Ramadan G, Schmidt R E, Schubert J
Department of Clinical Immunology, Hannover Medical School, Germany.
Clin Exp Immunol. 2001 Aug;125(2):237-44. doi: 10.1046/j.1365-2249.2001.01605.x.
The cytokine requirements to differentiate CD34+ progenitor cells from different origins either cord blood (CB) or peripheral blood (PB) into dendritic cells (DC) are known to be different. In addition to DC, macrophages and neutrophils are generated. On the other hand, phorbol esters such as PMA induce primary human CD34+ bone marrow (BM) progenitor cells to differentiate into functional DC and no other lineages are generated. In addition, FCS is used as culture supplement in most of the protocols described which contains additional foreign antigens potentially skewing the resulting immune response. Therefore, we evaluated the ability to differentiate CB- and PB-CD34+ progenitor cells into DC with PMA and under serum-free conditions. In this study, we delineate the maturation of cultured human blood DC by analysis of expression co-stimulatory molecule B7-2 (CD86). Human mature DC with typical morphology and surface antigen phenotype (CD1a-, CD83+ and CD86+) were obtained from CB- and PB-CD34+ progenitor cells after 1 week of culture in serum-free medium upon stimulation with PMA alone. The same result was obtained from ex vivo-expanded BM-CD34+ cells. CD86+ yield was increased by PMA compared to cytokine cocktails (28.0% +/- 7.0 versus 15.3% +/- 5.6 for CB and 44.6% +/- 7.5 versus 28.1% +/- 7.5 for PB, respectively). CD86 was most up-regulated in the presence of the calcium ionophore ionomycin. However, the number of viable cells after differentiation was decreased by PMA plus ionomycin (P < 0.05) or plus TNF-alpha (P > 0.05) as compared with that in PMA alone. We conclude that PMA is a potent activator to differentiate human CD34+ cells into mature DC in serum-free medium. This may be used for in vitro studies of primed or genetically modified DC against infectious and tumour-associated antigens.
已知将不同来源（脐带血（CB）或外周血（PB））的CD34 +祖细胞分化为树突状细胞（DC）所需的细胞因子不同。除了DC之外，还会生成巨噬细胞和中性粒细胞。另一方面，佛波酯如PMA可诱导原代人CD34 +骨髓（BM）祖细胞分化为功能性DC，且不会生成其他谱系。此外，在所述的大多数方案中都使用胎牛血清（FCS）作为培养补充剂，其中含有额外的外来抗原，可能会使产生的免疫反应发生偏差。因此，我们评估了在无血清条件下用PMA将CB -和PB - CD34 +祖细胞分化为DC的能力。在本研究中，我们通过分析共刺激分子B7 - 2（CD86）的表达来描绘培养的人血液DC的成熟过程。在用PMA单独刺激后，在无血清培养基中培养1周后，从CB -和PB - CD34 +祖细胞中获得了具有典型形态和表面抗原表型（CD1a -、CD83 +和CD86 +）的人成熟DC。从体外扩增的BM - CD34 +细胞中也获得了相同的结果。与细胞因子混合物相比，PMA可提高CD86 +的产量（CB分别为28.0%±7.0对15.3%±5.6，PB分别为44.6%±7.5对28.1%±7.5）。在存在钙离子载体离子霉素的情况下，CD86上调最为明显。然而，与单独使用PMA相比，PMA加离子霉素（P < 0.05）或加TNF -α（P > 0.05）后分化后的活细胞数量减少。我们得出结论，PMA是在无血清培养基中将人CD34 +细胞分化为成熟DC的有效激活剂。这可用于针对感染性和肿瘤相关抗原的致敏或基因改造DC的体外研究。
