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 细胞重编程技术的进步为可扩展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，即使有了先进的造血分化方法，造血原始波 主导多能干细胞 (PSC) 分化培养物和区分原始细胞和 明确的淋巴骨髓造血仍然很大程度上未知。因此，hPSC 的进一步翻译为 血液学临床需要更好地了解指导确定性淋巴细胞的分子程序 造血作用。在发育过程中，淋巴祖细胞和造血干细胞 (HSC) 产生于 造血内皮 (HE) 衬于动脉，但不衬于静脉。缺乏对 HSC 的静脉贡献 动脉命运获取和 HSC 发育所需的共同信号通路导致 假设动脉分化是 HSC 形成的关键先决条件。然而，直系祖先—— 动脉内皮和确定性淋巴骨髓造血之间的后代联系从未被证实 证明了。在本申请中，我们建议证明动脉规格是一个假设 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 生成淋巴细胞用于免疫治疗。在目标 1 中， 我们将鉴定 HE (AHE) 的动脉类型，并证明 AHE 和 HE 之间的直接祖代联系 使用动脉特异性增强子-Cre 示踪系统进行明确的淋巴骨髓造血。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骨髓细胞至关重要 造血计划。我们将证明 hPSC 的明确造血程序的增强可以 通过使用致动脉 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参与动脉生成的分子途径。相比之下，我们将 表明在 HE 规范后抑制动脉化会消除最终的造血作用。使用 RNAseq 和 ChipSeq 分析我们将确定连接动脉和决定性基因的基因调控网络 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 淋巴细胞生成的因素，我们将开发一种正向编程系统，用于从 hPSC 生成 T 细胞 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，建议 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 培养物中的动脉模式有助于体外 利用 hPSC 的淋巴潜能指导确定性造血的方法。此外，我们还将提供 一种新的系统，可以从 hPSC 中大规模生产现成的 T 细胞，用于免疫治疗。