Investigation of cellular cues for differentiation of ventricular cardiomyocytes

项目摘要 心力衰竭是部分由于心肌细胞（CM）死亡导致的心脏功能恶化。体外 将人多能干细胞（hPSC）分化为CM是开发细胞替代的关键方法 治疗心脏修复，因为心脏无法再生。尽管差异化有所改善， 尽管目前的方案效率低下，但在不同的条件下，目前的方案产生了功能不成熟的CM亚型的不同群体。 当移植到人体时，导致不利影响的发育状态。产生具体的，成熟的 CM亚型在体外心脏修复治疗中，了解细胞命运决定和 使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 在组织内的环境，特别是通过不同细胞类型之间的细胞-细胞相互作用。到 为了了解CM如何受到环境的影响，我们在2D中检查了CM的分化状态。 （单层）与3D（胚状体）细胞环境。在这里，我们观察到心室CM（vCM） 更有效地发展，CM在3D环境中表现出更成熟的细胞状态， 与2D环境相比。此外，3D系统包含更异质的细胞 环境，并具有与2D系统不同的信号提示。因此，这个问题的核心假设 建议是周围3D蜂窝环境的非蜂窝自主效应可以 通过产生更类似体内的环境来促进vCM分化。为此，我们将 研究2D与3D细胞环境对vCM分化的影响（目标1）。另外我们 将鉴定从非CM细胞类型分泌的影响vCM分化的旁分泌线索（Aim 2）。这些 关键的研究将阐明非CM细胞类型及其特定分子线索对产生的影响， vCM，从而允许产生更多体内样细胞类型的纯群体，其可用于 心力衰竭治疗和治疗发展。

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.