拟胚体直接诱导胚胎干细胞向树突状细胞的分化

Differentiation of ES-D3 embryonic stem cells into dendritic cells in vitro

直接从ES-D3胚胎干细胞(ESC)诱导分化出胚胎干细胞源性树突状细胞(esDC)。ESC单细胞悬液在低黏附性培养皿中悬浮培养形成拟胚体(EB)。取第14天的EB用含重组鼠粒-巨噬细胞集落刺激因子(rmGM-CSF)和重组鼠白细胞介素3(rmIL-3)的培养基在高黏附性组织培养瓶中培养10 d,消化细胞后传代培养。运用流式细胞仪检测细胞表面CD11c、MHCⅠ、MHCⅡ、CD86、CD80、CD83的表达情况。同时,运用脂多糖(LPS)刺激诱导细胞,观察其细胞形态学及MHCⅡ、CD80分子表达变化,并利用混合淋巴细胞培养观察其对同种淋巴细胞的刺激能力。结果显示在rmGM-CSF和rmIL-3细胞因子的作用下,EB在培养第4天开始出现大量早期的esDC,在接下来的培养中细胞大量扩增,常规消化后细胞保持很高的增殖活性并可传代培养。这些细胞表达高量的CD11c、低量的MHCⅠ,而不表达CD86、CD80、CD83及MHCⅡ。但当用LPS刺激作用后,该类细胞表现出成熟DC的细胞形态并上调MHCⅡ、CD80分子,并产生对同种淋巴细胞强烈的刺激作用(P<0.01)。因而,运用EB联合细胞因子rmGM-CSF和rmIL-3能从ESC中直接诱导分化出esDC。

In order to differentiate directly the ES-D3 embryonic stem cells（ESC）into dendritic cells, single ESC suspensions were plated onto bacteriological plastic to encourage the formation of EBs, and after 14 days in culture, EBs were plated onto tissue culture grade plastic in complete medium supplemented with 25 ng/ml of murine GM-CSF and 10 ng/ml of IL-3 and cul- tured for 10 days. The expressions of MHC Ⅰ and Ⅱ molecules, CDllc, CD80, CD86 and CD83 on the surface of the differ- entiated cells were evaluated by flow cytometry （FCM）. Meanwhile, LPS was used to induce maturation of these cells, and then the morphological changes and expressions of MHC Ⅱ molecule and CD80 of these cells were investigated. Also, the mixed leukocyte reactions （MLR） was used to study the allogeneic immunostimulatory function of matured or immature DCs derived from embryonic stem cells （esDCs）. By cultivation with IL-3 and GM-CSF, the early esDCs first appeared around days 4～5 of culture and rapidly expanded over the ensuing days. This population consistently regenerated following routine harvesting. Moderate expression of MHC Ⅰ molecule and high expression of CD11c could be demonstrated on these cells; but there was no expression of CD80, CD86, CD83 or MHC Ⅱ molecules, indicating that they were all immature DCs. However, when stimulated with LPS, these cells presented the morphology of matured DCs and could up-regulate the expressions of MHC Ⅱ and CD80 molecules on cell surface. Result of allogeneic MLR suggested that the mature esDCs showed a significant immunos- timulator activity （P〈0. 05）, but the immature esDCs did not show this activity（P〉0.05）. It is evident that the dendritic cells derived from the embryonic stem cells was successfully acquired though the direct differentiation ES-D3 embryonic stem cells into DCs by use of cytokine GM-CSF and IL-3.