Genetic analysis of stem cell maintenance in vivo

描述（由申请人提供）：造血干细胞（HSC）在整个生命过程中持续存在，并在损伤后通过依赖于细胞内在和细胞外在机制进行自我更新分裂来动态调节其数量。就细胞外在机制而言，HSC 被认为存在于由骨髓中的支持细胞创建的特殊微环境中，这些支持细胞表达促进 HSC 维持（生存和自我更新）并调节 HSC 迁移、静止和分化的膜结合和分泌因子。许多骨髓 HSC 位于骨内膜表面的成骨细胞处或附近，并且已提出成骨细胞分泌许多促进 HSC 维持的因子。许多 HSC 也位于骨髓中的窦状血管附近，并且血管或血管周围细胞也被认为可以分泌调节 HSC 维持的因子。尽管如此，这些因素都没有从任何候选利基细胞中有条件地删除。因此，这些因素的生理来源从未在功能实验中得到测试。血管生成素-1 (Ang-1)、干细胞因子 (SCF) 和 CXCL12 都是维持 HSC 正常数量所需的遗传因素，但这些因子均未从成骨细胞或血管/血管周围细胞中有条件删除，以鉴定这些因子的生物学重要来源。最终，如果不从基因上鉴定出分泌 HSC 维持所需因子的细胞，就不可能鉴定出创建 HSC 生态位的细胞。我们的初步表达数据表明巨核细胞是骨髓中Ang-1的主要来源，并且SCF有多种来源，包括内皮细胞和骨内膜细胞。为了测试哪些细胞是这些因子对 HSC 维持的功能性重要来源，我们生成了 Ang-1 和 Scf 的 floxed 等位基因，并建议将携带这些等位基因的小鼠与在成骨细胞、巨核细胞和内皮细胞特异性启动子控制下表达 Cre 重组酶的小鼠交配。这些实验还将测试单一细胞类型是否是 HSC 维持所需的多种因子的主要来源，或者不同的细胞类型是否产生不同的调节 HSC 的因子。这将首次对哪些细胞在体内调节 HSC 维持进行功能测试。 公共健康相关性：该项目旨在评估哪些细胞是生长因子的生理重要来源，这些生长因子对于造血干细胞的维持和调节至关重要。这将提供有关构成体内造血干细胞生态位的细胞身份的重要新信息。这些信息对于了解如何调节血细胞形成以及干细胞如何在整个生命过程中维持在该组织中至关重要。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) persist throughout life and dynamically regulate their numbers after injury by undergoing self-renewing divisions that depend upon both cell- intrinsic and cell-extrinsic mechanisms. With respect to cell-extrinsic mechanisms, HSCs are thought to reside within specialized microenvironments created by supporting cells in the bone marrow that express membrane-bound and secreted factors that promote HSC maintenance (survival and self-renewal), and that regulate HSC migration, quiescence, and differentiation. Many bone marrow HSCs reside at, or near, the osteoblasts at the endosteal surface and osteoblasts have been proposed to secrete a number of factors that promote HSC maintenance. Many HSCs also reside adjacent to sinusoidal blood vessels in the bone marrow, and vascular or perivascular cells have also been proposed to secrete factors that regulate HSC maintenance. Nonetheless, none of these factors have ever been conditionally deleted from any candidate niche cell. As a result, the physiological sources of these factors have never been tested in functional experiments. Angiopoietin-1 (Ang-1), Stem Cell Factor (SCF), and CXCL12 are all genetically required for maintenance of normal numbers of HSCs but none of these factors have been conditionally deleted from osteoblasts or from vascular/perivascular cells to identify the biologically important source(s) of these factors. Ultimately, it will not be possible to identify the cells that create HSC niches without genetically identifying the cells that secrete factors required for HSC maintenance. Our preliminary expression data suggest that megakaryocytes are the major source of Ang-1 in the bone marrow and that there are multiple sources of SCF including both endothelial cells and endosteal cells. To test which cells are functionally important sources of these factors for HSC maintenance we have generated floxed alleles of Ang-1 and Scf and propose to mate mice bearing these alleles with mice expressing Cre-recombinase under the control of promoters specific to osteoblasts, megakaryocytes, and endothelial cells. These experiments will also test whether a single cell type is the main source of multiple factors required for HSC maintenance or whether different cell types produce different factors that regulate HSCs. This will provide the first functional test of which cells regulate HSC maintenance in vivo. PUBLIC HEALTH RELEVANCE: This project is designed to assess which cells are the physiologically important sources of growth factors that are critical for the maintenance and regulation of hematopoietic stem cells. This will provide important new information regarding the identities of the cells that constitute the hematopoietic stem cell niche in vivo. Such information is critical to understand how blood cell formation is regulated and how stem cells are sustained in this tissue throughout life.