BACKGROUND Adipose-derived mesenchymal stem cells(ASCs)are characterized by long-term self-renewal and a high proliferation rate.Under adequate conditions,they may differentiate into cells belonging to mesodermal,endo...BACKGROUND Adipose-derived mesenchymal stem cells(ASCs)are characterized by long-term self-renewal and a high proliferation rate.Under adequate conditions,they may differentiate into cells belonging to mesodermal,endodermal or ectodermal lineages.Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level.The loss of pericytes,as occurs in diabetic retinopathy,results in a breakdown of the blood-retina barrier(BRB)and infiltration of inflammatory cells.In this context,the use of pericytelike differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage.AIM To test in vitro strategies to obtain pericyte-like differentiation of human ASCs(hASCs).METHODS Different culture conditions were tested:hASCs cultured in a basal medium supplemented with transforming growth factorβ1;and hASCs cultured in a specific pericyte medium(PM-hASCs).In a further sample,pericyte growth supplement was omitted from the PM.In addition,cultures of human retinal pericytes(hRPCs)were used for comparison.Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression ofα-smooth muscle actin(α-SMA)and neural/glial antigen 2(NG2).Interactions between human retinal endothelial cells(hRECs)and different groups of hASCs were investigated in co-culture experiments.In these cases,the expression of typical junctional proteins such as vascular endothelial-Cadherin,zonula occludens-1 and Occludin were assessed in hRECs.In an in vitro model of the BRB,values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs.The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone.Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization.RESULTS After 3-6 d of culture,α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium(PM-hASCs).In particular,α-SMA immunoreactivity,already visible at the basal level in pericytes and ASCs,was strongly increased only when transforming growth factor was added to the culture medium.NG2 expression,almost undetectable in most conditions,was substantially increased only in PMhASCs.Immunocytochemical results were confirmed by western blot analysis.The presence of pericyte growth supplement seems to increase NG2 expression rather thanα-SMA,in agreement with its role in maintaining pericytes in the proliferative state.In co-culture experiments,immunoreactivity of vascular endothelial-Cadherin,zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were also present.Supporting results were found by trans-endothelial electrical resistance measurements,gathered at 3 and 6 d of co-culture.The highest resistance values were obtained when hRECs were co-cultured with hRPCs or PM-hASCs.The pericyte-like phenotype of PM-hASCs was also confirmed in three-dimensional co-cultures in Matrigel,where PM-hASCs and hRPCs similarly localized around the tubular formations made by hRECs.CONCLUSION PM-hASCs seem able to strengthen the intercellular junctions between hRECs,likely reinforcing the BRB;thus,hASC-based therapeutic approaches may be developed to restore the integrity of retinal microcirculation.展开更多
The presence or absence of adult neural stem cells in the mammalian forebrain ependyma has been debated for two decades.In this study,we performed single-cell RNA sequencing to investigate the cellular composition of ...The presence or absence of adult neural stem cells in the mammalian forebrain ependyma has been debated for two decades.In this study,we performed single-cell RNA sequencing to investigate the cellular composition of the ependymal surface of the adult mouse forebrain using whole mounts of lateral walls of lateral ventricles.We identified 12 different cell subtypes in the ependymal surface.Immunocytochemical analyses revealed that CD133^(+)multi-ciliated cells comprised 67.6%of ependymal cells,while the remaining 32.4%were CD133^(-).CD133^(+)ependymal cells can be further classified into FOXJ1^(+)/SOX2^(+)/ACTA2^(+)cells,FLT1^(+)/CD31^(+)/CLDN5^(+)endothelial-like cells,and PDGFRB^(+)/VTN^(+)/NG2^(+)pericyte-like cells,as well as endothelial-pericyte-like cells and Foxj1^(+)endothelial-like cells.CD133^(-)ependymal cells can be further divided into endothelial-like cells,Foxj1^(+)ependymal cells,Foxj1^(+)endothelial-like cells,pericyte-like cells,endothelial-pericyte-like cells,VIM^(+)cells,and cells negative for all of these markers.This comprehensive profiling confirms the heterogeneity of the ependymal surface in the adult mouse forebrain.Debate regarding whether adult ependymal cells contain neural stem cells has arisen because different researchers have examined different populations of ependymal cells.Our study provides a new perspective for investigation of clinical endogenous neural stem cells,ultimately paving the way for stem cell therapies in neurological diseases.展开更多
基金“Piano Triennale per la Ricerca 2016-2018–Linea Intervento 2”,University of Catania,Italy,No.20722142118.
