Background: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands(SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprog...Background: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands(SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.Methods: The expression of the SG markers cytokeratin 5(CK5), cytokeratin 10(CK10), cytokeratin 18(CK18), carcinoembryonic antigen(CEA), aquaporin 5(AQP5) and α-smooth muscle actin(α-SMA) was assessed with quantitative PCR(qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells(iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs.BALB/c nude mice were randomly divided into normal group, SGM treatment group and iSGC transplantation group.Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.Results: Ectodermal dysplasia antigen(EDA) overexpression drove human dermal fibroblast(HDF) conversion into i SGCs in SG culture medium(SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18and CEA in iSGCs, and flow cytometry data demonstrated(4.18±0.04)% of iSGCs were CK5 positive and(4.36±0.25)%of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails,(23.05±2.49)% of iSGCs expressed CK5^(+) and(55.79±3.18)% of iSGCs expressed CK18^(+), respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79±7.71)% vs.(70.59±0.34)%, ns]. In vivo transplantation experiments showed approximately(5.2±1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.Conclusions: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.展开更多
Background:Focal segmental glomerulosclerosis (FSGS)is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function,which is characterized by podocyte injury and the depleti...Background:Focal segmental glomerulosclerosis (FSGS)is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function,which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments.The pathogenesis of FSGS has not been completely elucidated;however,recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis.Here,we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism. Methods:Using next-generation sequencing and Sanger sequencing,we screened the causative gene that was linked to FSGS in this study.The patient's total blood RNA was extracted to validate the messenger RNA (mRNA)expression of coenzyme Q10 monooxygenase 6(COQ6)and validated it by immunohistochemistry.COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then,F-actin was determined using immunofluorescence staining.Cell apoptosis was evaluated by flow cytometry,the expression of active caspase-3was determined by Western blot,and mitochondrial function was detected by MitoSOX. Results:Using whole-exome sequencing and Sanger sequencing,we screened a new causative gene,COQ6,NM_182480:exonl:c.G41A: p.W14X.The mRNA expression of COQ6 in the proband showed decreased.Moreover,the expression of COQ6,which was validated by immunohistochemistry,also had the same change in the proband.Finally,we focused on the COQ6 gene to clarify the mechanism of podocyte injury.Flow cytometry showed significantly increased in apoptotic podocytes,and Western blotting showed increases in active caspase-3in si-COQ6 podocytes.Meanwhile,reactive oxygen species (ROS)levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group. Conclusions:This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6,which could cause respiratory chain defect,increase the generation of ROS,destroy the podocyte cytoskeleton,and induce apoptosis.It provides basic theoretical basis for the screening of FSGS in the future.展开更多
基金This work was supported by the National Natural Science Foundation of China (Grant No. 81173625, 81373458) Thanks for the kind help of Dr. Wang (Pulmonary Division, Boston Children's Hospital, MA, USA), who was extremely helpful in the revision of the language.
基金supported in part by the National Natural Science Foundation of China (81871569, 81830064, 81721092, 61803250)the National Key Research and Development Plan (2018YFC1105704, 2017YFC1103304, 2016YFA0101000, 2016YFA0101002)+2 种基金the CAMS Innovation Fund for Medical Sciences (CIFMS, 2019-I2M-5-059)the Military Key Basic Research of Foundational Strengthening Program (2020-JCJQ-ZD-256-021)the Military Medical Research and Development Projects (AWS17J005, 2019-126)。
文摘Background: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands(SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin.Methods: The expression of the SG markers cytokeratin 5(CK5), cytokeratin 10(CK10), cytokeratin 18(CK18), carcinoembryonic antigen(CEA), aquaporin 5(AQP5) and α-smooth muscle actin(α-SMA) was assessed with quantitative PCR(qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells(iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs.BALB/c nude mice were randomly divided into normal group, SGM treatment group and iSGC transplantation group.Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs.Results: Ectodermal dysplasia antigen(EDA) overexpression drove human dermal fibroblast(HDF) conversion into i SGCs in SG culture medium(SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18and CEA in iSGCs, and flow cytometry data demonstrated(4.18±0.04)% of iSGCs were CK5 positive and(4.36±0.25)%of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails,(23.05±2.49)% of iSGCs expressed CK5^(+) and(55.79±3.18)% of iSGCs expressed CK18^(+), respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79±7.71)% vs.(70.59±0.34)%, ns]. In vivo transplantation experiments showed approximately(5.2±1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice.Conclusions: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.
文摘Background:Focal segmental glomerulosclerosis (FSGS)is a kidney disease that is commonly associated with proteinuria and the progressive loss of renal function,which is characterized by podocyte injury and the depletion and collapse of glomerular capillary segments.The pathogenesis of FSGS has not been completely elucidated;however,recent advances in molecular genetics have provided increasing evidence that podocyte structural and functional disruption is central to FSGS pathogenesis.Here,we identified a patient with FSGS and aimed to characterize the pathogenic gene and verify its mechanism. Methods:Using next-generation sequencing and Sanger sequencing,we screened the causative gene that was linked to FSGS in this study.The patient's total blood RNA was extracted to validate the messenger RNA (mRNA)expression of coenzyme Q10 monooxygenase 6(COQ6)and validated it by immunohistochemistry.COQ6 knockdown in podocytes was performed in vitro with small interfering RNA, and then,F-actin was determined using immunofluorescence staining.Cell apoptosis was evaluated by flow cytometry,the expression of active caspase-3was determined by Western blot,and mitochondrial function was detected by MitoSOX. Results:Using whole-exome sequencing and Sanger sequencing,we screened a new causative gene,COQ6,NM_182480:exonl:c.G41A: p.W14X.The mRNA expression of COQ6 in the proband showed decreased.Moreover,the expression of COQ6,which was validated by immunohistochemistry,also had the same change in the proband.Finally,we focused on the COQ6 gene to clarify the mechanism of podocyte injury.Flow cytometry showed significantly increased in apoptotic podocytes,and Western blotting showed increases in active caspase-3in si-COQ6 podocytes.Meanwhile,reactive oxygen species (ROS)levels were increased and F-actin immunofluorescence was irregularly distributed in the si-COQ6 group. Conclusions:This study reported a possible mechanism for FSGS and suggested that a new mutation in COQ6,which could cause respiratory chain defect,increase the generation of ROS,destroy the podocyte cytoskeleton,and induce apoptosis.It provides basic theoretical basis for the screening of FSGS in the future.