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目的探讨采用透明质酸材料的三维培养方法将人胚胎干细胞(h ESCs)来源造血干/祖细胞向红系细胞诱导分化的效果。方法将hESCs与小鼠主动脉-性腺-中肾基质细胞(AGM-S3)共培养(37℃、5%CO_2)12 d的细胞(1.0×10~6)分别种植进海藻酸钠水凝胶、胶原蛋白水凝胶、透明质酸水凝胶内,并以悬浮法培养h ESCs作对照。以流式细胞仪检测细胞表面抗原表达,比较不同材料建立的三维培养的优劣,优化红系诱导分化的三维培养体系。结果海藻酸钠组内细胞培养10 d的GPA~+细胞为2.3%,GPA~+CD36+细胞仅为0.9%而悬浮培养分别为15.8%和6.1%。胶原蛋白组内细胞培养10 d的GPA~+细胞为11.1%,GPA~+CD36+细胞为5.2%,而悬浮培养分别为35.7%和27.7%。透明质酸组内红系细胞GPA~+细胞量和比率分别为2.06×105个和19.6%,而悬浮培养的红系细胞GPA~+细胞量和比率分别为1.62×10~5个和15.7%,透明质酸组是悬浮培养的1.27倍,其中悬浮培养GPA~+CD36-为9.7%,而透明质酸水凝胶为GPA~+CD36-为13.1%。结论与悬浮培养相比,海藻酸钠水凝胶和胶原蛋白水凝胶未表现出明显的扩增红系细胞的效果。透明质酸凝胶建立的三维培养方法利于红系细胞生长,该培养方法诱导分化hESCs得到的红系细胞成熟度提高。
Objective To investigate the effect of three-dimensional culture of hyaluronic acid on differentiation of hematopoietic stem / progenitor cells derived from human embryonic stem cells (h ESCs) into erythroid cells. Methods Cells (1.0 × 10 ~ 6) co-cultured with mouse aorta-gonadal-mesangial cells (AGM-S3) for 12 d at 37 ℃ were cultured in sodium alginate hydrogel , Collagen hydrogel, hyaluronic acid hydrogel, and cultured h ESCs as a control by suspension method. The expression of cell surface antigens was detected by flow cytometry. The advantages and disadvantages of three-dimensional culture established by different materials were compared, and the three-dimensional culture system of erythroid differentiation was optimized. Results In the sodium alginate group, GPA ~ + cells were 2.3%, GPA ~ + CD36 + cells were only 0.9% and suspension culture were 15.8% and 6.1% respectively. In the collagen group, GPA ~ + cells were 11.1% and GPA ~ + CD36 + cells were 5.2% and 10%, respectively, while those cultured in suspension were 35.7% and 27.7% respectively. The amount and percentage of GPA ~ + cells in hyaluronic acid group were 2.06 × 105 and 19.6%, respectively. The amount and percentage of GPA ~ + cells in erythrocytes were 1.62 × 10 ~ 5 and 15.7% , Hyaluronic acid group was 1.27 times that of suspension culture, which suspension culture GPA ~ + CD36- 9.7%, while hyaluronic acid hydrogel GPA ~ + CD36- 13.1%. CONCLUSIONS Sodium alginate hydrogel and collagen hydrogel did not show significant erythroblastic expansion compared to suspension culture. The three-dimensional culture method established by hyaluronic acid gel is conducive to the growth of erythroid cells, and the culture method can improve the maturation of erythroid cells induced by differentiating hESCs.