Cardiac Induction by Small Molecule BMP Inhibitors

描述(由申请人提供)： 项目摘要由于心脏再生新组织以取代因损伤而失去的组织的内在能力可以忽略不计，目前除了器官移植外，还没有明确的心力衰竭治疗方法。最近的研究介绍了用多能干细胞来源的健康心肌细胞取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，包括肿瘤形成的可能性，心肌细胞形成率低，以及对心肌发生的机制理解不足。此外，由于缺乏提高干细胞治疗效果的药剂，转化努力受到阻碍。多索吗啡是第一个已知的骨形态发生蛋白(BMP)信号的小分子抑制剂，是小鼠胚胎干细胞(ES)诱导心肌生成的最有效的化学诱导剂之一。在胚胎干细胞分化的最初24至48小时内给予多索吗啡治疗，足以强大地诱导心肌细胞。值得注意的是，大量的心脏诱导明显发生在没有中胚层诱导的情况下，并以其他中胚层来源的谱系为代价，包括内皮、平滑肌和造血系。根据这些结果，我们假设及时的BMP信号抑制将原始的多能祖细胞转化为心肌细胞的发育。本研究利用这一独特而强大的心脏诱导模型来阐明心肌细胞的承诺和分化机制。在目标1中，我们详细研究了小分子BMP抑制剂对中胚层、心血管前体细胞和心肌细胞形成的影响。通过比较多索吗啡和一种专门的BMP抑制剂DMH1的效果，我们将测试多索吗啡对Flk1激酶的已知非靶点作用是否减少了整体中胚层的形成，以及纯BMP抑制剂是否可以诱导更大的心肌细胞形成。在目标2中，我们将检测DMH1处理的ES细胞中的Flk1中胚祖细胞是否优先定向为心肌细胞。此外，我们还将确定DMH1治疗所诱导的可能的心脏致病祖细胞的分子图谱。这些研究将有助于揭示心脏前体细胞的本质。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有力地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，以改善小鼠心肌损伤模型的心脏修复。利用小分子在成年动物体内阻断BMP信号的独特能力，我们将探索ES细胞移植后体内刺激心肌细胞形成的可能性。本研究利用小分子骨形态发生蛋白抑制剂来探讨心肌发生的机制，不仅将为未来干细胞治疗心脏病的策略提供参考，而且将为提高干细胞的治疗效果提供有价值的药物。

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.