Cardiac Induction by Small Molecule BMP Inhibitors

描述（由申请人提供）： 由于心脏再生新组织以替代因损伤而丧失的组织的内在能力可以忽略不计，因此除了器官移植外，目前还没有明确的心力衰竭治疗方法。最近的研究已经介绍了用来自多能干细胞的健康心肌细胞替代受损心脏组织的前景。然而，实现干细胞的全部治疗潜力面临着许多障碍，包括肿瘤形成的潜力，心肌细胞形成率低，以及对心肌发生机制的理解不足。此外，由于缺乏药剂来增强干细胞的治疗效果，翻译工作受到阻碍。Dorsomorphin是第一个已知的骨形态发生蛋白（BMP）信号传导的小分子抑制剂，是小鼠胚胎干细胞（ES细胞）中最有效的心肌发生化学诱导剂之一。在ES细胞分化的最初24至48小时期间，Dorsomorphin处理足以进行稳健的心肌细胞诱导。引人注目的是，大量的心脏诱导明显地在中胚层诱导的情况下发生，并且以其他中胚层衍生的谱系（包括内皮、平滑肌和造血谱系）为代价。根据这些结果，我们推测及时的BMP信号抑制使原始多能祖细胞向心肌细胞发育。本研究利用这种独特而强大的心脏诱导模型来阐明心肌样定向和分化的机制。在目标1中，我们详细研究了小分子BMP抑制剂对中胚层、心血管祖细胞和心肌细胞形成的影响。通过比较dorsomorphin和一种专门的特异性BMP抑制剂DMH1的作用，我们将测试dorsomorphin对Flk1激酶的已知脱靶作用是否会减少整体中胚层的形成，以及纯BMP抑制剂是否会诱导更大的心肌细胞形成。在目标2中，我们将检查来自DMH1处理的ES细胞的Flk 1+中胚层祖细胞是否优先定向成为心肌细胞。此外，我们将确定DMH1处理诱导的推定心脏定向祖细胞的分子特征。这些研究将揭示心脏祖细胞的本质。在目标3中，我们将采取下一个合乎逻辑的步骤来测试在体外强烈诱导心肌细胞形成的小分子是否可以对干细胞疗法产生有益的影响，以改善心肌损伤小鼠模型中的心脏修复。利用小分子阻断成年动物BMP信号传导的独特能力，我们将探索ES细胞移植后体内刺激心肌细胞形成的潜力。本研究利用小分子BMP抑制剂来探索心肌发生的机制，不仅将为未来基于干细胞的治疗心脏病的策略提供信息，还将提供有价值的药物来增强干细胞的治疗效果。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Because the heart has negligible intrinsic capacity to regenerate new tissues to replace those lost to injury, there is currently no definitive heart failure treatment, other than organ transplantation. Recent studies have introduced the prospect of replacing damaged heart tissues with healthy cardiomyocytes derived from pluripotent stem cells. However, realizing the full therapeutic potential of stem cells faces numerous hurdles, including the potential for tumor formation, a low rate of cardiomyocyte formation, and an inadequate mechanistic understanding of cardiomyogenesis. Additionally, translational efforts are hampered by a lack of pharmaceutical agents to boost therapeutic effects of stem cells. Dorsomorphin, the first known small molecule inhibitor of the bone morphogenetic protein (BMP) signaling, is one of the most potent chemical inducers of cardiomyogenesis in mouse embryonic stem (ES) cells. Dorsomorphin treatment during the initial 24 to 48 hours of ES cell differentiation was sufficient for robust cardiomyocyte induction. Strikingly, the massive cardiac induction occurs apparently in the absence of mesoderm induction and at the expense of other mesoderm-derived lineages, including endothelial, smooth muscle and hematopoietic lineages. From these results, we hypothesize that a timely BMP signal inhibition commits the primitive multipotent progenitor cells toward the cardiomyocyte development. The present study takes advantage of this unique and powerful model of cardiac induction to elucidate the mechanism of cardiomyogenic commitment and differentiation. In Aim 1, we examine in detail the effects of small molecule BMP inhibitors on mesoderm, cardiovascular progenitor and cardiomyocyte formation. By comparing the effects of dorsomorphin and an exclusively specific BMP inhibitor DMH1, we will test whether dorsomorphin's known off-target effects against the Flk1 kinase reduces overall mesoderm formation, and whether a pure BMP inhibitor could induce even greater cardiomyocyte formation. In Aim 2, we will examine whether the Flk1+ mesoderm progenitor cells from DMH1-treated ES cells are preferentially committed to become cardiomyocytes. In addition, we will determine the molecular profile of the putative cardiac-committed progenitors induced by the DMH1 treatment. These studies will shed much needed light on the nature of cardiac progenitor cells. In Aim 3, we will take the next logical step to test whether small molecules that robustly induce cardiomyocyte formation in vitro can have a beneficial impact on stem cell therapies to improve cardiac repair in a mouse model of myocardial injury. Utilizing the unique ability of small molecules to block BMP signaling in adult animals, we will explore the potential of in vivo stimulation of cardiomyocyte formation following ES cell transplantation. The present study, which utilizes the small molecule BMP inhibitors to probe the mechanism of cardiomyogenesis, will not only inform future stem cell-based strategies to treat heart disease, but provide valuable pharmaceutical agents to boost the therapeutic effects of stem cells.