两步法分离和培养人羊水来源胚胎间充质干细胞的生物学特征

背景:目前对干细胞的体外分离纯化技术大多是建立在对细胞表面标记识别的基础之上,包括单抗贴壁铺展法、流式细胞分选法、免疫磁珠分选法等,但操作复杂,价格昂贵.目的:观察两步法分离和培养人羊水来源胚胎间充质干细胞的生物学特征.设计、时间及地点:开放性实验,于2008-03/2009-03在新疆医科大学干细胞研究室完成.材料:10例羊水标本来自怀孕16～22周行产前诊断或自愿引产的孕妇,在超声引导下行羊膜腔穿刺取得20～40 mL羊水.方法:采用改进的两步培养法分离和培养人羊水间充质干细胞,首先同一步法将羊水标本离心、接种、培养至出现梭形细胞为主、类成纤维细胞的间充质干细胞集落.然后将上清培养液转移至新的25 cm~2培养瓶中继续培养,至出现梭形细胞为主、类成纤维细胞的间充质干细胞集落且达70%汇合后消化,改用α-MEM培养基,同样添加碱性成纤维细胞生长因子,接种培养,记为第1代.主要观察指标:①原代及传代羊水间充质干细胞形态学变化.②羊水间充质干细胞细胞核型、细胞周期、生长曲线和集落形成能力测定.③流式细胞仪、免疫荧光和RT-PCR检测表面抗原和细胞因子.结果:实验成功分离、培养和传代人羊水间充质干细胞.①孕中期羊水细胞原代培养平均7 d可见散在的梭形细胞为主、类成纤维细胞的间充质干细胞集落;传代细胞在12 h内完全贴壁,经过一两天潜伏期后增殖迅速,培养六七天可达90%融合,细胞排列有一定的方向性,呈漩涡状、辐射状排列,细胞为长梭形,相互之间界限不清.②两种方法得到的羊水间充质干细胞染色体核型为正常二倍体核型;生长曲线均呈S形,但两步法在第(6.1±0.5)天就达到高峰,显著快于一步法第(7.2±0.6)天(P=0.035).流式细胞仪检测结果显示两步法羊水P3细胞S期细胞占(14±2.3)%,显著多于一步法(9.0±1.4)%(P=0.031).两种方法获得的羊水间充质干细胞低密度种植7 d后,均可形成散在的细胞集落,但两步法集落形成率为(15.0±2.3)%,显著多于一步法(10.0±1.8)%(P=0.021).③流式细胞仪检测结果显示,羊水间充质干细胞表达CD44、CD29、CD105,而CD34、CD45、HLA-DR阴性;免疫荧光检测表明羊水中含有Oct-4阳性细胞,但两步法羊水间充质干细胞中Oct-4阳性细胞比例为(1.2±0.3)%,显著高于一步法(0.9±0.2)%(P=0.041);RT-PCR分析表明两种方法获得的羊水间充质干细胞均表达Oct-4.结论:人羊水中也存在间充质干细胞,两步培养法是一种高效、简便实用而且不干扰常规产前诊断程序的方法.

Biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells isolated and cultured using the two-step method

BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.