Expansion of Cardiac and Hematopoietic Pregenitors by Wnt and Notch

描述（由申请人提供）： 随着用于从胚胎干细胞（ESC）、诱导多能干细胞（iPSC）以及从分离的成体干细胞/祖细胞或直接“重编程”为谱系特异性干细胞/祖细胞的分化细胞衍生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。然而，实现这一目标将需要用于获得治疗上有用数量的细胞的方法，所述方法避免诱导永久性遗传改变，并确保所获得的谱系定向干细胞/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，以直接分化和扩增反映正常发育状态的细胞类型的群体。作为这种方法的模型，我们专注于Notch和Wnt途径和充分表征的造血系统，以产生造血干细胞（HSC）。扩增治疗上有用的干/祖细胞的可行性通过我们对脐带血衍生的干/祖细胞的扩增以及这些细胞在临床环境中的成功应用来证明。具体来说，我们将研究Notch和Wnt信号在胚胎中产生第一批HSC的需求和时间，以指导我们体外产生这些细胞的努力。我们将通过增强向永久造血干/祖细胞的生血内皮前体的分化，并通过促进这些多能干/祖细胞群的自我更新来产生ES-和iPS-衍生的HSC（项目1）。为了评估人类ES和iPS衍生的干/祖细胞的治疗有用性，我们将确定它们对转录、染色质和DNA甲基化和功能景观的保留（项目2）。这些研究将与关于Wnt和Notch在心脏干细胞的扩增和适当分化中的作用的协作关联申请中描述的研究相结合。

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.