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。这些自我更新的 存在于成人骨髓中的祖细胞一生中产生所有成熟的血液谱系。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骨髓瘤。 非恶性血液病供体的可用性不一致，移植材料的限制，以及可能的 使用体外衍生的HSC作为基因治疗和基因编辑方法的平台， 疾病引起了人们对HSC从多能前体（例如， 诱导多能干细胞（iPS）。但是，尽管几十年的研究，真正的HSC的产生与高 有效的植入和完整的多谱系潜力仍然是不可能的，这表明关键的规范， 信号仍有待确定。指导HSC体外规格的一种明显方法是尝试 重演正常的胚胎诱导过程。在整个脊椎动物门中，HSC被指定为从 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中，或其 同源，无羊膜脊椎动物如斑马鱼的背主动脉。在发电方面取得重大进展 造血干细胞的直接前体，“生血内皮”，近年来已经取得，但完整的 一组直接作用于该内皮细胞以启动最终造血程序的诱导信号 尚未确定。规范信号可能来自形成感应信号的相邻细胞。 规格“利基”。由于对这些细胞的起源或组成一无所知，我们开始定义 细胞类型，可能有助于规范生态位。造血编程高度保守， 从哺乳动物到斑马鱼，体内观察组织特化和造血发育是非常重要的。 在鱼类中可获得，由于它们的快速发育，在指定HSC时的光学透明度， 以及各种转基因动物的可用性，其中组织和细胞类型通过荧光标记 转基因在对斑马鱼的初步研究中，我们已经确定神经嵴来源的细胞是 第一个已知的细胞类型，有助于HSC的规格小生境。我们的研究结果表明神经嵴细胞 在HSC程序启动前不久物理接触生血内皮。多个单独的 引起神经嵴特化或形态发生缺陷的扰动导致HSC的损失， 这表明HSC的特化需要适当的神经嵴图案。这些数据建立了神经 嵴细胞作为诱导性HSC特化小生境的关键组成部分，表明它们呈现的信号 是指导造血程序启动的关键。在这里，我们建议识别未知的HSC 规范信号的神经嵴，并建立了保守的神经嵴贡献， HSC在哺乳动物中的特化生态位。

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.