Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors
Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors
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DOI：
10.3791/3736
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发表时间：
2012-07-01
影响因子：
1.2
通讯作者：
Grassi, Fabio
中科院分区：
文献类型：
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作者：
Frascoli, Michela;Proietti, Michele;Grassi, Fabio
骨髓是HSC和更成熟的血细胞谱系祖细胞在成年生物体中驻留和分化的主要场所。造血干细胞构成了一个多能细胞的微小细胞群，能够终生生成所有血细胞谱系（1）。骨髓中HSC稳态的分子解剖在造血、肿瘤学和再生医学中具有重要意义。我们描述了使用荧光抗体的标记方案和流式细胞术中的电子门控程序，以在个体小鼠中对造血祖细胞亚群和HSC分布进行评分（图1）。此外，我们描述了一种方法，以广泛丰富造血祖细胞以及长期（LT）和短期（ST）重建造血干细胞从合并的骨髓细胞悬浮液通过磁富集表达c-Kit的细胞。所得到的细胞制剂可用于分选选定的亚群，用于体外和体内功能研究（图2）。小梁成骨细胞（2，3）和窦状隙内皮细胞（4）构成支持骨髓中HSC的功能小生境。成骨细胞生态位中的几种机制，包括N-钙粘蛋白（+）成骨细胞的亚群（3）和HSC中表达的受体酪氨酸激酶Tie 2与其配体血管生成素-1（5）的相互作用，共同决定了HSC的静止。骨髓中的“冬眠”对于保护HSC免于复制和在过度循环活动时最终耗尽至关重要（6）。作用于先天免疫系统细胞的外源性刺激物如Toll样受体配体（7）和干扰素-α（6）也可诱导HSC增殖和分化为谱系定向祖细胞。最近，已描述了lin（-）c-Kit（+）Sca-1（+）CD 150（+）CD 48（-）CD 34（-）群体内的休眠小鼠HSC群体（8）。基于CD 34表达从如本文所述的造血祖细胞富集的细胞悬浮液中分选细胞允许分离静止的自我更新的LT-HSC和ST-HSC 9。先前已经描述了基于谱系阳性细胞的消耗和用CD 48和Flk 2抗体分选LT-HSC的类似程序（10）。在本报告中，我们提供了一个协议的表型表征和离体细胞周期分析的造血祖细胞，这可以是有用的监测造血在不同的生理和病理条件。此外，我们描述了一个流式细胞仪分选程序的HSC，它可以用来定义的因素和机制，调节他们的自我更新，扩增和分化的细胞生物学和信号转导试验，以及移植。
The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time(1). The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).Both trabecular osteoblasts(2,3) and sinusoidal endothelium(4) constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin(+) osteoblasts(3) and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-1(5) concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity(6). Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands(7) and interferon-alpha(6) can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin(-) c-Kit(+) Sca-1(+) CD150(+) CD48(-) CD34(-) population has been described(8). Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described(10). In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.