Construction And Vascularization Of Renal Organoids With Human Pluripotent Stem Cells

With the increasing incidence of chronic renal insufficiency,end-stage renal disease(ESRD)has become a worldwide social problem,which has a serious impact on public health.According to statistics,there are more than 1 million end-stage renal disease patients in China whose main treatment methods are hemodialysis and kidney transplantation.Despite recent technological advances,the mortality rate of hemodialysis patients remains high.However,the shortage of kidney source for kidneytransplantation is a difficult problem,and there is immune rejection inkidney transplantation,which needs lifelong treatment.In order to break through the limitations of renal replacement therapy,renal regeneration has become a research hotspot.With the development of stem cell technology and regenerative medicine,the application of stem cells in renal regeneration has brought new hope for patients with end-stage renal disease.kidney organoids,as one of the research directions of kidney regeneration,has gained extensive attention in recent years.It mimics the kidney development process in vivo.Stem cells and other seed cells go through various stages of differentiation from original strips to renal progenitor cells to construct 3D cell mass organization by control of Wnt and other signaling pathways.However,existing renal organoids generally lack vascular perfusion system,immature structure and other problems,which limit the application in the field of regeneration.Thus,the vascularization of renal organoids is an important step to break through these limitations.In this study,human pluripotent stem cells(hPSCs)were used as seed cells to construct a nephroid organ.The renal organoids with a higher degree of vascularization and a more mature structure was obtained via in vivo transplantation of the nephroid organ and in vitro vascularization.Part Ⅰ Vascularization and maturation of nephrons by implanting hPSC-derived kidney progenitors under kidney capsules of unilaterally nephrectomized micePurposeTo explore whether transplantation of Human pluripotent stem cells(hPSCs)-derived renal organoidsunder kidney capsules of unilaterally nephrectomized micecould improve vascularization and structural maturityMethods1.HPSCs were used as seed cells to induce kidney formation by renal epithelial differentiation kit.HE staining was used to observe the tubular structure of the kidney.Immunofluorescence was used to detect the expression of podocyte specific markers WT1 and PODXL,proximal tubule-specific marker LTL and distal tubule-specific marker CDH1 in the kidney.The ultrastructure of podocytes and renal tubules was observed by transmission electron microscopy(TEM).2.A single nephrectomy mouse model was established,and the transplantation of D15 organoids.SCID mice of 4-6 weeks were selected,and the left kidney was removed.D15 organoids were transplanted under the right renal capsule of mice.3.2 to 4 weeks after transplantation,the right kidney of mice and organoids specimens were collected for macroscopic observation,HE staining,immunofluorescence identification and TEM detection.(1)Macroscopic observation of the size and the distribution of renal organoids vessels.(2)HE staining was used to observe the glomerular,tubular,vascularized and non-renal structures of the kidney organoids.(3)Podocyte marker WT1,distal tubule marker CDH1,specific mouse endothelial cell marker MECA-32,endothelial cell marker CD31,human antinuclear antigen HNA and differentiation potential marker NANOG were detected by immunofluorescence.(4)The ultrastructure of glomeruli,renal tubules,blood vessels and red blood cells were examined by TEM.4.12 weeks of renal grafts were collected for macroscopic observation,HE and Masson staining,and immunofluorescence detection.(1)Macroscopic observation of the size and vascularization of the transplanted 12 weeks kidney(2)HE and Masson staining were used to observe the glomerular,tubular and non-renal structures of the grafts.(3)The expression of renal tubular marker CDH1 and podocyte marker WT1 was detected by Immunofluorescence.Results1.HE staining showed a large number of tubular structures in the in vitro induced renal organoids.Immunofluorescence showed that kidney specific markers WT1,PODXL,LTL and CDH1 were positive in renal organoids.TEM examination revealed the immature podocytes and tubular structures resembling kidneys.2.Identification of renal ornanoids after 2-4 weeks in vivo transplantation(1)After 2-4weeks in vivo transplantation,the size of the organoids were increased and several small blood vessels could be observed on the graft surface.(2)HE staining showed glomerulus-like structures,tubular structures and a large number of chondroid structures。A large number of red blood cells were observed in glomeruli and kidney-like matrix after 2-4weeks of renal organoids transplantation.With the prolongation of transplantation time,the renal vascularization became more abundant,and the chondroid components also increased.