Background:We previously reported that activation of the cell cycle in human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)enhances their remuscularization capacity after human cardiac muscle patch tr...Background:We previously reported that activation of the cell cycle in human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)enhances their remuscularization capacity after human cardiac muscle patch transplantation in infarcted mouse hearts.Herein,we sought to identify the effect of magnesium lithospermate B(MLB)on hiPSC-CMs during myocardial repair using a myocardial infarction(MI)mouse model.Methods:In C57BL/6 mice,MI was surgically induced by ligating the left anterior descending coronary artery.The mice were randomly divided into five groups(n=10 per group);a MI group(treated with phosphate-buffered saline only),a hiPSC-CMs group,a MLB group,a hiPSC-CMs+MLB group,and a Sham operation group.Cardiac function and MLB therapeutic efficacy were evaluated by echocardiography and histochemical staining 4 weeks after surgery.To identify the associated mechanism,nuclear factor(NF)-κB p65 and intercellular cell adhesion molecule-1(ICAM1)signals,cell adhesion ability,generation of reactive oxygen species,and rates of apoptosis were detected in human umbilical vein endothelial cells(HUVECs)and hiPSC-CMs.Results:After 4 weeks of transplantation,the number of cells that engrafted in the hiPSC-CMs+MLB group was about five times higher than those in the hiPSC-CMs group.Additionally,MLB treatment significantly reduced tohoku hospital pediatrics-1(THP-1)cell adhesion,ICAM1 expression,NF-κB nuclear translocation,reactive oxygen species production,NF-κB p65 phosphorylation,and cell apoptosis in HUVECs cultured under hypoxia.Similarly,treatment with MLB significantly inhibited the apoptosis of hiPSC-CMs via enhancing signal transducer and activator of transcription 3(STAT3)phosphorylation and B-cell lymphoma-2(BCL2)expression,promoting STAT3 nuclear translocation,and downregulating BCL2-Associated X,dual specificity phosphatase 2(DUSP2),and cleaved-caspase-3 expression under hypoxia.Furthermore,MLB significantly suppressed the production of malondialdehyde and lactate dehydrogenase and the reduction in glutathione content induced by hypoxia in both HUVECs and hiPSC-CMs in vitro.Conclusions:MLB significantly enhanced the potential of hiPSC-CMs in repairing injured myocardium by improving endothelial cell function via the NF-κB/ICAM1 pathway and inhibiting hiPSC-CMs apoptosis via the DUSP2/STAT3 pathway.展开更多
Engineering myocardium has shown great clinal potential for repairing permanent myocardial injury.However,the lack of perfusing blood vessels and difficulties in preparing a thick-engineered myocardium result in its l...Engineering myocardium has shown great clinal potential for repairing permanent myocardial injury.However,the lack of perfusing blood vessels and difficulties in preparing a thick-engineered myocardium result in its limited clinical use.We prepared a mixed gel containing fibrin(5 mg/ml)and collagen I(0.2 mg/ml)and verified that human umbilical vein endothelial cells(HUVECs)and human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)could fom microvascular lumens and myocardial cell clusters by harnessing the low-hardness and hyperelastic characteristics of fibrin.hiPSC-CMs and HUVECs in the mixed gel formed self-organized cell clusters,which were then cultured in different media using a three-phase approach.The successfully constructed vascularized engineered myocardial tissue had a spherical structure and final diameter of 1-2mm.The tissue exhibited autonomous beats that occured at a frequency similar to a normal human heart rate.The internal microvascular lumen could be maintained for 6 weeks and showed good results during preliminary surface re-vascularization in vitro and vascular remodeling in vivo.In summary,we propose a simple method for constructing vascularized engineered myocardial tissue,through phased cultivation that does not rely on high-end manufacturing equipment and cutting-edge preparation techniques.The constructed tissue has potential value for clinical use after preliminary evaluation.展开更多
Human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)have attracted attention in the field of regenerative medicine due to their potential ability to repair damaged hearts.However,the immature phenotyp...Human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)have attracted attention in the field of regenerative medicine due to their potential ability to repair damaged hearts.However,the immature phenotype of these cells limits their clinical application.Cardiomyocyte maturation is accompanied by changes in mitochondrial quality.PTEN-induced putative kinase 1(PINK1)has been linked to mitochondrial quality control.However,whether the changes in mitochondrial quality in hiPSC-CMs are associated with PINK1,and the impact of PINK1 on hiPSC-CMs development are not clear.In this study,we found that knockdown of PINK1 in hiPSC-CMs resulted in mitochondrial fragmentation and impaired mitochondrial functions such as mitophagy and mitochondrial biogenesis.PINK1 deletion also inhibited the maturation of hiPSC-CMs,reverting them to a naive structural and functional state.We found that restoring the mitochondrial structure did not completely rescue the mitochondrial dysfunction caused by PINK1 deletion,while activation of PINK1 kinase activity using kinetin promoted mitochondrial fusion,increased the mitochondrial membrane potential and ATP production,and maintained the development and maturation of hiPSC-CMs.In conclusion,PINK1 regulates the mitochondrial structure and function of hiPSC-CMs,and is essential for the maturation of hiPSC-CMs.展开更多
基金supported by the Natural Science Foundation of Hunan Province(Nos.2023JJ30793,2022JJ20088,and 2019JJ50858)the Science and Technology Innovation Program of Hunan Province(No.2021RC2106)+1 种基金the National Natural Science Foundation of China(No.82200323)the Scientific Research Launch Project for new employees of the Second Xiangya Hospital of Central South University.
