Novel hematopoietic stem cell specification signals from the neural crest

项目摘要 造血干细胞(HSCs)是成人造血系统的基础。这些自我更新 居住在成人骨髓中的祖细胞在一生中产生所有成熟的血统。在临床上， 造血干细胞是骨髓移植的治疗成分，用于治疗恶性肿瘤和 非恶性血液疾病。供者的可获得性不一致，移植材料的限制，以及可能的 利用体外来源的造血干细胞作为基因治疗的平台和基因编辑方法治疗慢性粒细胞白血病 疾病引起了人们对定向分化HSC与多能前体细胞的兴趣，例如 诱导多能干细胞。但尽管经过几十年的研究，真正的HSCs的产生具有高 效率植入和充分的多系潜力仍然是不可能的，这表明关键规范 信号仍有待确定。在体外指导HSC规范的一个明显的方法是尝试 重述正常的胚胎诱导过程。在脊椎动物门中，造血干细胞是从 发育中的动脉内皮，最明显的是在哺乳动物胚胎的原始降主动脉或其 同源，无羊膜脊椎动物的背主动脉，如斑马鱼。迈向新一代的重大进展 造血干细胞的直接前体，“血源性内皮细胞”，是近年来制造出来的，但完整的 一组直接作用于该内皮细胞以启动明确的造血程序的诱导信号 还没有确定。规范信号可能来自形成感应器的相邻细胞 详细说明是“利基”。由于对这种细胞的起源或组成一无所知，我们开始定义 可能对规范利基做出贡献的细胞类型。造血编程是高度保守的 哺乳动物到斑马鱼，并在体内观察组织规格和造血发育情况高度一致 在鱼类中，由于其快速发展，在指定HSC时的光学透明度， 以及用荧光标记组织和细胞类型的各种转基因动物的可用性 转基因。在对斑马鱼的初步研究中，我们已经确定神经脊来源的细胞是 第一个对HSC规范利基做出贡献的已知细胞类型。我们的结果表明，神经脊细胞 在HSC计划启动前不久，身体接触血液内皮细胞。多个独立的 导致神经脊规范或形态发生缺陷的扰动会导致HSCs的丧失， 证明HSC规范需要适当的神经脊模式。这些数据建立了神经 作为诱导性HSC规范的关键组件的CREST细胞，表明它们提供的信号 对指导造血程序的启动至关重要。在这里，我们建议识别未知的HSC 说明了神经峰所呈现的信号，并建立了神经峰对守恒性的贡献 哺乳动物中的HSC规格利基。

Project Summary Hematopoietic stem cells (HSCs) are the foundation of the adult hematopoietic system. These self-renewing progenitors that reside in the adult bone marrow generate all mature blood lineages through life. Clinically, HSCs are the therapeutic component of bone marrow transplants, used in the treatment of both malignant and non-malignant blood disorders. Inconsistent availability of donors, limitations in graft material, and the possible use of in vitro derived HSCs as a platform for gene therapy and gene editing approaches to treatment of disease has heightened interest in directed differentiation of HSCs from pluripotent precursors, such as induced pluripotent stem (iPS) cells. But despite decades of research, generation of true HSCs with high efficiency engraftment and full multilineage potential remains impossible, suggesting that key specification signals remain to be determined. An obvious means of instructing HSC specification in vitro is by attempting to recapitulate the normal embryonic inductive processes. Across vertebrate phyla, HSCs are specified from developing arterial endothelium, most notably in the primitive descending aorta of mammalian embryos, or its cognate, the dorsal aorta in anamniotic vertebrates such as zebrafish. Significant progress towards generation of the immediate precursor to HSCs, the “hemogenic endothelium”, has been made in recent years, but the full set of inductive signals that work directly on this endothelium to initiate the definitive hematopoietic program has not been determined. Specification signals likely come from neighboring cells that form an inductive specification “niche.” As nothing is known about the origin or composition of such cells, we set out to define cell types that might contribute to the specification niche. Hematopoietic programming is highly conserved from mammals to zebrafish, and in vivo observation of tissue specification and hematopoietic development is highly accessible in fish, due to their rapid development, optical transparency at the times when HSCs are specified, and the availability of diverse transgenic animals in which tissue and cell types are labeled by fluorescence transgenes. In preliminary studies with zebrafish, we have determined that neural-crest-derived cells are the first known cell type contributing to the HSC specification niche. Our results show that neural crest cells physically contact hemogenic endothelium shortly before the HSC program initiates. Multiple separate perturbations causing defects in neural crest specification or morphogenesis lead to a loss of HSCs, demonstrating that proper neural crest patterning is required for HSC specification. These data establish neural crest cells as a key component of the inductive HSC specification niche, indicating that signals they present are critical to instruct initiation of the hematopoietic program. Here we propose to identify the unknown HSC specification signals presented by neural crest, and establish the conservation of neural crest contribution to the HSC specification niche in mammals.