Regulation of Cardiac Progenitor Maintenance

心脏祖细胞维持的调节

• 批准号: 9750751
• 负责人: Chulan Kwon
• 金额: $ 33.02万
• 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: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750751
• 关键词: Affect; Branchial arch structure; Cardiac; Cardiac Myocytes; Cell Maintenance; Cells; Complex; Congenital Abnormality; Congenital Heart Defects; Development; Diagnostic; Disease model; Embryo; Environment; Environmental Risk Factor; Erinaceidae; Etiology; Fetal Development; Genes; Goals; Growth; Heart; Heart Abnormalities; In Vitro; Instruction; Integrin beta Chains; Intervention; Intrinsic factor; Knowledge; Lead; Maintenance; Mediating; Mosaicism; Numbness; Oranges; Phenotype; Play; Pluripotent Stem Cells; Population; Preventive; Preventive Medicine; Proliferating; Protein Family; Proteins; Regulation; Research; Role; Second branchial arch structure; Signal Pathway; Source; Specific qualifier value; Stem Cell Factor; Stem cells; Testing; Therapeutic; Undifferentiated; Work; base; cardiac regeneration; cardiac repair; cardiogenesis; congenital heart disorder; embryo cell; in vivo; infant death; insight; migration; mutant; prevent; progenitor; self-renewal; smoothened signaling pathway; stem cell niche

Cardiac progenitor cells (CPCs)—identified in early embryos—are cell sources to make the heart during fetal development, and abnormal CPC development is closely associated with the etiology of congenital heart defects. In particular, proper regulation of their number and fate is essential for the ensuing heart growth. However, it remained unknown if they undergo self-renewal and if there is a dedicated environment for their maintenance. We have identified a renewing population of CPCs and their niche during development, and this proposal aims to elucidate the mechanisms governing the self-renewal and expansion of CPCs in vivo. Numb family proteins (NFPs) are conserved endocytic proteins with critical roles in cell-fate decisions. We found that precardiac deletion of NFPs depletes CPCs in the second pharyngeal arch (PA2) and is associated with a hypoplastic heart and early embryonic lethality. Based on this phenotype, we hypothesized that CPCs expand in the PA2 before their cardiac differentiation. Confirming the hypothesis, CPCs normally remained undifferentiated and expansive in the PA2, and differentiated into cardiac cells soon as they migrated out of the PA2. CPCs co-cultured with PA2 cells formed distinct colonies that continued to grow without cardiac differentiation, and differentiated into cardiac cells when PA2 cells were removed. These suggested that CPCs proliferate without differentiation in the PA2, suggesting the presence of a stem cell–niche paradigm. The proliferation was promoted by Hedgehog (Hh) proteins, crucial developmental and stem cell regulators enriched in the PA2, implying the Hh signaling may mediate the environmental role of PA2 cells. To determine the CPC-autonomous role of NFPs in the PA2, we generated lineage-specific mosaicism that allowed tracing of CPCs lacking NFPs without causing the lethality. The NFP-deleted mutant CPCs normally populated in the PA2, but failed to expand in the PA2 and progressed to cardiac cells. The mutant CPCs showed dramatically decreased levels of the conserved stem cell regulator Itgb1. Itgb1 physically associated with NFP in CPCs, and precardiac deletion of Itgb1 resulted in CPC depletion in the PA2, similar to the phenotype of NFP-deleted embryos. These findings set the stage for a mechanistic exploration of CPC maintenance by intrinsic and extrinsic factors. The specific aims of this proposal are (1) to determine if NFPs play an instructive role for CPC renewal in the PA2, (2) to determine if NFPs prevent endolysosomal degradation of Itgb1 for CPC maintenance, and (3) To investigate if PA2 cells affect CPC renewal and expansion via Hh signaling. With these aims, I expect to elucidate the factors and mechanisms by which CPCs are maintained in a renewing state in their microenvironment. This knowledge will provide first insights into the mechanistic understanding of the self-renewal of CPCs in their microenvironment during heart development, which will open up new avenues of research in congenital heart disease and may allow us to maintain and expand CPCs in a homogeneous and undifferentiated state in vitro for heart regeneration research.

心脏祖细胞（CPC）-在早期胚胎中鉴定-是胎儿时期制造心脏的细胞来源 CPC发育异常与先天性心脏病的病因密切相关 缺陷特别是，适当调节它们的数量和命运对随后的心脏生长至关重要。 然而，仍然不清楚它们是否进行自我更新，以及是否有一个专门的环境供它们使用。 上维护我们已经确定了一个更新的人口的CPC和他们的利基在发展过程中，这 该提案旨在阐明CPC在体内自我更新和扩增的机制。麻木 家族蛋白（NFP）是在细胞命运决定中具有关键作用的保守的内吞蛋白。我们发现 心前区NFP的缺失会使第二咽弓（PA 2）中的CPC减少，并与 心脏发育不全和早期胚胎致死。基于这种表型，我们假设CPC扩增 在心脏分化之前的PA 2中。证实了这一假设，CPC通常保持在 在PA 2中未分化和扩张，并在迁移出PA 2后立即分化为心肌细胞。 PA 2.与PA 2细胞共培养的CPC形成了独特的集落，在没有心脏刺激的情况下继续生长。 分化，并且当去除PA 2细胞时分化为心肌细胞。这表明，CPC 在PA 2中增殖而不分化，表明存在干细胞生态位范例。的 Hedgehog（Hh）蛋白促进增殖，Hh蛋白是关键的发育和干细胞调节剂 提示Hh信号可能介导了PA 2细胞的环境作用。以确定 NFP在PA 2中的CPC自主作用，我们产生了允许追踪的谱系特异性镶嵌 缺乏NFP的CPC不会导致致命性。NFP缺失的突变型CPC通常在细胞中聚集， PA 2中扩增，但未能在PA 2中扩增并进展到心肌细胞。突变的CPC显著地表现出 降低了保守的干细胞调节因子Itgb 1的水平。Itgb 1与CPC中的NFP物理相关，以及 Itgb 1的心前缺失导致PA 2中CPC耗尽，与NFP缺失的表型相似。 胚胎这些发现为CPC维护的内在机制探索奠定了基础， 外在因素这项建议的具体目的是（1）确定国家联络点是否对产品总分类发挥指导作用 PA中的更新2，（2）以确定NFP是否阻止CPC的Itgb 1的内溶酶体降解 （3）研究PA 2细胞是否通过Hh信号通路影响CPC的更新和扩增。与 这些目标，我希望阐明的因素和机制，CPC是维持在一个更新的 在他们的微环境中。这一知识将提供第一次深入了解机械的理解， 心脏发育过程中CPC在其微环境中的自我更新，这将开辟新的途径 先天性心脏病的研究，并可能使我们能够维持和扩大CPC在一个同质的 和未分化状态进行体外心脏再生研究。