体外诱导人类诱导性多能干细胞分化为造血干/祖细胞的研究

Differentiation of In Vitro Induced Human Pluripotent Stem Cells into Hematopoietic Stem/Progenitor Cells

本研究探讨体外诱导人诱导性多能干细胞(induced pluripotent stem cell,iPSC)分化为造血干/祖细胞的能力。在体外用小鼠骨髓基质细胞OP9与人类iPSC共培养的方法,将iPSC诱导分化为造血干/祖细胞;用流式细胞术检测造血干/祖细胞表面标志物的表达水平;用实时定量PCR检测分化过程中iPSC及造血干/祖细胞的相关基因mRNA表达水平的变化;用免疫磁珠法分离CD34+造血干/祖细胞并进行半固体集落形成实验检测细胞的集落形成能力。结果表明,iPSC与OP9细胞共培养诱导造血分化的第4天即可观察到iPSC形态变化;流式细胞术检测显示,分化得到的细胞表达已知的造血干/祖细胞相关表面标志物CD34和CD43分子。在体外分化过程中多能性的标志基因Oct4的表达逐渐下降,造血相关转录因子Gata-2的表达逐渐升高,而Runx-1的表达量则呈波浪式变化,CD34表达量逐渐升高。集落培养14 d能够得到红系集落(CFU-E),粒系集落(CFU-G),巨核系集落(CFU-M),粒-巨核系集落(CFU-GM)和混合系集落(CFU-GEMM)。结论:iPSC细胞能够在体外通过与OP9细胞共培养分化为造血干/祖细胞。

This study was aimed to explore the differentiation of in vitro induced human pluripotent stem cells （iPSC）into hematopoietic stem progenitor cells.The human iPSC were induced to differentiate into hematopoietic stem/progenitor cell by co-culturing with OP9 bone marrow stromal cells.The expression of hematopoietic stem/progenitor cell surface markers were detected by flow cytometry.The regulation gene expressions of iPSC and hematopoietic stem/ progenitor cells were measured by real-time PCR.The CD34 ＋ hematopoietic stem/progenitor cells were isolated by using immunomagnetic beads,and were used for colony formation assay.The results showed that after iPSC were cocultured with OP9 cells for 4 days,the morphological changes of iPSC could be observed.Hematopoietic stem/progenitor cell surface markers CD34 and CD43 could be detected by flow cytometry after differentiation.The pluripotent marker gene OCT4 expression gradually decreased and blood-related transcription factor Gata-2 expression gradually increased,while Runx-1 expression was wavily changed,CD34 expression gradually increased.The erythroid colony （CFU-E）,granulocyte colony（CFU-G）,megakaryocytic colony（CFU-M）,granulocyte-megakaryocytic colony（CFU-GM）,and mixed colony（CFU-GEMM） were obtained after cultures for 14 d.It is concluded that the human iPSC cells can be induced to differentiase into hematopoietic stem/progenitor cells in vitro by co-culture with OP9 ceils.