de Bruijn M F, Ploemacher R E, Mayen A E, Voerman J S, Slieker W A, van Ewijk W, Leenen P J
Department of Immunology, Erasmus University, Rotterdam, The Netherlands.
Eur J Immunol. 1996 Dec;26(12):2850-8. doi: 10.1002/eji.1830261208.
Studies on the early events in the differentiation of the nonspecific immune system require the identification and isolation of myeloid-committed progenitor cells. Using the monoclonal antibodies (mAb) ER-MP12 and ER-MP20, generated against immortalized macrophage precursors, we have shown previously that the earliest macrophage colony-stimulating factor (M-CSF)-responsive cells in the bone marrow have the ER-MP12hi 20- phenotype. In addition, we found that the ER-MP12hi 20- subset (comprising about 2 % of total nucleated marrow) contains progenitor cells of all hematopoietic lineages. Aiming at the identification and purification of the myeloid progenitor cells within the ER-MP12hi 20-subset, we used ER-MP58, a marker expressed at high level by all M-CSF-responsive bone marrow progenitors. With this marker the ER-MP12hi 20- cell population could be divided into three subfractions: one with absent or low level ER-MP58 expression, one with intermediate, and one with high ER-MP58 expression. These subfractions were isolated by fluorescence-activated cell sorting and tested in vitro and in vivo for their differentiation capacities. In addition, the expression of ER-MP58 on stem cell subsets was examined in the cobblestone area-forming cell (CAFC) assay. Our data indicate that in the ER-MP12hi 20- subpopulation myeloid-committed progenitors are characterized by high-level expression of the ER-MP58 antigen, whereas cells with other or broader differentiation capacities have an ER-MP58 negative/low or intermediate phenotype. These myeloid-committed progenitors have no significant repopulating ability in vivo, in contrast to the ER-MP58 intermediate cells. Primitive CAFC-28/35, corresponding to cells providing long-term hematopoietic engraftment in vivo, also did not express the ER-MP58 Ag at a high level. Thus, cells committed to the myeloid lineage can be separated from progenitor cells with other differentiation capacities by means of multiparameter cell sorting using ER-MP58 in combination with ER-MP12 and ER-MP20.
对非特异性免疫系统分化早期事件的研究需要鉴定和分离髓系定向祖细胞。利用针对永生化巨噬细胞前体产生的单克隆抗体（mAb）ER-MP12和ER-MP20，我们先前已表明骨髓中最早对巨噬细胞集落刺激因子（M-CSF）有反应的细胞具有ER-MP12hi 20-表型。此外，我们发现ER-MP12hi 20-亚群（约占骨髓有核细胞总数的2%）包含所有造血谱系的祖细胞。为了鉴定和纯化ER-MP12hi 20-亚群中的髓系祖细胞，我们使用了ER-MP58，这是一种在所有对M-CSF有反应的骨髓祖细胞中高表达的标志物。利用该标志物，ER-MP12hi 20-细胞群体可分为三个亚组分：一个亚组分中ER-MP58表达缺失或水平低，一个亚组分中ER-MP58表达中等，另一个亚组分中ER-MP58表达高。通过荧光激活细胞分选分离这些亚组分，并在体外和体内测试它们的分化能力。此外，在鹅卵石区形成细胞（CAFC）试验中检测了ER-MP58在干细胞亚群上的表达。我们的数据表明，在ER-MP12hi 20-亚群中，髓系定向祖细胞的特征是ER-MP58抗原高表达，而具有其他或更广泛分化能力的细胞具有ER-MP58阴性/低或中等表型。与ER-MP58中等细胞相比，这些髓系定向祖细胞在体内没有显著的再增殖能力。对应于在体内提供长期造血植入的细胞的原始CAFC-28/35也没有高水平表达ER-MP58抗原。因此，通过使用ER-MP58与ER-MP12和ER-MP20相结合的多参数细胞分选，可以将定向到髓系谱系的细胞与具有其他分化能力的祖细胞分离。
