Generation of engraftable HSPCs from hemogenic endothelial cells

从造血内皮细胞生成可移植的 HSPC

• 批准号: 9104360
• 负责人: ZACK Z. WANG
• 金额: $ 29.16万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9104360
• 关键词: 5' -Nucleotidase; Address; Adenosine; Animal Model; Animals; Aorta; Autologous; Blood Vessels; Bone Marrow Transplantation; Cell Culture System; Cell Maturation; Cell Therapy; Cell physiology; Cell surface; Cells; Chemicals; Coculture Techniques; Cues; Derivation procedure; Development; Embryo; Embryonic Development; Endothelial Cells; Engraftment; Enhancers; Environment; Future; Generations; Genes; Gonadal structure; Growth; Hematological Disease; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Immunophenotyping; In Vitro; Instruction; Investigation; Lead; Mesonephric structure; Metabolism; Modeling; Molecular; Monitor; Mus; Pathway interactions; Patients; Play; Pluripotent Stem Cells; Procedures; Process; Proto-Oncogene Proteins c-akt; Purinergic P1 Receptors; Receptor Signaling; Regulation; Reporter; Research; Role; Signal Pathway; Signal Transduction; Source; Specific qualifier value; Staging; Stem cells; System; Testing; Therapeutic; Time; Transplantation; Vascular Endothelium; Viral Genes; Wiskott-Aldrich Syndrome; Zebrafish; clinical application; embryonic stem cell; extracellular; gene therapy; genetic manipulation; human embryonic stem cell; in vivo; induced pluripotent stem cell; insight; interest; novel; novel strategies; pre-clinical; preclinical study; progenitor; programs; promoter; public health relevance; response; stem; stem cell differentiation; stem cell therapy; tool

 DESCRIPTION (provided by applicant): This is a single central aim project to investigate the role of autologous endothelial cells (ECs) in regulating the generation of definitive hematopoietic stem/progenitor cells (HSPCs) from human pluripotent stem cells (hPSCs). The transplantation of hematopoietic stem cells (HSCs) allows for the cure of patients with hematological disorders. Because of limited availability of HSCs from donors, derivation of HSCs from hPSCs provides an alternative source of HSCs for bone marrow transplantation. However, generation of hPSCs-derived HSCs that are capable of long-term engraftment and differentiation to multilineage hematopoietic cells remain significant challenges, which limits progress of the clinical applications of stem cell therapy and gene therapy using patient-specific induced pluripotent stem cells (hiPSCs). The difficulty to efficiently generate HSCs from hPSCs is possibly due to a restricted temporal window and demanding niche cues for HSC emergence and development from hPSCs. During embryonic development, hemogenic ECs, a specified subset of ECs in vascular endothelium of aorta-gonad-mesonephros (AGM), give rise to multipotent HSCs. We hypothesized that during hPSC differentiation, autologous ECs provide vascular niche to promote the generation of definitive HSCs from hemogenic ECs in multiple stages, including hemogenic EC-to-hematopoietic transition, pre-HSC maturation to HSCs, and HSC expansion. We have developed a novel reporter system to monitor definitive HSC emergence from hemogenic ECs by utilizing Runx1 enhancer and minimal promotor of Wiskott-Aldrich Syndrome (WAS) gene. Using this reporter system and cell immunophenotypes, we will test our hypothesis, and investigate the role of extracellular ATP/adenosine from vascular niche in regulating the generation of definitive hematopoietic cells from hemogenic ECs. CD73, an ecto-5'-nucleotidase that convert AMP to adenosine at cell surface, plays a key role in extracellular ATP metabolism and the generation of adenosine. Adenosine-receptor signaling promotes definitive HSPC generation in AGM. However, CD73 is absent in hemogenic ECs. We will use CD73-deficient hiPSCs and pharmacological regents to investigate how extracellular ATP/adenosine regulates definitive HSPC generation. To evaluate their functionalities of repopulation and differentiation in vivo, we will characterize hematopoietic stem and progenitor cells derived from hemogenic ECs by a human-to-mouse xenogeneic transplantation model, which represents powerful tools and essential preclinical platform for assessing human HSC function. Our investigation will provide new insight into the human HSPC generation regulation mechanism, accelerate the establishment of a novel strategy to generate hematopoietic stem and progenitor cells, and lead to a preclinical study for cell-based therapy for hematological disorders.

 描述（由申请人提供）：这是一个单一的中心目标项目，旨在研究自体内皮细胞（EC）在调节最终造血细胞生成中的作用 来自人类多能干细胞 (hPSC) 的干细胞/祖细胞 (HSPC) 造血干细胞 (HSC) 的移植可以治愈血液疾病患者，因为来自捐赠者的 HSC 来源有限。然而，hPSC 衍生的 HSC 的产生能够长期植入和分化。多系造血细胞仍然是重大挑战，这限制了使用患者特异性诱导多能干细胞（hiPSC）进行干细胞治疗和基因治疗的临床应用的进展，从 hPSC 有效生成 HSC 的困难可能是由于时间窗口和限制。 hPSC 的 HSC 出现和发育需要生态位线索，在胚胎发育过程中，造血 EC 是血管内皮细胞中特定的 EC 子集。主动脉-性腺-中肾 (AGM)，产生多能 HSC，在 hPSC 分化过程中，自体 EC 提供血管生态位，促进多个阶段的造血 EC 生成最终的 HSC，包括造血 EC 到造血的转变。 HSC 前成熟为 HSC 以及 HSC 扩增 我们开发了一种新型报告系统，可通过以下方式监测从造血 EC 中确定的 HSC 出现。 Wiskott-Aldrich 综合征 (WAS) 基因的 Runx1 增强子和最小启动子使用该报告系统和细胞免疫表型，我们将检验我们的假设，并研究来自血管生态位的细胞外 ATP/腺苷在调节终形造血细胞生成中的作用。 CD73 是一种胞外 5'-核苷酸酶，可在细胞表面将 AMP 转化为腺苷，在造血 EC 中发挥关键作用。细胞外 ATP 代谢和腺苷受体信号传导促进 AGM 中最终 HSPC 的生成。然而，造血 EC 中不存在 CD73。我们将使用 CD73 缺陷的 hiPSC 和药理学试剂来研究细胞外 ATP/腺苷如何调节最终 HSPC 的生成。为了评估它们在体内的再增殖和分化功能，我们将表征源自 的造血干细胞和祖细胞。通过人-小鼠异种移植模型制备造血 EC，这是评估人类 HSC 功能的强大工具和重要的临床前平台，我们的研究将为人类 HSPC 生成调控机制提供新的见解，加速建立新的生成策略。造血干细胞和祖细胞，并引发了针对血液疾病的细胞疗法的临床前研究。