The Effect Of Substrate Stiffness On The Maturation Of Human IPSC-derived Cardiomyocytes

Myocardial infarction(MI)and other cardiovascular diseases are currently the leading cause of death in the world,despite remarkable advances in interventional cardiology,cardiac surgery and modern drug therapy.A significantly irreversible loss of cardiomyocytes mainly result in all those diseases.Owing to the non-regenerative nature of terminally differentiated cardiomyocytes(CMs),myocardial repair remains severely limited by the source of viable CMs for replacement.Pluripotent stem cells(PSCs),including embryonic stem cells(ESCs)and induced pluripotent stem cells(iPSCs),have self-renewal ability and can differentiate into virtually all cell types,including CMs.Therefore,ESCs/iPSCs provide an unlimited ex vivo source of CMs for clinical application,drug discovery and cardiotoxicity screening.What's more,human iPSCs(hiPSCs)are human origin and have no ethical limitations are more widely practical application than hESCs.Therefore,exploreing the differentiation methods of of CMs induced from hiPSC-CMs can provide a potential cell source for MI therapy.However,some reports shows hPSC-CMs resemble fetal or embryonic CMs,which appear far different in structure and function from adult CMs.Its immaturity becomes a major bottleneck hindered its clinical application,which may cause arrhythmia and metabolism dysfunction.Strategies currently being used to improve maturation,which include long term culture,electrical stimulation,mechanical strain,and chemical factors,and so on.But the results have not achieved success.As we all know,The heart growth is modulated by not only biochemical and paracrine,but also mechanical factors(such as stiffness).The elastic modulus of the heart tissue gradually increases from the embryo to the newborn and to the adult.MI will also increased the elastic modulus of myocardium which affect the physiology and function of the heart.It has been reported that substrate stiffness can promote the functional maturation of rats neonatal cardiomyocytes on well-organized sarcomere and enhanced calcium transient.This indicates that substrate stiffness may affect the maturation of hPSC-CMs.Before considering the maturation strategy,we used the characteristics difference between mature(adult)and immature(fetal)CMs as a benchmark for judging the maturation state of hPSC-CMs.The structure and function of mature CMs are mainly based on(1)wel-organized sarcomere which are structural and functional unit of morphology and contraction of CMs,(2)strong calcium transient which is functional foundation for excitation-contraction coupling and a most widely characterized functional parameter,(3)lower proliferation,(4)gene expression profile with increased adult structure gene expression,(5)mature powerhouse – mitochondria which structural and functional changes are critical components of maturation.This study based on "substrate stiffnes is an important factor on maturation of stem cell-derived cardiomyocytes",studied The effect of substrate stiffnes on the structure and function of sarcomere,calcium transients,proliferation,gene expression and mitochondria of hiPSC-CMs,to evaluate the effect of stiffnes on the maturation of hiPSC-CMs,to explore the mature method of hiPSC-CMs,to provide experimental evidences for the treatment of cardiac diseases by stem cell transplantation,promote the clinical application of stem cell therapy.Part 1 The maturity of CMs induced from hiPSC using chemical defined methodAim: Using established chemical defined method induced hi PSCs into cardiomyocytes,to evaluate the maturity of hiPSC-CMs.Methods: hiPSCs(cell line >p20)were seeded on the materigel coated dishes in the mTeSR1 medium.Change medium everyday.hiPSCs were split using Versene.The pluripotency of hiPSCs was detected with SSEA-4 and OCT3/4 expression by immunofluorescence staining and flow cytometery.hiPSCs were differentiated into cardiomyocytes by chemical defined method which is in the RPMI1640 and B27 medium by adding Wnt pathway activiator CHIR99021 and inhibitor IWR-1.The CMs were identified with structural cardiac markers cardiac troponin T(cTnT)and a-Actinin expression by immunofluorescence staining and flow cytometery.?