Molecular Determinants of Hemogenic Endothelium

造血内皮的分子决定因素

• 批准号: 9975885
• 负责人: Igor I. Slukvin
• 金额: $ 49.51万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9975885
• 关键词: Adult; Aorta; Applications Grants; Arteries; Blood; Blood Cells; Blood Vessels; CAR T cell therapy; CRISPR/Cas technology; Cells; Clinic; Development; Drug Delivery Systems; Embryo; Endothelium; Engraftment; Enhancers; Ensure; Erythro; Erythrocytes; Erythropoiesis; Event; Generations; Gonadal structure; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Immunotherapy; In Vitro; Knowledge; Link; Lymphoid; Lymphoid Cell; Lymphopoiesis; Megakaryocytes; Mesonephric structure; Messenger RNA; Methods; Molecular; Myelopoiesis; Pathway interactions; Patients; Pattern; Pluripotent Stem Cells; Polyploidy; Production; Regulator Genes; Role; Signal Pathway; Site; Somatic Cell; Stem Cell Development; System; T-Lymphocyte; Technology; Time; Transfusion; Translations; Transplantation; Umbilical cord structure; Venous; Yolk Sac; base; cadherin 5; chimeric antigen receptor T cells; endonuclease; gene therapy; genome editing; hematopoietic differentiation; hematopoietic stem cell formation; hemogenic endothelium; human pluripotent stem cell; improved; in vivo; induced pluripotent stem cell; innovation; novel; novel strategies; pluripotency; progenitor; programs; small molecule; stem cell differentiation; transcription factor; transcriptome sequencing

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.

抽象的 蜂窝重编程技术的进展已创建了替代平台，用于可扩展的生产 血细胞通过诱导体细胞多能性，用于输血，免疫疗法和移植。 但是，即使有进步的造血分化方法，造血的原始波 主导多能干细胞（PSC）分化培养物和分化原始和标记 确定的淋巴细胞造血基本上仍然未知。因此，将HPSC进一步翻译为 血液学诊所需要更好地了解分子程序指导确定的淋巴细胞 造血。在发育期间，淋巴样祖细胞和造血干细胞（HSC）来自 血液内皮（HE）衬里动脉，而不是静脉。缺乏对HSC的静脉贡献 动脉命运采集和HSC发展所需的常见信号通路，导致 假设动脉规范是HSC形成的关键先决条件。但是，直接祖先 - 动脉内皮与确定淋巴细胞造血之间的后代联系从未有过 证明。在当前的应用中，我们建议证明动脉规范是一个假设 确定造血的基本先决条件，并证明促进他的动脉模式 可以提供一种新的策略，以帮助从HPSC中产生免疫疗法的淋巴样细胞。在AIM 1中， 我们将确定HE（AHE）的动脉类型，并展示AHE和 使用动脉特异性增强子-CRE痕迹系统，确定的淋巴细胞造血。在AIM 2中，我们将 证明动脉程序激活对于建立明确的淋巴细胞至关重要 造血计划。我们将表明，HPSC的确定造血计划的增强可以 通过激活动脉生成ET和SOXF转录因子（TF），可实现 并使用小分子对参与动脉生成的分子途径进行调节。相比之下，我们将 表明他规范后抑制动脉化可以消除明确的造血。使用 RNASEQ和CHIPSEQ分析我们将确定连接动脉和确定性的基因调节网络 造血计划。在AIM 3中，基于了解动脉生成的作用而获得的知识 淋巴管的因素，我们将开发一个从HPSC的T细胞生成的正向编程系统 使用改良的mRNA并评估其对体内CAR-T细胞疗法的适用性。总体而言，提议 研究将首次建立动脉编程和确定性之间的分子联系 造血，并提供证据表明在HPSC培养物中促进动脉模式可以帮助体外 指导HPSC的淋巴电势的定向造血的方法。此外，我们将提供 一种新型系统，允许从HPSC从HPSC进行可扩展的T细胞进行免疫疗法。