Expansion of Cardiac and Hematopoietic Pregenitors by Wnt and Notch

描述(由申请人提供)： 随着从胚胎干细胞(ESC)、诱导多能干细胞(IPSC)以及从分离的成体干细胞/祖细胞或分化的细胞中获得此类细胞的方法的开发，用于治疗目的的所需细胞类型的产生正在成为现实，这些细胞直接被重新编程为谱系特异性的干/祖细胞。然而，实现这一目标将需要一些方法来获得治疗上有用的细胞数量，以避免导致永久性的基因改变，并确保衍生的血统承诺的干细胞/祖细胞的行为保真度。为了解决这个问题，我们建议测试我们的假设，即调节正常发育的途径可以被操纵来指导反映正常发育状态的细胞类型的群体的分化和扩张。作为这一方法的模型，我们关注Notch和Wnt途径以及生成造血干细胞(HSC)的特征良好的造血系统。我们对脐带血来源的干细胞/祖细胞的扩增以及这些细胞在临床环境中的成功应用证明了扩增治疗有用的干细胞/祖细胞的可行性。具体地说，我们将研究Notch和Wnt信号在胚胎中产生第一批HSCs的需求和时机，以指导我们在体外生产这些细胞的努力。我们将通过促进特定造血干/祖细胞向造血内皮祖细胞的分化，以及通过促进这些多能干/祖细胞群体的自我更新来产生ES-和iPS来源的HSC(项目1)。为了评估人类ES和iPS来源的干细胞/祖细胞的治疗有效性，我们将确定它们在转录、染色质和DNA甲基化以及功能方面的保存情况(项目2)。这些研究将与关于Wnt和Notch在心脏干细胞扩增和适当分化中的作用的协作链接申请中描述的那些研究相结合。

DESCRIPTION (provided by applicant): The generation of desired cell types for therapeutic purposes is becoming a reality with the development of methods for deriving such cells from embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), as well as from isolated adult stem/progenitor cells or differentiated cells that are directly "reprogrammed" into lineage-specific stem/progenitor cells. Realizing this goal, however, will require methods for deriving therapeutically useful numbers of cells that avoid inducing permanent genetic alterations, and ensure the behavioral fidelity of derived lineage-committed stem/progenitor cells. To address this issue, we propose to test our hypotheses that pathways regulating normal development can be manipulated to direct differentiation and expansion of populations of cell types that reflect normal developmental states. As a model for this approach, we focus on the Notch and Wnt pathways and the well-characterized hematopoietic system to generate hematopoietic stem cells (HSC). The feasibility of expanding therapeutically useful stem/progenitor cells is demonstrated by our expansion of cord blood-derived stem/progenitor cells and by our successful application of these cells in a clinical setting. Specifically, we will examine the requirement for and timing of Notch and Wnt signaling in generating the first HSCs in the embryo, to guide our efforts to produce these cells ex vivo. We will generate ES- and iPS-derived HSC by enhancing differentiation towards hemogenic endothelial precursors of definitive hematopoietic stem/progenitor cells, and by promoting selfrenewal of these multipotent stem/progenitor populations (Project 1). To assess the therapeutic usefulness of human ES- and iPS-derived stem/progenitor cells, we will determine their preservation of the transcriptional, chromatin and DNA methylation and functional landscapes (Project 2). These studies will interface with those described in the collaborative linked application on the role of Wnt and Notch in expansion and proper differentiation of cardiac stem cells.