Gupta Dipti, Shah Hetavi Parag, Malu Krishnakumar, Berliner Nancy, Gaines Peter
Department of Biological Sciences, University of Massachusetts Lowell, Lowell, Massachusetts.
Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
Curr Protoc Immunol. 2014 Feb 4;104:22F.5.1-22F.5.28. doi: 10.1002/0471142735.im22f05s104.
Ex vivo differentiation of myeloid cells begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34(+) antigen (human) or Sca-1(+) (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid-induced myeloid development; however, both cell lines exhibit defects in the up-regulation of late-expressed neutrophil-specific genes. Multiple murine factor-dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation; EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid; and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradiol in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR.
髓系细胞的体外分化始于通过谱系清除和/或对CD34(+)抗原（人类）或Sca-1(+)（小鼠）细胞进行阳性选择产生的富集骨髓来源造血干细胞群体，然后将其扩增并随后在体外诱导，该过程模拟正常髓系发育。髓系细胞系包括两个人类白血病细胞系，NB-4和HL-60，它们已被证明可经历视黄酸诱导的髓系发育；然而，这两个细胞系在晚期表达的中性粒细胞特异性基因上调方面均表现出缺陷。也有多种小鼠因子依赖性骨髓生成细胞模型，它们表达全套中性粒细胞成熟标志物，包括：32Dcl3细胞，其经历G-CSF诱导的髓系成熟；EML/EPRO细胞，其在细胞因子和视黄酸作用下发育为成熟中性粒细胞；以及ER-Hoxb8细胞，其在维持培养基中去除雌二醇后经历髓系成熟。在本单元中，描述了这些模型系统中每个系统的髓系成熟诱导过程，包括在适用情况下它们向中性粒细胞或巨噬细胞的分化。还描述了用于测试发育中细胞髓系特征的常用技术，包括流式细胞术和实时RT-PCR。
