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

 描述(申请人提供)：由于难以找到匹配的供者，以及供者和受者之间的免疫耐受，造血干细胞(HSC)移植在遗传性或获得性血液病中的治疗应用经常受到限制。因此，迫切需要寻找新的方法来从人体组织中产生高通量、临床级的功能性HSCs。这项应用的目的是分析刺激从血源性内皮细胞到造血干细胞的发育转变的新的细胞-外在机制。我们最近证实，尽管血液循环受损，但在cdh5缺失的斑马鱼胚胎中出现了有功能的造血干细胞。这些发现引导我们研究了不依赖于剪应力的生物力学机制，刺激内皮细胞向HSC转变。利用超声心动图、微血管造影术、3D多普勒和共聚焦成像，我们证明了心跳调节脉压，从而周向拉伸刺激HSC的形成。药物抑制拉伸激活的离子通道进一步减少HSC的形成。因此，我们假设，脉压介导的周向拉伸通过刺激拉伸激活的离子通道来刺激内皮细胞向HSCs的转变。我们将使用培养皿器官和细胞工程学的进展来总结小鼠血液内皮细胞的原位周向拉伸条件。由于沉默的心脏(tnnt2，sih)沉默的胚胎没有心跳和血液循环，我们还将研究药物刺激拉伸激活的离子通道是否可以挽救sih沉默的胚胎的造血缺陷。此外，我们将使用转基因斑马鱼、体外小鼠胚胎和外植体培养方法，以及共聚焦成像方法来分析拉伸激活的离子通道TRPV4和/或Piezo1的刺激如何影响功能性HSCs的出现。我们的目标是在HSC形成过程中建立一个新的保守的细胞外在作用和周向拉伸的机制。识别刺激内皮细胞造血特性的新因素将加速内皮细胞作为造血干细胞来源的发展，用于治疗人类血液疾病。

 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.