猪诱导多能干细胞的制备和无饲养层培养

Preparation of feeder-free porcine induced pluripotent stem cells

目的建立猪诱导多能干细胞（piPSCs）系，优化培养条件。方法猪皮肤成纤维细胞（PF）经慢病毒介导表达人多能性因子Oct4（hOct4）、人性别决定区Y框蛋白2（hSox2）、人Kruppel样因子4（hKlf4）和he—Myc，诱导成为piPSCs，挑选克隆培养于piPSCs培养基中，观察piPSCs生长状态；免疫荧光法、逆转录聚合酶链反应（RT-PCR）分别检测piPSCs多能性基因表达；观察拟胚体（EB）、畸胎瘤形成能力。结果piPSCs呈典型的克隆状生长，克隆呈圆形或椭圆形，边界清晰。克隆内细胞排列紧密。细胞体积小，核大，细胞核／质比高；piPSCs高表达人多能性基因，以转染2d后基因表达水平为标准值1，hOct4、hc-Myc、hSox2、hKlf4的相对表达水平分别为1．14、0．89、0．74和0．86，而PF几乎不表达这些人源性基因；piPSCs也高表达猪多能性标记Sox2（pSox2）、pNanog和pLin28（以转染2d后基因表达水平为标准值1，相对表达水平分别为29．62、2．81、7．46）；piPSCs还表达碱性磷酸酶（ALP）活性；体外悬浮培养能形成EB；接种体内能形成畸胎瘤。结论成功获得piPSCs系，能稳定增殖，保持自我更新及多向分化潜能。

Objective To establish a stable culture system for porcine induced pluripotent stem cells （piPSCs）. Methods Pig fibroblasts were seeded on Matrigel-coated plate, and then infected with lentivirus expressing human Oct4 （hOct4） , human kruppel-like factor 4 （hKlf4） , human sex determining region Y-box 2 （hSox2） and hc-Myc. Seven to ten days later, piPSCs clones were picked up under invert microscope and cultured in KnockoutTM DMEM medium supplemented with 20% KnockoutTM serum replace- ment. piPSCs clones were detached and dissociated by Accutase digestion. Total mRNA was extracted and pluripotent-associated genes expression of hOot4, hKlf4, hSox2 and hc-Mye and pig Sox2 （pSox2）, pNanog and pLin28 was detected by using reverse transcriptase-polymerase chain reaction （RT-PCR）. Quantification was performed using the comparative Ct method normalized with the [5-actin. The expression of each target gene at each stage was normalized to its level in PF two days after transduction. The immun- oflurescence analysis of pluripotency marker （hOct4） and alkaline phosphatase （ALP） activity was also conducted. Results piPSCs were maintained on Matrigel and formed condensed clones with clear borders. Cells in clones had high nucleus/cytoplasm ratio and predominant nuclei. All piPSCs strongly expressed endogenous pOct4, pNanog and pSox2, and the relative expression level was 29. 62, 2. 81 and 7.46, re- spectively, piPSCs also expressed exogenous hOct4, hc-Myc, hSox2, hKlf4, and the relative expression level was 1.14, 0. 89, 0. 74 and 0. 86, respectively, piPSCs also expressed ALP activity. All these data suggest the pluripotent state of piPSCs, piPSCs formed embryoid bodies in vitro and teratoma in vivo, indi- cating the piPSCs proliferated stably and kept high self-renewal and differentiation potency. Conclusion The culture system is suitable for making long-term subculture of piPSCs.