Cellular and molecular analyses of hematopoietic stem cell [HSC] interactions with bone marrow niches to improve HSC engraftment for transplantation and tolerance induction

项目摘要 我们已经证明，我们首先分离的纯化造血干细胞（HSC）可以同种异体移植 没有GvH（他们缺乏T细胞），阻止自身免疫性1型糖尿病（和系统性红斑狼疮）， 诱导对HSC供体组织/器官移植耐受。然而，纯化的HSC移植不被使用， 部分原因是预处理方案的毒性。因此，至关重要的是，我们要提高对 造血干细胞如何与其局部支持环境-造血生态位相互作用。本研究的目的 进一步鉴定和表征骨髓HSC的细胞和分子成分， niche，并了解HSC及其niche之间正在进行的细胞间通讯 调节它们在健康和血液疾病中的归巢、锚定、存活和功能。在以前版本的 这项赠款，我们建立了抗体，以取代有毒方案，以条件接受者，并将进一步修改 改善植入和加速重建的方案。此外，了解HSC利基 我们在病理学中阐明了相互作用对干细胞竞争的影响，例如， 造血系统疾病，如骨髓增生异常综合征（MDS）、骨髓增生性肿瘤（MPN），和 扩增突变克隆以产生克隆造血，一些引起急性髓性白血病 （AML）。在白血病和MDS的发展过程中，具有癌前突变的造血干细胞克隆的竞争力超过了正常造血干细胞。 HSC，但HSC的总数似乎没有变化，这意味着HSC数量的控制由生态位。 HSC的自然再循环允许高度竞争性克隆在整个细胞中占据主导地位。 因此，我们对正常HSC发育和归巢的了解也可能适用于各种各样的疾病。 造血障碍，包括克隆性造血和白血病。这里的实验涉及 HSC归巢、锚定、支持和利基竞争。在目标1中，我们将利用长期（LT）-HSC报告基因 小鼠探索生态位的特异性，鉴定与LT-HSC形成直接接触的细胞类型， 分析负责HSC和生态位串扰的粘附分子、细胞因子和趋化因子。在Aim中 2我们建议了解HSC周围BM细胞的作用-关注相邻的正弦信号 内皮细胞和基本的BM基质亚群及其表达因子。建立在我们的 骨骼干细胞（SSC）的发现和表征，其克隆生成骨，软骨， 几种不同的骨髓基质细胞支持造血。我们计划培育小鼠，其中每个细胞 小生境中的一个子集，包括HSC，可以被工程化以敲除已识别的基因，以破译复杂性。 niche-HSC的相互作用。这可能导致一项谅解，如果有办法增加 功能性小生境，分子相互作用的调节是否可以明确小生境， 促进HSC植入，并提供调节克隆HSC扩增的靶点。 !

Project Summary We have shown that purified hematopoietic stem cells (HSC), first isolated by us, can be allotransplanted without GvH (they lack T cells), block autoimmune type 1 diabetes (and Systemic Lupus Erythematosus), and induce transplant tolerance to HSC donor tissues/organs. However, purified HSC transplantation is not used, in part due to the toxicity of the conditioning regimens. Therefore, it is vital that we improve our understanding of how HSCs interact with their local supporting environment—the hematopoietic niche. The goal of this research is to further identify and characterize the cellular and molecular components of the bone marrow (BM) HSC niche, and to understand how the ongoing inter-cellular communication between HSCs and their niche regulates their homing, anchoring, survival and function in health and in blood diseases. In previous versions of this grant we established antibodies in place of toxic regimens to condition recipients and will further modify the regimens to improve engraftment and accelerate reconstitution. Further, understanding the HSC-niche interaction has implications for stem cell competitions we have elucidated in pathology, for example, in hematopoietic disorders such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), and expansion of mutated clones to give clonal hematopoiesis, some that give rise to acute myeloid leukemia (AML). In the run-up to leukemia and MDS, clones of HSCs with pre-cancer mutations outcompete normal HSCs, but the total number of HSCs appears not to change, implying control of HSC numbers by the niche. The natural recirculation of HSCs allows highly competitive clones to dominate HSC niches throughout the body, so what we learn about normal HSC development and homing will also likely apply to a variety of disorders of hematopoiesis, including clonal hematopoiesis and leukemias. The experiments here concern HSC homing, anchoring, support, and niche competition. In Aim 1, we will utilize long-term (LT)-HSC reporter mice to explore the specificity of the niche, identify the cell types that form direct contact with LT-HSC, and analyze the adhesion molecules, cytokines and chemokines responsible for HSC and niche cross-talk. In Aim 2 we propose to understand the role of HSC-surrounding BM cells—focusing on adjacent sinusoidal endothelial cells and the essential BM stromal subsets and their expressed factors. That builds on our discovery and characterization of the skeletal stem cells (SSC), which clonally generate bone, cartilage, and several distinct BM stromal cells that support hematopoiesis. We plan to produce mice in which each cell subset in the niche, including HSC, can be engineered to knock out identified genes to decipher the complexity of niche-HSC interactions. This could lead to an understanding if there are means to increase the number of functional niches, whether modulation of the molecular interactions could clear the niche specifically to promote the HSC engraftment, and to provide targets for modulating clonal HSC expansions. !