Berthier R, Valiron O, Schweitzer A, Marguerie G
Laboratoire d'Hématologie, INSERM U 217, Département de Biologie Moléculaire et Structurale, CEN Grenoble, France.
Stem Cells. 1993 Mar;11(2):120-9. doi: 10.1002/stem.5530110207.
The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition. The megakaryocytes were identified by using an immunocytochemical procedure after labeling with an anti-GPIIb-IIIa monoclonal antibody. High percentages (15 to 20%) of megakaryocytes were present in serum-free cultures stimulated by IL-3 alone or combined with IL-6. The absolute number of megakaryocytes in serum-free medium exceeds by 3.3 (IL-3 plus IL-6) to 4.4 (IL-3 alone) times the corresponding number of megakaryocytes observed in human plasma supplemented cultures. The optimal concentration of IL-3 alone was 5 ng/ml, and an optimal synergistic effect of IL-6 (5 ng/ml) was obtained when combined with a suboptimal dose of IL-3 (1 ng/ml). The poor growth of megakaryocyte colonies from CD34+ BM cells in human plasma suggested the presence of an inhibitory factor. When a neutralizing monoclonal antibody against transforming growth factor beta (TGF beta) is present in human plasma supplemented cultures of CD34+ BM cells, the number of megakaryocyte colonies is increased to the level observed in corresponding serum-free cultures. The high efficiency of this serum-free medium to promote the growth of human megakaryocytes will be useful to study the effects of regulators and platelet agonists acting on human megakaryocytes, without interference from factors in the serum.
使用添加正常人血浆或无血清培养基的甲基纤维素半固体测定法，比较了源自人骨髓（BM）细胞的人巨核细胞祖细胞的生长情况。与添加人血浆的培养物相比，在无血清培养基中观察到，白细胞介素3（IL-3）和白细胞介素6（IL-6）刺激的CD34+ BM细胞产生的巨核细胞集落生长得更好。使用相同的无血清培养基成分在液体培养中证实了这些结果。在用抗GPIIb-IIIa单克隆抗体标记后，通过免疫细胞化学程序鉴定巨核细胞。单独用IL-3或与IL-6联合刺激的无血清培养物中存在高百分比（15%至20%）的巨核细胞。无血清培养基中巨核细胞的绝对数量比添加人血浆的培养物中观察到的相应巨核细胞数量多出3.3倍（IL-3加IL-6）至4.4倍（单独IL-3）。单独IL-3的最佳浓度为5 ng/ml，与次最佳剂量的IL-3（1 ng/ml）联合使用时，可获得IL-6（5 ng/ml）的最佳协同效应。人血浆中CD34+ BM细胞的巨核细胞集落生长不佳表明存在抑制因子。当在添加人血浆的CD34+ BM细胞培养物中存在抗转化生长因子β（TGFβ）的中和单克隆抗体时，巨核细胞集落的数量增加到相应无血清培养物中观察到的水平。这种无血清培养基促进人巨核细胞生长的高效率将有助于研究作用于人巨核细胞的调节剂和血小板激动剂的作用，而不受血清中因子的干扰。
