II原型胶原的IIA异构体(Col2a1)对NKx2.5+心脏祖细胞分化调控机理及应用

Despite significant therapeutic advances, heart failure remains the predominant cause of mortality in worldwide. Cardiac Progenitor/stem cell (CPC) biology holds great promise for a new era of cell-based therapy to salvage the failing heart. But there are major challenges. First, while major advances have been made towards achieving the directed differentiation of stem cells to cardiac lineages, the efficiency and preferential directed differentiation to the individual different cardiac lineages is still a challenge. Second is overcoming fibrotic reactions which result in scar formation. The best environmental cues for promoting successful engraftment of cardiomyocytes are unclear. The long-term outcome of current stem cell therapy for cardiac repair has been disappointing, partly because the destruction of the native myocardial extracellular matrix (ECM) exerts a critical negative influence on the myocardial regeneration. Identification of appropriate ECM niche components instructing and regulating CPC differentiation and engraftment is urgently needed. ..Recently we have found that a IIA isoform of procollagen II (named as Col2al), plays as a novel regulator of cardiac morphogenesis and differentiation of cardiac progenitor cells (CPC). We generated COL2a1-null mice and found they display severe defects reminiscent of human congenital heart disease (CHD) associated with defects in the morphogenesis of the outflow tract and ventricles. We discovered a novel COL2a1-dependent positive feedback loop that reinforces BMP/SMAD signaling-dependent activities in cardiac specification and morphogenesis through regulating the expression of cardiac transcription factors, Isl1 and Nkx2.5, These studies implicate COL2a1 in the extracellular niche as a regulator of cardiac morphogenesis and differentiation of CPCs..We hypothesize that ECM procollagen COL2a1 plays a critical role in instructing and regulating Nkx2.5+cardiac progenitor cell (NKx2.5+CPC)-mediated myocardial repair. The central aims is to define and understand the role of COL2a1 in determining successful differentiation and survival of cardiac progenitors ex vivo and during the regenerative process in vivo. The study will focus on the following objectives..1. Using pluripotent stem cells to elucidate COL2a1 in regulating the generation and differentiation of Nkx2.5 expressing CPCs..2. Examining in vivo interdependent interactions of COL2a1 and cardiac progenitor cells mediated.myocardial regeneration following infarction..3. Development of a COL2A1 scaffold to promote NKx2.5+CPC-mediated myocardial regeneration...This study will not only provide new insight into mechanisms of ECM COL2a1 in the regulation of CPC biology, but also will provide a basis for the development of COL2a1 as a novel agent to promote the potential of progenitor/stem cells for myocardial regeneration.

心肌祖细胞(Cardiac Progenitor Cell,CPC）对心脏修复极具潜力，但距临床应用还面临很多挑战。首先，心肌祖细胞产生的效率极低。其次，心梗后原细胞外基质损毁,严重影响了心肌祖细胞存活和心肌修复。我们前期实验结果表明：在心肌祖细胞产生和分化的过程中，细胞外基质II型原胶原的IIA异构体（以下简称Col2a1）起到至关重要的作用。Col2a1不仅与心脏发育相关,并且能够诱导胚胎干细胞向心肌细胞的分化。本研究拟利用携带报告基因的多能干细胞, 基因敲除小鼠及诱导心肌梗塞和再生模型等手段, 进一步明确Col2a1对心脏祖细胞的体内外发育、分化和心肌再生过程的调控作用, 有望为临床干细胞的应用提供新的思路，成为新的治疗策略。

心梗后原细胞外基质损毁, 心脏纤维化严重影响了心肌祖细胞存活和心肌的修复。明确心肌细胞或者心肌祖细胞(CPC)和 细胞外基质之间的相互作用关系对于心肌组织再生具有非常重要的意义。目前干细 胞治疗对心肌的修复，长期疗效依然有限，重要的原因在于我们对支持心肌细胞或者CPC 再生的合适细胞外基质研究中，仍然有很多问题尚未明确。找到细胞外基质巢中有利于 心肌再生的组分是当前研究中急需解决的问题。细胞外基质基Col2a1在心形态发中不仅与心发关,且可够导干 向心异性分化，并且在心梗死后心主导心修复中 到作。本课题组发现细胞外基质（如II型原胶原的IIA异构体Col2a1）不仅与心脏发育相关,并且能够诱导胚胎干细胞向心肌细胞的分化。 在本课题的研究中我们主要有以下一些重要发现:1) Col2a1条件性基因敲除小鼠发现心脏发育NKx2.5+CPC 生产和心肌分化和hnrnpa1 调控有关 。hnrnpa1突变在缺少细胞外基质Col2a1支持下NKx2.5+CPC产生障碍（JCI Insight 2018）。2) 细胞外基质Fibronectin对NKx2.5+CPC体内外存活和分化过程的有重要影响， Fibronectin通过激活PI3K-ATK和Integrin-Stat3信号通道，促进NKx2.5+CPC体内外存活和分化， 缺少Fibronectin，移植的NKX2.5-CPC无法存活。进一步说明细胞外基质对NKx2.5+心脏祖细胞有重要影响。（ Gao Fei。Ph. D Thesis: The Essential Role of Fibronectin for Cardiac Progenitor Cells Mediated Repairs following Myocardial Infarction.） （ Manuscript in Preparation）本课题在阐明细胞外基质对心脏祖细胞的体内外存活和分化过程的调控机理，为临床细胞治疗提供理论依据和实践基础。为将来临床试验提供宝贵的数据。