Liu Aichun, Takahashi Masuhiro, Narita Miwako, Zheng Zhiyin, Kanazawa Naoko, Abe Takashi, Nikkuni Kohji, Furukawa Tatsuo, Toba Ken, Fuse Ichiro, Aizawa Yoshifusa
First Department of Internal Medicine, Faculty of Medicine, Niigata University, Niigata, Japan.
J Immunol Methods. 2002 Mar 1;261(1-2):49-63. doi: 10.1016/s0022-1759(01)00545-2.
The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.
本研究的目的是探索一种培养方法，以从人CD34+造血祖细胞中大量生成功能性成熟树突状细胞（DC）。在本研究中，我们采用两步法并结合钙离子载体，从脐血（CB）或正常人骨髓（BM）CD34+祖细胞诱导生成DC。两步法包括第一步在SCF、IL-3、IL-6、G-CSF存在下培养10天，用于CD34+造血祖细胞的扩增和增殖；第二步在GM-CSF、IL-4和TNF-α存在下培养7-11天，用于诱导DC。通过两步培养，与培养开始时的CD34+细胞数量相比，CB或BM细胞培养物中的总核细胞分别增加了208±66（±SD，n = 13）倍或94±29（n = 5）倍。在总核细胞中，CB细胞培养物中有23±10.4%的细胞以及BM细胞培养物中有25±5%的细胞获得了DC特征性表型，其特征为CD1a、HLA-DR的显著表达，共刺激分子如CD80、CD40以及黏附分子如CD58的表达。在同种异体混合淋巴细胞反应（MLR）中，两步培养的细胞显示出强大的同种异体刺激能力。与使用SCF、GM-CSF和TNF-α从CD34+细胞生成DC的常用一步培养法相比，通过这种两步培养，共表达HLA-DR、CD80、CD40和CD58的CD1a+细胞的绝对数量在CB细胞培养物中增加了约3倍，在BM细胞培养物中增加了1.9倍。然而，在两步培养生成的这些DC上，共刺激分子CD86和成熟DC标志物CD83的表达并不充分。通过用钙离子载体试剂（A23187）处理两步培养的细胞，共刺激分子如CD86和CD80（尤其是CD86）的表达上调。此外，通过短时间（24小时）用A23187处理可显著诱导成熟DC标志物CD83的表达。与表面分子CD86、CD80和CD83的上调一致，用A23187处理的两步培养细胞与未用A23187处理的细胞相比，也显示出更强的同种异体刺激能力。总之，本研究表明两步培养法有效地提高了从CD34+细胞生成的CD1a+ DC的产量，并且通过用钙离子载体试剂处理可有效增强这些CD1a+ DC的表型和功能。