(3)Four weeks after the renal organoids transplantation,immunofluorescence assay showed that WT1 was positively expressed in the glomerular structure,CDH1 in the renal tubular structure,and CD31 in vascular endothelial cells.Meca-32 positive cells were scattered in the graft tissue and glomerular structure,suggesting that the vascular endothelial cells of the graft might be partially derived from the host.The positive expression of human specific antibody HNA in the graft confirmed that the graft was HPSC origin.The positive expression of the graft stemness marker NANOG indicated that the graft still has differentiation ability 4w after transplantation.(4)4 weeks after renal organoids transplantation,TEM showed evidence of renal organ maturation,including presence of GBM,porous endothelium,podocyte hiatus and capillaries.The ultrastructure of mature renal tubules was also revealed by TEM,including the presence of tight junctions of monolayer epithelial cells,peritubular capillary network,polarization of open tubular lumen and epithelial cells,and mitochondria and microvilli of renal tubular epithelial cells.3.Identification of renal organoids after 12 weeks in vivo transplantation(1)After 12 weeks of renal organoidstransplantation,the graft size continued to increase compared with 2-4 weeks transplantation,and the vascular structure could still be observed on the graft surface.(2)HE staining and Masson staining showed the disappearance of cartilage structures,the presence of a large number of mesenchymal tissues,the disappearance of glomerular structures,and only scattered tubular structures were observed.(3)Immunofluorescence showed that CDH1 was positively expressed in the scattered tubular structures.While,WT1 was not positively expressed in the grafts.ConclusionHPSCs can be used as seed cells to induce immature kidney organoids structures in vitro.In the in vivo environment,vascularization of the renal organoids was promoted by chimeric host vessels,thus,could improve the graft development and maturation.However,long-term transplantation will lead to the replacement of nephron tissue by non-renal tissue.Part Ⅱ Vascularization of renal organoids by co-culturing renal organoids with hPSC-derived vascular endothelial cellsPurposesTo construct the kidney organoids with more abundant blood vessel components by co-culturing hPSCs-derived kidney organoids with HiPSCs induced the blood vessels organoids in vitro..MethodsHPSCs were used as seed cells to induce kidney organoids through renal epithelial differentiation kit.Kidney specific markers WT1,PODXL,LTL and CDH1,vascular endothelial cell markers CD31 and UEA-1,and vascular smooth musclemarker α-SMA were detected by immunofluorescence.HPSCs were used as seed cells to induce blood vessel organoids.The purity of vascular endothelial cells(CD31+CD144+)was detected by flow cytometry.The vascular endothelial cell marker VECAD and vascular smooth muscle marker α-SMA,endothelial cell marker CD31 and UEA-1,and pericyte marker PDGFR-β were detected by immunofluorescence.Day7 renal progenitor cells were co-cultured with endothelial cells for 11 days.Rt-qPCR was used to analyze the expression of kidney specific genes in kidney organoids and blood vessels organoids co-cultures.The expressions of CDH1,acetylated-tubulin,VECAD and PODXL were detected by immunofluorescence.Immunofluorescence analysis of VECAD and PODXL expression in kidney organoids and blood vessels organoids co-cultures and kidney organoids alone.ResultsKidney organoids identification:Immunofluorescence showed that CD31 and UEA-1 were positively expressed in kidney organoids,indicating that vascular endothelial structure was generated during the differentiation of kidney organoids.The positive expression of the vascular smooth muscle marker α-SMA suggests that the vascular smooth muscle component is produced in the kidney organoids.Blood vessels organoids detection:Immunofluorescence showed that VECAD,CD31 and UEA-1 were positively expressed in the blood vessels organoids,indicatingthe blood vessels organoids contains vascular endothelial component.The expression of α-SMA was negative,indicating that pure vascular endothelial cells were obtained without the formation of arterial smooth muscle components.The positive expression of PDGFR-β was detected in the blood vessels organoids,suggesting that pericyte component was generated in the blood vessels organoids.Flow cytometry showed that the third generation of vascular endothelial cells(CD31+CD144+)accounted for more than 95%.Detection of kidney organoids and blood vessels organoids co-culture:light microscope can observe the renal tubular structure with obvious differentiation structure.RT-qPCR showed the expression of nephroid specific genes in the kidney organoids and blood vessels organoids co-cultures.Immunofluorescence detected the expression of CDH1 and acetylated-tubulin,suggesting the formation of renal tubules in the co-cultures.Both VECAD and PODX were positively expressed,suggesting that podocyte components and vascular endothelial cell components were generated in the co-cultures.Comparison of vascularization between co-cultures and kidney organoids:VECAD positive cells in co-cultures were significantly more than those in renal organoids.These results suggest that the co-culture produces more vascular endothelial cell components than the renal organoids alone.ConclusionIn the process of inducing kidney,kidney organoids could have richer vascular components by co-culture with HPSCs-derived blood vessels organoids.