Transwell 接触共培养促进单散iPSCs 生长及分化

Transwell contact co-culture promotes growth and differentiation of single-dissociated iPSCs

目的：观察Transwell接触共培养促进单散人诱导多能干细胞（inducedpluripotentstemcells， iPSCs）生长及分化的作用。方法：将1～2代牛角膜内皮细胞（corneal endothelial cells， CECs）接种在Transwell 小室底面培养8 h后，应用Accutase消化及40μm过滤处理获得单散iPSCs，将其接种到已有CECs的Transwell小室内共培养14 d，前3 d使用mTeSR1培养基，第4天开始用含10％胎牛血清的低糖DMEM培养基。分别进行实时荧光定量聚合酶链式反应（ real-time fluorescence quantitative polymerase chain reaction ， qPCR ）、免疫荧光、死活细胞染色及碱性磷酸酶（ alkaline phosphatase ， ALP）染色，对iPSCs多能特性表达及分化进行鉴定。设定单散iPSCs共培养组为实验组，常规培养iPSCs组为对照组（一），非共培养单散iPSCs组为对照组（二）。结果：培养牛CECs形态呈典型的六边形铺路石样外观。人iPSCs呈克隆样生长，共培养3 d后iPSCs贴壁呈单散细胞生长，免疫荧光检测未分化标志Nanog和Oct4呈阳性。 qPCR检测Nanog、Oct4和Sox2 mRNA表达，实验组与对照组（一）比较差异无统计学意义（P＞0.05）。死活细胞染色显示，实验组死细胞明显减少，与对照组（二）比较差异有统计学意义（P＜0.01）。共培养14 d后，人iPSCs形态比较均一，呈多边形，体积增大，无明显克隆团块；ALP染色阴性；免疫荧光染色ZO-1、AQP1和CD31表达阳性，CD34和CD133表达阴性。 qPCR检测Oct4、Nanog和Sox2 mRNA表达明显下调，与对照组（一）比较差异有统计学意义（P＜0．01）。结论：与牛CECs共培养可增强人单散iPSCs活性，使iPSCs形态上向内皮样细胞转化，表达部分CECs的标志。 Transwell接触共培养模型可以促进单散iPSCs生长及分化。

AIM：ToinvestigatethepromotingroleofTranswellcontactco-culturesysteminthegrowthand differentiation of single-dissociated induced pluripotent stem cells （iPSCs）.METHODS：Bovine corneal endothelial cells （CECs） at passage 1~2 （P1~2） were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃and 5%CO2 for 8 h.Accutase digestion and 40μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs , and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR 1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks.The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction （ qPCR ） , immunofluorescence staining, live＆amp;dead cell staining and alkaline phosphatase （ALP） staining.The group of iPSCs cultured in conventional medium was used as control group 1.The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2.RESULTS： The bovine CECs showed typical hexagonal cobblestone shape .iPSCs showed colony-like growth , while became single-dissociated cells after Tran-swell contact co-culture with bovine CECs for 3 d.The single-dissociated iPSCs positively expressed the undifferentiated＆amp;nbsp;markers, Nanog and Oct4.The mRNA expression levels of Nanog , Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference （P＆gt;0.05）.The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 （P＆lt;0.01）.After 14 d of induced differentiation co-culture , the single-dissociated iPSCs showed rather uniform polygonal morphology , increased dimension and no obvious colony existence .Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed.The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased , and had statistically significant difference compared with control group 1 （P＆lt;0.01）.CONCLUSION： When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs .Transwell contact co-culture system not only enhances the growth of single-dis-sociated iPSCs , but also promotes their differentiation .