TGF-b family signaling in cardiomyocyte differentiation from embryonic stem cells

描述（由申请人提供）：对心肌组织修复和组织再生的细胞治疗有相当大的兴趣。胚胎干细胞可以在培养中分化为心肌细胞，因此被认为是心脏修复的无限细胞来源。更好地了解这一分化过程的调控将极大地有助于心肌细胞的产生，并可能导致提高产量，控制质量和均匀性以及直接心肌细胞亚型特征的方法。对控制人胚胎干细胞分化潜能和心肌细胞分化的信号通路和转录因子知之甚少。Oct4， Sox2和Nanog是自我更新和多能性的重要转录因子，但也可能在向心肌细胞分化的起始过程中发挥关键作用。TGF-2家族蛋白的信号传导控制着干细胞的自我更新、多能性和分化，TGF-2家族蛋白的自分泌信号传导可能在胚胎干细胞的自我更新和分化中发挥关键作用。TGF-2家族蛋白通过Smads进行基因表达应答，Smads可增强或抑制靶基因上转录因子的转录活性，从而发挥细胞内在分化介质的作用。Smads可能通过这种作用方式调控Oct4、Sox2、Nanog等胚胎干细胞转录因子的表达水平和功能，调控胚胎干细胞对心肌细胞的选择和分化进程。本提案的总体目标是：(1)评估TGF-2家族/Smad信号对Oct4， Sox2和Nanog的表达和活性的调节，(2)将这种水平的控制与心肌细胞谱系的分化潜力和分化特征相关联，(3)利用这些知识生成具有高效和明确特征的心肌细胞祖细胞。我们假设：(1)TGF-2家族蛋白介导的Smad信号传导调节胚胎干细胞转录因子的表达和活性；(2)Smad信号传导和Oct4、Sox2和/或Nanog活性的改变可改变细胞的分化能力，特别是心肌细胞的分化。我们提出三个目标：(1)研究TGF-2家族信号对胚胎干细胞转录因子表达和活性的调控，并将这些发现与心肌细胞谱系分化联系起来；(2)研究单个Smads对Oct4、Sox2和Nanog的调控作用，以及这些Smads与Oct4、Sox2和Nanog本身在心肌细胞谱系分化中的作用；(3)研究胚胎干细胞衍生的心肌细胞前体的体内分化和组织整合特征，这些细胞在培养过程中有利于这些细胞的产生。公共卫生相关性：用于心脏组织修复和组织再生的细胞疗法，例如在心脏梗死后，可能提供巨大的希望，人类胚胎干细胞被认为是这些细胞（称为心肌细胞）的大提琴来源。我们现在提出了一项研究计划，我们试图了解可能指导心肌细胞分化的信号通路。我们将探索如何修改这些途径，并希望设计出增加和改善心肌细胞生成和特征的方法。这将通过细胞培养实验和一种将心肌细胞注射到心脏梗塞小鼠心肌中的新方法来验证。

DESCRIPTION (provided by applicant): There is considerable interest in cell-based therapies for the repair and tissue regeneration of myocardial tissue. Embryonic stem cells are explored as an unlimited source of cells for cardiac repair, as they can differentiate into cardiomyocytes in culture. A better understanding of the regulation of this differentiation process would greatly aid in the generation of cardiomyocytes, and may lead to methods to improve the yield, control the quality and homogeneity, and direct subtype characteristics of the cardiomyocytes. Little is known about the signaling pathways and transcription factors that control the differentiation potential and cardiomyocyte differentiation of human embryonic stem cells. Oct4, Sox2 and Nanog function as essential transcription factors for self-renewal and pluripotency, yet may also play key roles in the initiation of differentiation into cardiomyocytes. Signaling by TGF-2 family proteins controls self-renewal, pluripotency and differentiation of stem cells, and autocrine signaling by TGF-2 family proteins is likely to play a key role in the self-renewal and differentiation of embryonic stem cells. TGF-2 family proteins exert gene expression responses through Smads, which enhance or repress the transcription activities of transcription factors at target genes, and thus function as cell-intrinsic mediators of differentiation. Through this mode of action, Smads are likely to regulate the expression levels and functions of embryonic stem cell transcription factors, such as Oct4, Sox2 and Nanog, and regulate the selection and progression of differentiation of cardiomyocytes from embryonic stem cells. The overall goals of this proposal are to (1) evaluate the regulation of expression and activities of the Oct4, Sox2 and Nanog by TGF-2 family/Smad signaling, (2) correlate this level of control with the differentiation potential and characteristics of differentiation along the cardiomyocyte lineage, (3) to use this knowledge to generate cardiomyocyte progenitors with high efficiency and defined characteristics. We hypothesize that (1) Smad signaling by TGF-2 family proteins regulates the expression and activities of the embryonic stem cell transcription factors, (2) alterations in Smad signaling and Oct4, Sox2 and/or Nanog activities modify the differentiation capacity of the cells and specifically cardiomyocyte differentiation. We propose three Aims: (1) to examine the regulation of embryonic stem cell transcription factor expression and activities by TGF-2 family signaling and to correlate these findings with cardiomyocyte lineage differentiation, (2) to study the roles of individual Smads in the regulation of Oct4, Sox2 and Nanog, and the roles of these Smads and Oct4, Sox2 and Nanog themselves in cardiomyocyte lineage differentiation, (3) To examine the in vivo differentiation and tissue integration characteristics of cardiomyocyte precursors derived from embryonic stem cells following manipulations that favor the generation of these cells in culture. PUBLIC HEALTH RELEVANCE: Cell-based therapies for the repair and tissue regeneration of heart tissue, for example following heart infarct, may provide great promise, and human embryonic stem cells are being considered as a cello source for these cells, called cardiomyocytes. We now propose a research plan in which we try to understand signaling pathways that may direct the differentiation of cardiomyocytes. We will explore how to modify these pathways and hope to design approaches that increase and improve the generation and characteristics of the cardiomyocytes. This will be tested through a combination of cell culture experiments and a new method in which the cardiomyocytes are injected into the heart muscle of mice with a heart infarct.