Biomechanical Stimulation of Hemogenic Endothelial Cells to Develop Clinical-Grade Hematopoietic Stem Cells

 描述（由申请人提供）：由于难以找到HLA匹配的供体以及供体和受体之间的免疫不耐受，造血干细胞（HSC）移植用于遗传性或获得性血液病的治疗用途通常受到限制。因此，迫切需要鉴定新方法以从人组织产生高通量、临床级功能性HSC。本申请的目的是分析新的细胞外源性机制，刺激造血内皮细胞向HSC的发育转变。我们最近确定，功能性HSC出现在cdh 5缺失的斑马鱼胚胎中，尽管血液循环受损。这些发现使我们研究剪切应力独立的生物力学机制刺激内皮细胞向HSC的转变。使用超声心动图，微血管造影，三维多普勒，共聚焦成像，我们证明，心跳介导的脉搏压力，从而周向拉伸刺激HSC的形成。牵张激活离子通道的药理学抑制进一步减少了HSC的形成。因此，我们假设，脉压介导的周向牵张通过刺激牵张激活的离子通道来刺激内皮细胞向HSC的转变。我们将利用器官培养皿和细胞工程学的进展来重现小鼠生血内皮细胞的原位周向拉伸条件。由于沉默心脏（tnnt 2，sih）沉默胚胎缺乏心跳和血液循环，我们也将研究是否牵张激活离子通道的药理学刺激可以挽救sih沉默胚胎的造血缺陷。此外，我们将使用转基因斑马鱼，离体小鼠胚胎和外植体培养方法，以及共聚焦成像来分析如何刺激拉伸激活离子通道，Trpv 4和/或Piezo 1，影响功能性HSC的出现。我们的目标是建立一个新的保守的细胞外源性的作用和机制的周向拉伸在HSC的形成。发现新的刺激内皮细胞造血特性的因子将促进内皮细胞作为治疗人类血液病的HSC来源的发展。

 DESCRIPTION (provided by applicant): The therapeutic use of hematopoietic stem cell (HSC) transplantation for inherited or acquired blood diseases is often restricted due to the difficulty with finding HLA-matched donors and immunologic intolerance between donor and recipient. Therefore, there is a critical need to identify new methods to generate high throughput, clinical-grade functional HSCs from human tissues. The objective of this application is to analyze novel cell-extrinsic mechanisms that stimulate the developmental transition from hemogenic endothelial cells to HSCs. We recently established that functional HSCs emerge in the cdh5-deleted zebrafish embryos, despite an impaired blood circulation. These findings led us to investigate shear-stress independent biomechanical mechanisms stimulating endothelial to HSC transition. Using echocardiography, micro-angiography, 3D Doppler, and confocal imaging, we demonstrate that heartbeat mediated pulse-pressure, and thus circumferential stretch stimulates HSC formation. Pharmacological inhibition of stretch-activated ion channels further reduced HSC formation. We, therefore, hypothesize that pulse-pressure mediated circumferential stretch stimulates the transition of endothelial cells to HSCs by stimulating stretch-activated ion channels. We will use advances in organ-in-a-dish and cell-engineering to recapitulate in situ circumferential stretch conditions on murine hemogenic endothelial cells. Since the silent-heart (tnnt2, sih)-silenced embryos lack heartbeat and blood circulation, we will also investigate if pharmacological stimulation of stretch-activated ion channels could rescue hematopoietic deficiency in the sih-silenced embryos. In addition, we will use transgenic zebrafish, ex vivo mouse embryo and explant culture methods, as well as confocal imaging to analyze how stimulation of stretch activated ion channels, Trpv4 and/or Piezo1, influences the emergence of functional HSCs. Our aims will establish a new conserved cell-extrinsic role and mechanisms of circumferential stretch during HSC formation. Identification of new factors stimulating hemogenic properties of endothelium would expedite the development of endothelial cells as a source of HSCs for the treatment of human blood diseases.