Harada Sachio, Kimura Takafumi, Fujiki Hiroshi, Nakagawa Hitoshi, Ueda Yuji, Itoh Tsuyoshi, Yamagishi Hisakazu, Sonoda Yoshiaki
Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Osaka, Japan.
Int J Oncol. 2007 Jun;30(6):1461-8.
Current in vitro culture systems allow the generation of human dendritic progenitor cells (CFU-DCs). The aim of this study was to assess the effect of Flt3 ligand (FL) on the proliferation of human peripheral blood-derived myeloid CFU-DCs and their differentiation into more committed precursor cells (pDCs) using in vitro culture systems. Immunomagnetically separated CD34+ cells were cultured in serum-free, as well as in serum-containing, liquid suspension cultures to investigate the expansion and/or proliferation/differentiation of CFU-DCs, pDCs, and more mature dendritic cells (DCs). FACS-sorted CD34+Flt3+/- cells were cultured in methylcellulose to assay hematopoietic progenitors, including CFU-DCs. In the clonal cell culture supplemented with granulocyte/macrophage (GM) colony-stimulating factor (CSF), interleukin-4, and tumor necrosis factor alpha, the frequency of CFU-DCs was significantly higher in the CD34+Flt3+ fraction than in the CD34+Flt3- population, thus suggesting functional Flt3 expression on CFU-DCs. Serum-free suspension culture of CD34+ cells revealed the potent effect of FL on the expansion of CFU-DCs in synergy with GM-CSF and thrombopoietin (TPO). In addition, FL strongly induced the maturation of CFU-DCs into functional CD1a+ pDCs in serum-containing liquid suspension culture. Moreover, these FL-generated pDCs showed remarkable potential to differentiate into mature DCs with surface CD83/CD86 expression, which induced a distinct allogeneic T-cell response. These results clearly demonstrate that FL supports not only the proliferation of early hematopoietic progenitor cells, but also the maturation process of committed precursor cells along with the DC-lineage differentiation. Therefore, it is possible to develop a more efficient DC-based cancer immunotherapy using this specific cytokine combination, GM-CSF+TPO+FL in vitro in the near future.
目前的体外培养系统能够生成人类树突状祖细胞（CFU-DCs）。本研究的目的是利用体外培养系统评估Flt3配体（FL）对源自人外周血的髓样CFU-DCs增殖及其分化为更定向的前体细胞（pDCs）的影响。对免疫磁珠分离的CD34+细胞进行无血清以及含血清的液体悬浮培养，以研究CFU-DCs、pDCs和更成熟树突状细胞（DCs）的扩增和/或增殖/分化。对通过荧光激活细胞分选术（FACS）分选的CD34+Flt3+/-细胞进行甲基纤维素培养，以检测包括CFU-DCs在内的造血祖细胞。在补充有粒细胞/巨噬细胞（GM）集落刺激因子（CSF）、白细胞介素-4和肿瘤坏死因子α的克隆细胞培养中，CD34+Flt3+组分中CFU-DCs的频率显著高于CD34+Flt3-群体，从而表明CFU-DCs上存在功能性Flt3表达。CD34+细胞的无血清悬浮培养显示，FL与GM-CSF和血小板生成素（TPO）协同作用对CFU-DCs的扩增具有强大作用。此外，在含血清的液体悬浮培养中，FL强烈诱导CFU-DCs成熟为功能性CD1a+pDCs。而且，这些由FL生成的pDCs显示出分化为具有表面CD83/CD86表达的成熟DCs的显著潜力，后者可诱导明显的异基因T细胞反应。这些结果清楚地表明，FL不仅支持早期造血祖细胞的增殖，还支持定向前体细胞的成熟过程以及DC谱系分化。因此，在不久的将来，有可能利用这种特定的细胞因子组合GM-CSF+TPO+FL在体外开发出更有效的基于DC的癌症免疫疗法。
