小鼠骨髓间充质干细胞、骨髓单核细胞和胚胎成纤维细胞重编程为诱导性多能干细胞的效率比较

Reprogramming of mouse bone marrow mesenchymal stem cells,mouse bone marrow mononuclear cells and mouse embryonic fibroblasts into induced pluripotent stem cells:a comparison of efficiencies

目的比较小鼠骨髓间充质干细胞(BMSCs)、小鼠骨髓单核细胞(BM-MNCs)和小鼠胚胎成纤维细胞(MEFs)重编程为诱导性多能干细胞(iPS细胞)的效率。方法用慢病毒LV-ef1a-mOct4-IRES-EGFP、LV-ef1amSox2-IRES-EGFP、LV-ef1a-mKlf4-IRES-EGFP和LV-ef1a-mc-Myc-IRES-EGFP感染BMSCs、BMMNCs和MEFs,通过计算碱性磷酸酶染色阳性克隆数比较这3种细胞重编程为iPS细胞的效率。用胚胎干细胞表面标记检测、胚胎干细胞内源基因检测、拟胚体形成实验和畸胎瘤形成实验验证重编程获得的iPS细胞的多能性。结果起源于小鼠BMSCs、BM-MNCs及MEFs的3种iPS细胞均能形成边缘光整的致密克隆,可表达干性基因Nanog、Rex-1、SSEA-1,并能在体内外分化为三胚层组织。但是BMSCs来源的碱性磷酸酶阳性克隆数低于BM-MNCs和MEFs来源的克隆数。结论小鼠BMSCs、BM-MNCs及MEFs均可重编程为iPS细胞,但BMSCs重编程为iPS细胞的效率低于BM-MNCs和MEFs。

Objective To compare the reprogramming efficiencies of mouse bone marrow mesenchymal stem cells （BMSCs）, mouse bone marrow mononuclear cells （BM-MNCs） and mouse embryonic fibroblasts （MEFs） into induced pluripotent stem cells （iPS cells）. Methods BMSCs, BM MNCs and MEFs were infected with lentivirus （LV-efla-mOct4- IRES-EGFP, LV-efla mSox2 IRES-EGFP, LV-eIla-mKl f4-IRES-EGFP and LV-efla mc-Myc IRE-EGFP） at a multiplicity of infection. Reprogramming efficiencies of BMSCs, BM-MNCs and MEFs were compared by counting the number of alkaline phosphatase （AP） positive clones. Pluripotency of the clones was evaluated by detecting the expression of embryonic stem cells markers and assessing their ability to form embryoid bodies and teratomas. Results iPS cells derived from BMSCs, BM-MNCs and MEFs were all able to grow into clones with clear borders, to express enbryonic stem cell specific cell surface markers （Nanog, Rea-1 and SSEA-1）, and to express characteristic genes of all three germ layers both in vitro and vivo. The AP positive clones derived from BMSCs were notably less than those from BM MNCs and MEFs. Conclusion BMSCs, BM MNCs and MEFs can all reprogram into iPS cells, but the reprogramming efficiency of BMSCs in adherent culture is lower than those of BM MNCs and MEFs.