人多能干细胞诱导类肾的实验研究

An experimental study on kidney organoids induced by human pluripotent stem cells

目的:体外诱导人多能干细胞(human pluripotent stem cell,hPSC)来源的类肾体,探讨类肾的结构成熟度、血管化和非肾系分化现象。方法:通过肾上皮分化试剂盒诱导hPSC成类肾,用免疫荧光观察类肾元结构中特异性标志物CDH1、LTL和PODX的表达,透射电镜(transmission electron microscopy,TEM)观察肾小球和肾小管微观结构;第15天类肾移植到SCID小鼠肾包膜下,4周后处死动物,取移植物行组织形态学观察,免疫荧光法观察移植物中人抗核抗原(human nuclear antigen,HNA)、CDH1、PODX和MECA-32的表达。结果:肾脏特异性标志物CDH1、LTL和PODX呈阳性表达,电镜观察到不成熟的足细胞和肾小管结构;移植后的类肾体积增大,观察到肉眼可见的血管,HE染色移植物内可见红细胞和类软骨组织,HNA、CDH1、PODX和MECA-32呈阳性表达。结论:hPSC可体外诱导结构不成熟的类肾,在体内环境下,类肾可完成血管化并持续生长,但存在大量非肾系结构。

Objective:To investigate the maturity,vascularization and nonrenal differentiation of kidney organoids using induced renal organoids derived human pluripotent stem cells(h PSCs). Methods:hPSCs were induced into renal organoids cells through a renal epithelial cell differentiation kit. The renal organoids structures were highly expressed with specific kidney markers CDH1,LTL,and PODXl by immunofluorescence method. The microstructures of glomeruli and tubules were observed by transmission electron microscopy;The animals were sacrificed under the renal capsule of SCID mice for 4 weeks,the grafts were taken for histomorphological observation,and the expressions of HNA,CDH1,PODX and MECA-32 were observed by immunofluorescence method. Results:The kidney markers CDH1,LTL,and PODX were positively expressed. Immature podocytes and tubular structures were observed by transmission electron microscopy. The kidney organoid volume increased after transplantation,blood vessels were visible to the naked eye,and HE stained grafts were visible red blood cells and cartilage-like tissues,HNA,CDH1,PODX and MECA-32 were positively expressed. Conclusion:hPSC can induce immature kidney organoids structures in vitro. In vivo,renal orgaoids structures can complete vascularization and continue to grow,but there were a large number of non-renal structures.