文摘BACKGROUND Adipose-derived mesenchymal stem cells(ASCs)are characterized by long-term self-renewal and a high proliferation rate.Under adequate conditions,they may differentiate into cells belonging to mesodermal,endodermal or ectodermal lineages.Pericytes support endothelial cells and play an important role in stabilizing the vessel wall at the microcirculation level.The loss of pericytes,as occurs in diabetic retinopathy,results in a breakdown of the blood-retina barrier(BRB)and infiltration of inflammatory cells.In this context,the use of pericytelike differentiated ASCs may represent a valuable therapeutic strategy for restoring BRB damage.AIM To test in vitro strategies to obtain pericyte-like differentiation of human ASCs(hASCs).METHODS Different culture conditions were tested:hASCs cultured in a basal medium supplemented with transforming growth factorβ1;and hASCs cultured in a specific pericyte medium(PM-hASCs).In a further sample,pericyte growth supplement was omitted from the PM.In addition,cultures of human retinal pericytes(hRPCs)were used for comparison.Pericyte-like differentiation of hASCs was tested by immunocytochemical staining and western blotting to evaluate the expression ofα-smooth muscle actin(α-SMA)and neural/glial antigen 2(NG2).Interactions between human retinal endothelial cells(hRECs)and different groups of hASCs were investigated in co-culture experiments.In these cases,the expression of typical junctional proteins such as vascular endothelial-Cadherin,zonula occludens-1 and Occludin were assessed in hRECs.In an in vitro model of the BRB,values of trans-endothelial electrical resistance were measured when hRECs were co-cultured with various groups of pretreated hASCs.The values observed were compared with co-cultures of hRECs and hRPCs as well as with cultures of hRECs alone.Three-dimensional co-cultures of hRECs and hRPCs or pericyte-like hASCs in Matrigel were designed to assess their reciprocal localization.RESULTS After 3-6 d of culture,α-SMA and NG2 immunocytochemistry showed that the closest pericyte-like phenotype was observed when hASCs were cultured in Pericyte Medium(PM-hASCs).In particular,α-SMA immunoreactivity,already visible at the basal level in pericytes and ASCs,was strongly increased only when transforming growth factor was added to the culture medium.NG2 expression,almost undetectable in most conditions,was substantially increased only in PMhASCs.Immunocytochemical results were confirmed by western blot analysis.The presence of pericyte growth supplement seems to increase NG2 expression rather thanα-SMA,in agreement with its role in maintaining pericytes in the proliferative state.In co-culture experiments,immunoreactivity of vascular endothelial-Cadherin,zonula occludens-1 and Occludin was considerably increased in hRECs when hRPCs or PM-hASCs were also present.Supporting results were found by trans-endothelial electrical resistance measurements,gathered at 3 and 6 d of co-culture.The highest resistance values were obtained when hRECs were co-cultured with hRPCs or PM-hASCs.The pericyte-like phenotype of PM-hASCs was also confirmed in three-dimensional co-cultures in Matrigel,where PM-hASCs and hRPCs similarly localized around the tubular formations made by hRECs.CONCLUSION PM-hASCs seem able to strengthen the intercellular junctions between hRECs,likely reinforcing the BRB;thus,hASC-based therapeutic approaches may be developed to restore the integrity of retinal microcirculation.
基金supported by the State Key Program of the National Natural Science Foundation of China,No.82030035(to YES)Peak Disciplines(Type IV)of Institutions of Higher Learning in Shanghai(to LZ).
文摘The presence or absence of adult neural stem cells in the mammalian forebrain ependyma has been debated for two decades.In this study,we performed single-cell RNA sequencing to investigate the cellular composition of the ependymal surface of the adult mouse forebrain using whole mounts of lateral walls of lateral ventricles.We identified 12 different cell subtypes in the ependymal surface.Immunocytochemical analyses revealed that CD133^(+)multi-ciliated cells comprised 67.6%of ependymal cells,while the remaining 32.4%were CD133^(-).CD133^(+)ependymal cells can be further classified into FOXJ1^(+)/SOX2^(+)/ACTA2^(+)cells,FLT1^(+)/CD31^(+)/CLDN5^(+)endothelial-like cells,and PDGFRB^(+)/VTN^(+)/NG2^(+)pericyte-like cells,as well as endothelial-pericyte-like cells and Foxj1^(+)endothelial-like cells.CD133^(-)ependymal cells can be further divided into endothelial-like cells,Foxj1^(+)ependymal cells,Foxj1^(+)endothelial-like cells,pericyte-like cells,endothelial-pericyte-like cells,VIM^(+)cells,and cells negative for all of these markers.This comprehensive profiling confirms the heterogeneity of the ependymal surface in the adult mouse forebrain.Debate regarding whether adult ependymal cells contain neural stem cells has arisen because different researchers have examined different populations of ependymal cells.Our study provides a new perspective for investigation of clinical endogenous neural stem cells,ultimately paving the way for stem cell therapies in neurological diseases.
