Cardiac Differentiation from Human Embryonic Stem Cells

现在有可能以适合大型实验或小型临床试验的规模指导多能人类干细胞分化为心肌细胞。尽管取得了这一成功，但我们对信号通路如何汇聚在细胞核上以控制心脏分化的理解仍存在很大的差距。此外，干细胞衍生的心肌细胞目前处于成熟的胎儿阶段，这可能不太适合模拟成人疾病或组织修复。项目3以下列目标处理这些问题。在目标1中，我们探索了通过ChIPseq实验鉴定的心脏发育的三种候选新调节剂的生物学。这些包括两个转录因子（MEIS 2和H 0XB 2）和一个新的Wnt/b-连环蛋白（b-catenin）。 调节蛋白（TMEM 88）。这些基因将在分化的hESC和发育中的斑马鱼胚胎中被敲低，以确定它们在心脏分化和形态发生中的作用。在目标2中，我们将利用两种遗传修饰的hESC系，当它们分化为中胚层（BRACHYURY敲入）或第一心野细胞（TBX 5敲入）时，它们激活荧光蛋白表达。我们将确定它们的命运如何在2D和3D培养中分化期间以及体内移植后受到限制。此外，我们将使用Wnt/β-连环蛋白报告基因来跟踪该途径如何积极和消极地调节这些关键细胞类型的形成。目标3解决了心肌细胞成熟的问题， 探索在成熟过程中上调的4种候选miRNAs的作用。接下来，我们将通过RNA-seq鉴定新的成熟相关miRNA，并使用过表达，耗尽和新的miRNA传感器评估它们的功能。

It is now possible to direct the differentiation of pluripotent human stem cells into cardiomyocytes at a scale suitable for large experiments or small clinical trials. Despite this success, there are large gaps in our understanding of how signaling pathways converge on the nucleus to control cardiac differentiation. Furthermore, stem cell-derived cardiomyocytes are currently at a fetal stage of maturity, which may be poorly suited for modeling adult diseases or tissue repair. Project 3 addresses these issues with the following aims. In Aim 1, we explore the biology of three candidate novel regulators of heart development identified by ChlPseq experiments. These include two transcription factors (MEIS2 and H0XB2) and a novel Wnt/b-catenin regulating protein (TMEM88). The genes will be knocked down in differentiating hESCs and in developing zebrafish embryos to determine their roles in cardiac differentiation and morphogenesis. In Aim 2 we will utilize two lines of genetically modified hESCs that activate fluorescent protein expression when they differentiate to mesoderm (BRACHYURY knock-in) or first heart field cells (TBX5 knock-in). We will determine how their fates become restricted during differentiation in 2D and 3D culture and after transplantation in vivo. Additionally, we will use a Wnt/b-catenin reporter to track how this pathway positively and negatively regulates formation of these key cell types. Aim 3 addresses the issue of cardiomyocyte maturation, beginning by exploring the role of 4 candidate miRNAs that are up-regulated during maturation. Next we will identify novel maturation-associated miRNAs through RNA-seq, and we will assess their function using over-expression, depletion and novel miRNA sensors.