Paracrine interactions during cardiac and endothelial co-differentiation of hPSCs

 产品说明：人类多能干细胞（hPSC）提供无限自我更新潜力和多能性的独特组合，这两种特性赋予用于产生用于发育研究、毒性测试和细胞疗法的人类体细胞和组织的强大系统。hPSC是一种特别有前途的心脏组织来源，因为心肌细胞（CM）不能容易地从初级来源获得，并且在疾病和药物评价中具有巨大的重要性。在当前的项目期间，我们开发了一种有效的、完全定义的方案，通过使用小分子对经典Wnt信号进行时间调节来从hPSC产生人类CM，解决了hPSC心脏应用中的主要挑战之一。然而，目前的CM分化方案未能获得具有成人样表型的成熟细胞，限制了这些hPSC衍生的CM在再生应用中的潜力。在先前的工作中，我们还发现Wnt信号的不同时间调节将hPSC引导至血管内皮细胞（EC）。在心脏发育期间，发育中的EC和CM之间的旁分泌相互作用指导心脏组织结构和功能的形成，但这些信号不存在于当前的CM分化平台中。因此，在所提出的项目中，我们将开发新的2D和3D平台以将hPSC共分化为空间图案化的EC和CM，并使用这些平台来测试EC-CM共分化期间的旁分泌相互作用增强CM成熟的假设。我们建议利用我们团队在生物材料，干细胞生物学，心脏细胞生物学，基因组编辑和心血管发育方面的专业知识，通过Wnt信号的时间调节将纯CM和EC群体与hPSC区分开来，以包括Wnt信号的空间调节，使CM和EC能够以精确设计的模式共分化。此外，我们将使用共分化平台，以确定EC产生的旁分泌机制介导CM成熟。我们检验本申请假设的具体目的是：1。采用Wnt信号传导的光遗传学空间和时间调节以在2D中使hPSC共分化为EC和CM模式化。共分化将通过光介导的时空激活和Wnt信号传导的抑制来实现。 2.实施Wnt调节剂的释放以在3D中将hPSC共分化模式化为EC和CM。通过Wnt抑制剂在分化3D hPSC聚集体中的局部释放将实现共分化。 3.阐明共分化过程中EC信号传导至CM的机制。将采用生物化学和遗传机制来鉴定在目标1和2中开发的2D和3D共分化平台中刺激CM成熟的旁分泌信号传导。

 DESCRIPTION: Human pluripotent stem cells (hPSCs) provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating human somatic cells and tissues for developmental studies, toxicity testing, and cellular therapies. hPSCs are a particularly promising source of cardiac tissues since cardiomyocytes (CMs) cannot easily be attained from primary sources and are of tremendous importance in disease and pharmaceutical evaluation. In the current project period we developed an efficient, completely defined protocol to produce human CMs from hPSCs via temporal modulation of canonical Wnt signaling using small molecules, solving one of the major challenges in cardiac applications of hPSCs. However, current CM differentiation protocols fail to achieve mature cells possessing adult-like phenotypes, limiting the potential of these hPSC-derived CMs in regenerative applications. In prior work we also found that a different temporal modulation of Wnt signaling directs hPSCs to vascular endothelial cells (ECs). During heart development, paracrine interactions between developing ECs and CMs guide formation of cardiac tissue structure and function, but these signals are not present in current CM differentiation platforms. Thus, in the proposed project we will develop novel 2D and 3D platforms to co-differentiate hPSCs to spatially patterned ECs and CMs and use these platforms to test the hypothesis that paracrine interactions during EC-CM co- differentiation enhance CM maturation. We propose to use our team's expertise in biomaterials, stem cell biology, cardiac cell biology, genome editing, and cardiovascular development to extend our advances on differentiating pure populations of CMs and ECs from hPSCs via temporal modulation of Wnt signaling to include spatial regulation of Wnt signaling, enabling co-differentiation of CMs and ECs in precisely-designed patterns. In addition, we will use co-differentiation platforms to identify EC-generated paracrine mechanisms mediating CM maturation. Our specific aims to test the hypothesis of this application are: 1. Employ optogenetic spatial and temporal regulation of Wnt signaling to pattern hPSC co- differentiation to ECs and CMs in 2D. Co-differentiation will be enabled by light-mediated spatio-temporal activation and repression of Wnt signaling. 2. Implement release of Wnt modulators to pattern hPSC co-differentiation to ECs and CMs in 3D. Co- differentiation will be enabled by localized release of Wnt inhibitors in differentiating 3D hPSC aggregates. 3. Elucidate mechanisms of EC signaling to CMs during co-differentiation. Biochemical and genetic mechanisms will be employed to identify paracrine signaling that stimulates CM maturation in the 2D and 3D co-differentiation platforms developed in Aims 1 and 2.