Genetic analysis of stem cell maintenance in vivo

描述（由申请人提供）：造血干细胞（hsc）在生命中持续存在，并在损伤后通过依赖于细胞内在和细胞外在机制的自我更新分裂来动态调节其数量。关于细胞外源机制，造血干细胞被认为存在于由骨髓中支持细胞创造的特殊微环境中，这些细胞表达膜结合和分泌因子，促进造血干细胞的维持（生存和自我更新），并调节造血干细胞的迁移、静止和分化。许多骨髓造血干细胞位于或靠近内皮表面的成骨细胞，并且成骨细胞被认为可以分泌许多促进造血干细胞维持的因子。许多造血干细胞也存在于骨髓窦状血管附近，血管或血管周围细胞也被认为分泌调节造血干细胞维持的因子。尽管如此，这些因子都没有被有条件地从任何候选生态位细胞中删除。因此，这些因素的生理来源从未在功能实验中得到验证。血管生成素-1 （ang1）、干细胞因子（SCF）和CXCL12都是维持正常造血干细胞数量所必需的遗传因子，但这些因子都没有从成骨细胞或血管/血管周围细胞中有条件地删除，以确定这些因子的重要生物学来源。最终，如果没有基因鉴定分泌维持造血干细胞所需因子的细胞，就不可能鉴定产生造血干细胞壁龛的细胞。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，包括内皮细胞和内皮细胞。为了测试哪些细胞是这些维持HSC因子的重要功能来源，我们产生了Ang-1和Scf的固定等位基因，并提出在成骨细胞、巨核细胞和内皮细胞特异性启动子的控制下，将携带这些等位基因的小鼠与表达cre -重组酶的小鼠进行配对。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，或者不同细胞类型是否产生不同的调节HSC的因子。这将提供第一个细胞在体内调节HSC维持的功能测试。

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.