文摘Background:We previously reported that activation of the cell cycle in human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)enhances their remuscularization capacity after human cardiac muscle patch transplantation in infarcted mouse hearts.Herein,we sought to identify the effect of magnesium lithospermate B(MLB)on hiPSC-CMs during myocardial repair using a myocardial infarction(MI)mouse model.Methods:In C57BL/6 mice,MI was surgically induced by ligating the left anterior descending coronary artery.The mice were randomly divided into five groups(n=10 per group);a MI group(treated with phosphate-buffered saline only),a hiPSC-CMs group,a MLB group,a hiPSC-CMs+MLB group,and a Sham operation group.Cardiac function and MLB therapeutic efficacy were evaluated by echocardiography and histochemical staining 4 weeks after surgery.To identify the associated mechanism,nuclear factor(NF)-κB p65 and intercellular cell adhesion molecule-1(ICAM1)signals,cell adhesion ability,generation of reactive oxygen species,and rates of apoptosis were detected in human umbilical vein endothelial cells(HUVECs)and hiPSC-CMs.Results:After 4 weeks of transplantation,the number of cells that engrafted in the hiPSC-CMs+MLB group was about five times higher than those in the hiPSC-CMs group.Additionally,MLB treatment significantly reduced tohoku hospital pediatrics-1(THP-1)cell adhesion,ICAM1 expression,NF-κB nuclear translocation,reactive oxygen species production,NF-κB p65 phosphorylation,and cell apoptosis in HUVECs cultured under hypoxia.Similarly,treatment with MLB significantly inhibited the apoptosis of hiPSC-CMs via enhancing signal transducer and activator of transcription 3(STAT3)phosphorylation and B-cell lymphoma-2(BCL2)expression,promoting STAT3 nuclear translocation,and downregulating BCL2-Associated X,dual specificity phosphatase 2(DUSP2),and cleaved-caspase-3 expression under hypoxia.Furthermore,MLB significantly suppressed the production of malondialdehyde and lactate dehydrogenase and the reduction in glutathione content induced by hypoxia in both HUVECs and hiPSC-CMs in vitro.Conclusions:MLB significantly enhanced the potential of hiPSC-CMs in repairing injured myocardium by improving endothelial cell function via the NF-κB/ICAM1 pathway and inhibiting hiPSC-CMs apoptosis via the DUSP2/STAT3 pathway.
基金Key projects of the National Natural Science Foundation of China(81830055,82230073)the Postdoctoral Research Special Support Projects of Chongqing(2020-2010010006263477)Thanks for the support of Yonghong Fan's the Science and Technology Innovation Enhancement Projects of Third Military Medical University(2020xQN03).
文摘Engineering myocardium has shown great clinal potential for repairing permanent myocardial injury.However,the lack of perfusing blood vessels and difficulties in preparing a thick-engineered myocardium result in its limited clinical use.We prepared a mixed gel containing fibrin(5 mg/ml)and collagen I(0.2 mg/ml)and verified that human umbilical vein endothelial cells(HUVECs)and human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)could fom microvascular lumens and myocardial cell clusters by harnessing the low-hardness and hyperelastic characteristics of fibrin.hiPSC-CMs and HUVECs in the mixed gel formed self-organized cell clusters,which were then cultured in different media using a three-phase approach.The successfully constructed vascularized engineered myocardial tissue had a spherical structure and final diameter of 1-2mm.The tissue exhibited autonomous beats that occured at a frequency similar to a normal human heart rate.The internal microvascular lumen could be maintained for 6 weeks and showed good results during preliminary surface re-vascularization in vitro and vascular remodeling in vivo.In summary,we propose a simple method for constructing vascularized engineered myocardial tissue,through phased cultivation that does not rely on high-end manufacturing equipment and cutting-edge preparation techniques.The constructed tissue has potential value for clinical use after preliminary evaluation.
基金supported by the National Natural Science Foundation of China(No.81970244)General Basic Research Project from the Ministry of Education Key Laboratory of Child Development and Disorders,China(No.GBRP-202108).
文摘Human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)have attracted attention in the field of regenerative medicine due to their potential ability to repair damaged hearts.However,the immature phenotype of these cells limits their clinical application.Cardiomyocyte maturation is accompanied by changes in mitochondrial quality.PTEN-induced putative kinase 1(PINK1)has been linked to mitochondrial quality control.However,whether the changes in mitochondrial quality in hiPSC-CMs are associated with PINK1,and the impact of PINK1 on hiPSC-CMs development are not clear.In this study,we found that knockdown of PINK1 in hiPSC-CMs resulted in mitochondrial fragmentation and impaired mitochondrial functions such as mitophagy and mitochondrial biogenesis.PINK1 deletion also inhibited the maturation of hiPSC-CMs,reverting them to a naive structural and functional state.We found that restoring the mitochondrial structure did not completely rescue the mitochondrial dysfunction caused by PINK1 deletion,while activation of PINK1 kinase activity using kinetin promoted mitochondrial fusion,increased the mitochondrial membrane potential and ATP production,and maintained the development and maturation of hiPSC-CMs.In conclusion,PINK1 regulates the mitochondrial structure and function of hiPSC-CMs,and is essential for the maturation of hiPSC-CMs.
