不同的胞外基质对人胚胎干细胞分化为造血祖细胞的影响

Effects of Different Extracellular Matrix on Differentiation of Human Embryonic Stem Cells into Hematopoietic Progenitor Cells

本研究建立人胚胎干细胞分化为造血细胞的新诱导体系,并探讨不同的胞外基质对产生造血祖细胞的影响。选择3种胞外基质——matrigel、纤维黏连蛋白(fibronectin)和IV型胶原蛋白(collagen IV)包被培养板,采用直接贴壁培养的诱导方法,分步添加造血生长因子诱导人胚胎干细胞向造血祖细胞分化,用造血集落形成试验检测集落形成细胞数,流式细胞术以及实时定量PCR方法检测造血特异性标志物的表达,比较这3种胞外基质对生成造血祖细胞的差异。结果表明,诱导14 d时IV型胶原蛋白组形成造血集落形成细胞数目、产生CD34阳性细胞数以及造血特异性基因SCL和CD34的表达量均明显高于matrigel和纤维黏连蛋白两组(P<0.05)。结论:人胚胎干细胞在胞外基质上直接贴壁诱导能有效地分化为造血祖细胞,且IV型胶原蛋白更有利于向造血系的分化。

This study was purposed to establish a new inducing system for differentiation of human embryonic stem cells into hematopoietic progenitor cells and to explore the effect of different extracellular matrices （DEM） on production of hematopoietic cells.The 3 kinds of extracellular matrices——matrigel,fibronectin and Ⅳ type collagen （collagen Ⅳ）were chosen to package cultured plates,the direct adherent culture on extracellular mafrixs was used,and the hematopoietic growth factors were added into cultured plates to induce the differentiation of human embryonic stem cells into hematopoietic progenitor cells.The hematopoietic colony forming unit assay was used to determine the yielded colony forming cells,the flow cytometry and real-time quantitative PCR were used to detect the expression of markers specific to hematopoiesis and the effect of 3 extracellular matrices on production of hematopoietic progenior cells was compared.The results showed that after being induced for 14 days,the total yield of colony forming cells,the ratio of CD34 ＋ cells and the expression level of SCL and CD34 on collogen Ⅳ were significantly higher than those on matrigel and fibronectin groups （P ＜ 0.05）.It is concluded that human embryonic stem cells can efficiently differentiate into hematopoietic progenitor cells by direct adherent culture on extracellular matrices,and the collagen Ⅳ can improve the hematopoietic differentiation of human embryonic stem cells.