| Objective: This study aims to provide new light on the reconstruction failure caused by a poor blood supply in the repair area of urinary system tissue engineering by promoting the angiogenesis ability of exosomes derived from human induced pluripotent stem cells(hiPSCs-Exo),and exploring the preliminary molecular mechanism.Methods:(1)The expression of pluripotent markers of hiPSCs was identified by immunofluorescence staining and flow cytometry.We differentiated hiPSCs into urothelial cells using a two-step approach,and the expression of cells differentiation markers were verified by q RT-PCR,western blot,and immunofluorescence.(2)The Ultracentrifugation obtained hiPSCs-Exo,the biological characteristics were observed through the transmission electron microscopy,and particle size analyzer was used to detect the diameter,and Western blot was used to identify the expression of exosomespecific markers.(3)PKH26 staining was used to label hiPSCs-Exo and observe whether hiPSCs-Exo could be taken up by human umbilical vein endothelial cells(HUVECs).hiPSCs-Exo was co-cultured with HUVECs,and the proliferation,migration,and pro-angiogenic abilities of HUVECs were detected by CCK-8 assay,scratch assay,and angiogenesis assay.The angiogenesis-related gene expression was examined by q RT-PCR.(4)The C57/BL6 J mice were randomly divided into three groups:(1)sham group;(2)model group;(3)hiPSCs-Exo group.The sham group only exposed the femoral artery without ischemia treatment.The mouse hindlimb ischemia model was established by ligation of the femoral artery in the model group and hiPSCsExo group,and PBS and hiPSCs-Exo were injected into each mouse quadriceps once on days 0,4,8.The recovery of blood flow in the lower extremities of mice was examined by laser Doppler flowmetry on days 0,7,14,and 21.On days 14 and 21,mice were randomly selected from each group for immunofluorescence staining of the ischemic quadriceps muscle to detect the density of new microvessels in the lower limb muscles.The degree of lower limb ischemia in the three groups of mice was observed on day 21,and hematoxylin-eosin staining(H&E staining)was performed on the muscles of the ischemic lower limbs of the remaining mice to observe the morphology of myocytes.(5)The high throughput sequencing comprehensive detection for miRNA,was used to screen miRNAs that are associated with the angiogenesis.The q RT-PCR was used to validate the sequencing results.hiPSCs-Exo was extracted after transfection of Cy3-labeled miR-103a-3p mimics into hiPSCs to observe whether miR-103a-3p could be taken up by HUVECs.The HUVECs uptake of Cy3-labeled miR-103a-3p mimicswas examined using confocal microscopy.After overexpressing and knocking down miR-103a-3p in HUVECs using lentiviral transfection,we examined the effects of the miR-103a-3p on the proangiogenic capacity of HUVECs by the matrigel tube formation experiment.Results:(1)The immunofluorescence staining and flow cytometry results showed that hiPSCs expressed specific proteins Nanog and Oct4,which met the typical characteristics of hiPSCs.The q RT-PCR and western blot results showed that the hiPSCs expressed the urothelial cells marker(UPK Ib and UPK IIIb)after 18 days of differentiation induction(P<0.001).(2)Under transmission electron microscopy,the hiPSCs-Exo were round or oval,ranging from 40~150 nm in diameter,and had a typical "The saucer shape" structure.The particle size analysis results showed that hiPSCs-Exo had a mean diameter of 95.28 nm.Western blot results showed that hiPSCs-Exo expressed exosome-specific proteins CD9,CD63,and CD81,which met the characteristics of exosomes.(3)PKH26 staining results showed that hiPSCs-Exo was successfully taken up by HUVECs and entered the cytoplasm.CCK8 experiment results showed that the proliferation ability of HUVECs was significantly enhanced after coculture with the hiPSCs-Exo(P<0.05).The scratch experiment results showed that the migration ability of HUVECs was significantly enhanced after co-culture with the hiPSCs-Exo(P<0.01).The matrigel tube formation experiment results showed that HUVECs tube-forming ability was significantly enhanced(P<0.01).The q RT-PCR results showed that angiogenesis-related gene(VEGF,Ang1,Ang2)expression was elevated in HUVECs(P<0.01).(4)Laser Doppler images showed that the mice in the hiPSCs-Exo group had better blood flow recovery by 21 days after surgery than control mice(P<0.05).Immunofluorescence staining results showed that the expression levels of CD31 and CD34 in the hiPSCs-Exo group were markedly higher compared with those in the model group.H&E staining of quadriceps muscle showed that cells in the model group appeared shrunken and apoptotic seriously,while those in the hiPSCs-Exo group appeared mild shrunken and apoptotic.(5)MiRNAs high-throughput sequencing results showed that many miRNAs were present in hiPSCs-Exo.According to the sequencing results,miR-103a-3p was selected as the follow-up study object.Cy3-labeled miR-103a-3p was successfully uptaken by HUVECs.The matrigel tube formation experiment results showed that angiogenesis ability of HUVECs was significantly enhanced after overexpressing miR-103a-3p(P<0.01).Conclusions:(1)Directed differentiation of hiPSCs into urothelial cells could be achieved in vitro.(2)hiPSCs-Exo could promote HUVECs proliferation,migration and tube formation,and angiogenesis-related gene expression.(3)The hiPSCs-Exo may help improve blood flow in ischemic lower limbs and promote angiogenesis in the ischemic muscle in mice.(4)MiR-103a-3p participated in the angiogenesis of hiPSCsExo.Significance: This study provides the theoretical basis for clarifying the molecular mechanisms underlying the pro-angiogenic function of hiPSCs-Exo and provides new ideas for addressing poor vascularization and easy infection of urinary tissueengineered grafts to achieve the clinical transformation of hiPSCs-Exo better. |
