Genetic analysis of stem cell maintenance in vivo

描述(申请人提供)：造血干细胞(HSCs)在一生中持续存在，并在受伤后通过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。在细胞-外在机制方面，HSC被认为存在于特殊的微环境中，这些微环境由骨髓中的支持细胞创造，表达膜结合和分泌的因子，促进HSC的维持(存活和自我更新)，并调节HSC的迁移、静止和分化。许多骨髓HSC位于或靠近骨内膜表面的成骨细胞，成骨细胞被认为可以分泌许多促进HSC维持的因子。许多HSC也与骨髓中的血窦血管相邻，血管或血管周围细胞也被认为分泌调节HSC维持的因子。尽管如此，这些因素都没有被有条件地从任何候选利基单元中删除。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在基因上都是维持正常数量的HSC所必需的，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(S)。最终，如果不对分泌维持HSC所需的因子的细胞进行基因鉴定，就不可能鉴定出创造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，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的重要功能来源，我们产生了Ang-1和SCF的等位基因，并建议在成骨细胞、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重组酶的小鼠配对。这些实验还将测试一种细胞类型是否是维持HSC所需多种因素的主要来源，或者不同的细胞类型是否会产生不同的调节HSC的因素。这将为体内调节HSC维持的细胞提供第一个功能测试。 公共卫生相关性：该项目旨在评估哪些细胞是生理上重要的生长因子来源，这些生长因子对造血干细胞的维持和调节至关重要。这将为体内构成造血干细胞生态位的细胞的特性提供重要的新信息。这些信息对于了解血细胞形成是如何调控的，以及干细胞是如何在这个组织中维持一生的至关重要的。

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.