导具有来自hPSC的淋巴潜能的确定性造血的方法。此外，我们将提供 一种新的系统，其允许从hPSC可规模化地现成生产用于免疫治疗的T细胞。

ABSTRACT Progress in cellular reprogramming technologies has created alternative platforms for scalable production of blood cells for transfusion, immunotherapies and transplantation through inducing pluripotency in somatic cells. However, even with advances hematopoietic differentiation methods, primitive wave of hematopoiesis dominates pluripotent stem cell (PSC) differentiation cultures and markers that distinguish primitive and definitive lymphomyeloid hematopoiesis remains largely unknown. Thus, further translation of hPSCs to hematology clinic requires a better understanding of the molecular program guiding definitive lymphomyeloid hematopoiesis. During development, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. The lack of venous contribution to HSCs along with the common signaling pathways required for both arterial fate acquisition and HSC development, led to the hypothesis that arterial specification is a critical prerequisite for HSC formation. However, a direct progenitor- progeny link between arterial endothelium and definitive lymphomyeloid hematopoiesis has never been demonstrated. In present application, we propose to prove the hypothesis that arterial specification is an essential prerequisite for definitive hematopoiesis and demonstrate that promotion of arterial patterning of HE can provide a novel strategy to aid in generating of lymphoid cells from hPSCs for immunotherapies. In aim 1, we will identify arterial type of HE (AHE) and demonstrate a direct progenitor-progeny link between AHE and definitive lymphomyeloid hematopoiesis using arterial-specific enhancer-Cre tracing system. In aim 2, we will demonstrate that arterial program activation is essential for establishing definitive lymphomyeloid hematopoietic program. We will show that enhancement of definitive hematopoietic program from hPSCs can be achieved through activation of arterial program with arteriogenic ETS and SOXF transcription factors (TF), and modulation of the molecular pathways involved in arteriogenesis using small molecules. In contrast, we will show that inhibiting arterialization following HE specification abrogates definitive hematopoiesis. Using RNAseq and ChipSeq analysis we will identify a gene regulatory network connecting arterial and definitive hematopoietic programs. In aim 3, based on the knowledge gained in understanding the role of arteriogenic factors in lymphopoiesis, we will develop a forward programming system for T cell generation from hPSCs using modified mRNA and assess their suitability for CAR-T cell therapies in vivo. Overall, the proposed studies will establish for the first time a molecular link between arterial programming and definitive hematopoiesis, and provide evidence that promoting arterial patterning in hPSC cultures can aid to in vitro approaches to instruct definitive hematopoiesis with lymphoid potentials from hPSCs. In addition, we will offer a novel system allowing for scalable off-the-shelf production of T cells from hPSCs for immunotherapies.