McIver Skye C, Hewitt Kyle J, Gao Xin, Mehta Charu, Zhang Jing, Bresnick Emery H
Department of Cell and Regenerative Biology, Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, 4009 WIMR, 1111 Highland Ave, Madison, WI, 53705, USA.
UW-Madison Blood Research Program, University of Wisconsin, Madison, WI, 53705, USA.
Methods Mol Biol. 2018;1698:67-89. doi: 10.1007/978-1-4939-7428-3_4.
Multipotent hematopoietic stem cells differentiate into an ensemble of committed progenitor cells that produce the diverse blood cells essential for life. Physiological mechanisms governing hematopoiesis, and mechanistic aberrations underlying non-malignant and malignant hematologic disorders, are often very similar in mouse and man. Thus, mouse models provide powerful systems for unraveling mechanisms that control hematopoietic stem/progenitor cell (HSPC) function in their resident microenvironments in vivo. Ex vivo systems, involving the culture of HSPCs generated in vivo, allow one to dissociate microenvironment-based and cell intrinsic mechanisms, and therefore have considerable utility. Dissecting mechanisms controlling cellular proliferation and differentiation is facilitated by the use of primary cells, since mutations and chromosome aberrations in immortalized and cancer cell lines corrupt normal mechanisms. Primary erythroid precursor cells can be expanded or differentiated in culture to yield large numbers of progeny at discrete maturation stages. We described a robust method for isolation, culture, and analysis of primary mouse erythroid precursor cells and their progeny.
多能造血干细胞分化为一群定向祖细胞,这些祖细胞产生生命所必需的各种血细胞。在小鼠和人类中,调控造血的生理机制以及非恶性和恶性血液系统疾病背后的机制异常通常非常相似。因此,小鼠模型为揭示体内控制造血干细胞/祖细胞(HSPC)在其驻留微环境中功能的机制提供了强大的系统。涉及体内产生的HSPC培养的体外系统使人们能够区分基于微环境的机制和细胞内在机制,因此具有相当大的实用性。使用原代细胞有助于剖析控制细胞增殖和分化的机制,因为永生化细胞系和癌细胞系中的突变和染色体畸变会破坏正常机制。原代红系前体细胞可以在培养中扩增或分化,以在离散的成熟阶段产生大量后代。我们描述了一种分离、培养和分析原代小鼠红系前体细胞及其后代的可靠方法。