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 我们首次分离纯化的造血干细胞(HSC)可以进行同种异体移植。 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)，以及 诱导对HSC供体组织/器官的移植耐受。然而，纯化的HSC移植并未使用，在 部分原因是调理疗法的毒性。因此，重要的是我们要提高对 造血干细胞如何与其本地支持环境--造血利基--相互作用。这项研究的目的是 是进一步鉴定和表征骨髓(BM)HSC的细胞和分子组成 利基，并了解HSC和它们的利基之间正在进行的细胞间通信 调节它们在健康和血液疾病中的归宿、锚定、生存和功能。在以前的版本中 这笔赠款我们建立了抗体来代替有毒的方案来治疗接受者，并将进一步修改 改善植入率和加速重建的养生法。此外，了解HSC-利基市场 相互作用对我们在病理学中阐明的干细胞竞争有影响，例如，在 造血疾病，如骨髓增生异常综合征(MDS)、骨髓增生性肿瘤(MPN)和 扩增突变克隆以获得克隆性造血，其中一些会导致急性髓系白血病 (AML)。在白血病和MDS的前期，带有癌前突变的HSC克隆比正常情况下竞争更激烈 但HSC的总数似乎没有变化，这意味着HSC的数量由生态位控制。 造血干细胞的自然再循环使具有高度竞争力的克隆在整个 因此，我们所学到的关于正常的HSC发育和归巢也可能适用于各种 造血障碍，包括克隆性造血和白血病。这里的实验涉及到 HSC归位、锚定、支持和利基竞争。在目标1中，我们将使用Long Term(LT)-HSC记者 小鼠探索该生态位的特异性，确定与LT-HSC形成直接接触的细胞类型，并 分析导致HSC和小生境串扰的黏附分子、细胞因子和趋化因子。在AIM 2我们建议理解HSC周围的BM细胞的作用--专注于邻近的正弦 内皮细胞和基本骨髓基质亚群及其表达因子。这是建立在我们的 骨骼干细胞(SSC)的发现和鉴定，它能克隆生成骨、软骨和 支持造血的几种不同的骨髓基质细胞。我们计划培育出小鼠，在其中每个细胞 利基中的子集，包括HSC，可以被设计成敲除已识别的基因来破译复杂性 利基-HSC的相互作用。这可能会导致理解，如果有办法增加数量 功能生态位，分子相互作用的调节是否可以清除特定的生态位 促进HSC植入，为调控克隆HSC扩增提供靶点。 好了！

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. !