Molecular Determinants of Hemogenic Endothelium

摘要 细胞重新编程技术的进步为可扩展的生产创造了替代平台 通过诱导体细胞的多能性用于输血、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，原始的造血波 主导多能干细胞(PSC)分化培养和区分原始和 确切的淋巴髓性造血在很大程度上仍不清楚。因此，进一步将hPSC翻译为 血液学临床需要更好地理解指导确诊淋巴瘤的分子程序 造血术。在发育过程中，淋巴祖细胞和造血干细胞(HSCs)从 血液内皮(HE)衬里动脉，但不是静脉。缺乏静脉对HSCs的贡献 动脉命运获得和HSC发育所需的共同信号通路导致了 假设动脉规范是形成HSC的关键前提。然而，一个直接的祖先- 动脉内皮细胞和明确的淋巴髓性造血之间的后代联系从未被发现 演示了。在目前的应用中，我们建议证明动脉规范是一种 明确造血的基本前提，并证明促进HE的动脉构型 可以提供一种新的策略来帮助从hPSC产生淋巴样细胞用于免疫治疗。在目标1中， 我们将确定动脉类型的HE(AHE)，并展示AHE和AHE之间的直接祖代-后代联系 使用动脉特异性增强子-Cre示踪系统确定淋巴髓样造血。在目标2中，我们将 证明动脉程序激活对于建立明确的淋巴髓性疾病是必不可少的 造血程序。我们将展示增强来自hPSCs的明确的造血程序可以 通过激活动脉生成ETS和SOXF转录因子(TF)的动脉程序来实现， 以及使用小分子调节参与动脉形成的分子通路。相比之下，我们将 结果表明，根据HE规范抑制动脉化可取消明确的造血。vbl.使用 RNASeq和ChipSeq分析我们将确定一个连接动脉和最终的基因调控网络 造血计划。在目标3中，基于在理解动脉形成的作用方面所获得的知识 淋巴生成的因素，我们将开发一个从hPSC产生T细胞的正向编程系统 使用修饰的mRNA，并评估它们在体内用于CAR-T细胞治疗的适宜性。整体而言，建议的 研究将首次建立动脉编程和确定性之间的分子联系 造血功能，并提供证据表明促进hPSC培养中的动脉构型有助于体外 利用hPSCs的淋巴潜能指导明确的造血。此外，我们还将提供 一种新的系统，允许从hPSCs中大规模现成地生产T细胞用于免疫治疗。

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.