Investigation of cellular cues for differentiation of ventricular cardiomyocytes

项目总结 心力衰竭是部分由于心肌细胞(CM)死亡而导致的心功能恶化。离体 将人多能干细胞分化为CMS是开发细胞替代的关键方法 心脏修复的治疗，由于心脏不能再生。尽管差异化有所改善 效率，当前的协议导致功能不成熟、不同类型的CM亚型在不同的 当移植到人类体内时，导致不利影响的发育状态。以产生特定的、成熟的 CM亚型在体外用于心脏修复治疗，必须了解细胞命运的决定和 允许细胞成为特定细胞类型的发育线索。细胞的发育高度依赖于 对组织内环境的影响，特别是通过不同细胞类型之间的细胞-细胞相互作用。至 为了了解CMS是如何受到环境的影响的，我们在2D中研究了CMS的分化状态 (单层)与3D(类胚体)细胞环境。在这里，我们观察到室性CMS(VCM) 开发效率更高，CMS在3D环境中显示出更成熟的细胞状态，因为 与2D环境相比。此外，3D系统包含一个更加异质的细胞 与2D系统相比，具有不同的信号提示。因此，这一点的中心假设 建议是，周围3D蜂窝环境的非细胞自主效应可能 通过创造一个更接近体内的环境来促进VCM的分化。为此，我们将 研究2D和3D细胞环境对VCM分化的影响(目标1)。此外，我们 将识别影响VCM分化的非CM细胞类型分泌的旁分泌信号(目标2)。这些 关键研究将阐明非CM细胞类型及其特定的分子线索对 VCM，从而允许产生更类似于体内的细胞类型的纯群体，可用于 心力衰竭的治疗和治疗进展。

PROJECT SUMMARY Heart failure is the deterioration of cardiac function in part due to cardiomyocyte (CM) death. In vitro differentiation of human pluripotent stem cells (hPSCs) into CMs is a key method for developing cell-replacement therapies for heart repair, due to the heart’s inability to regenerate. Despite improvements in differentiation efficiencies, current protocols give rise to functionally immature, diverse populations of CM sub-types at different developmental states leading to unfavorable effects when transplanted in humans. To generate specific, mature CM sub-types in vitro for heart repair treatment, it is essential to understand the cell fate decisions and developmental cues that allow a cell to become a particular cell type. A cell’s development is highly dependent on its environment within a tissue, particularly through cell-cell interactions between distinct cell types. To understand how CMs are influenced by their environment, we examined the differentiation states of CMs in a 2D (monolayer) versus 3D (embryoid body) cellular environment. Here, we observed that ventricular CMs (vCMs) develop more efficiently and CMs exhibit a more developmentally mature cellular state in the 3D environment as compared to the 2D environment. Additionally, the 3D system contains a more heterogenous cellular environment and has differing signaling cues versus the 2D system. Therefore, the central hypothesis of this proposal is that the non-cell autonomous effects of the surrounding 3D cellular environment may promote vCM differentiation by generating a more in vivo-like environment. Toward this end, we will investigate the impact of the 2D versus 3D cellular environments on vCM differentiation (Aim 1). Additionally, we will identify paracrine cues secreted from non-CM cell types that influence vCM differentiation (Aim 2). These key studies will elucidate the impact of non-CM cell types and their specific molecular cues on the generation of vCMs, thus allowing for the generation of pure populations of more in vivo-like cell types that can be used for heart failure treatment and therapeutic development.