-SMA(a immature cardiac marker)was assessed by flow cytometery.Using transmission electron microscope(EM)to show ultrastruature of CMs.The proliferation of CMs was detected by Edu staining.Results: hiPSCs had a quick growth in typically colony morphology.Immunofluorescence staining showed SSEA-4 expressed on the surface and OCT3/4 expressed in the nucleus of cell.The positive expression of SSEA-4 and OCT3/4 were 99.3% and 98.0% by Flow cytometery.cTnT and a-Actinin were positive expression by immunofluorescence staining,and flow cytometery showed cTnT had 90% positive expression,while the ?-SMA had 99.2% positive expression.CMs morphology is heterogeneous with disorganized myofibrils distribution.The fragmented myofibrils were connected with fragmented Z line.No T-tubule.There also showed some Edu positive cells in hiPSC-CMs.Conclusions: hiPSCs can be differentiated into cardiomyocytes by chemical defined method with 90% differentiation efficiency.And the cardiomyocytes are immature with disorgnized myofibrils and high positive expression of ?-SMA.Part 2 The effect of substrate stiffness on the maturation of hiPSC-CMsAim: Based on substrate mimicing the stiffness of normal and infarcted myocardium,we detected the effect of substrate stiffness on myofibrils orgnization,sarcomere structure,adult structural proteins and genes expression,calcium transients and cell proliferation of hiPSC-CMs,to evaluated the structural and functional maturation of hiPSC-CMs.Methods: Silicone elastomer and curing agent(PDMS)were mixed thoroughly in 30:1 and 60:1 ratios to make the Stiff and Soft matrix,respectively.Then using alternate layer-by-layer coating with polyethylenimine/Polystyrene sulfonic acid(PEI/PSS)to create a hydrophilic surface on the PDMS matrix for cell attachment.The hiPSCs attachment and proliferation were measured by cell attachment rate and doubling time.hiPSCs were seeded on Soft and Stiff PDMS,on TCPs as control.Differentiated hiPSCs into cardiomyocytes by chemical defined method.After 30 days differentaiton,cell beating was detected by inverted phase contrast microscope.The myofibril morphology and structures can be observed by immunofluorescence staining and EM.Using flow cytometery to detect the coexpression efficiency of cTnT and ?-Actinin.Western Blot(WB)and HTA2.0 to detect the cardiac proteins and genes expression.The cell cycling activity of hiPSC-CMs can be showed by EdU labelled staining and ki67 flow cytometery.Calcium transient can be detected by loading fura-2 dye,and the related calcium handling proteins and genes expression to be detected by WB and HTA 2.0.Results: The cell attachment rate of hiPSCs on Soft and Stiff PDMS reached 99%.The doubling time of hiPSCs on TCPs,Stiff PDMS and Soft PDMS which were 19.28±0.48 h,19.67±1.05 h and 18.56±0.72 h,respectively,have no significant difference.The beating clusters of cardiomyocytes can be observed at the differentiation day 8-10,while the robust beating can be observed at day12.The mean beating rate of hiPSC-CMs on TCPs and Stiff PDMS were about 35 bpm,while it was 60 bpm on Soft PDMS.The immunofluorescence staining of ?-Actinin combined with cTnT and EM showed the myofibrils had more well-organization on Soft PDMS,the length of Z band increased on Soft PDMS which was 0.56±0.15?m compared with TCPs which was 0.28±0.09?m,but there was no changed in saromere length.The cardiac structural genes,mRNA and proteins were increased,no significant,expression on Soft matrix,while ?-SMA had significantly decreased.The hiPSC-CMs in mitosis were 21.82±1.46%,12.75±1.68% and 7.97±2.89% on TCPs,Stiff PDMS and Soft PDMS,respectively by Ed U staining,while Ki67 positive expression of hiPSC-CMs were 27.08±2.45%,26.13±1.76% and 17.61±1.42% by flow cytometery.hiPSC-CMs had a robust and lower transient curves,they rised and declined at a substantially slower rate.The amplitude and departure velocity of the calcium release was highest on the Soft matrix and progressively lower on stiffer substrates.Calcium handling proteins and genes were increased.Conclusions: The stiffness had no significant influence on cell attachment,proliferation of hiPSCs.Soft PDMS improved the hiPSC-CMs maturation on cardiac morphology with well-organized myofibrils,cardiac sturctures with enlongated Z band and increased cardiac structure markers,cardiac function with enhanced calcium transient and increased calcium handling proteins and genes,and decreased proliferation.Part 3 The effect of substrate stiffness on mitochondrial maturation in hiPSC-CMsAim: Mitochondria are the powerhouse of heart,to maintain continuous cell beating and physiology of CMs.This part we focused on the effect of stiffness on structure and function in mitochondria of hiPSC-CMs,to explore the relationship between substrate stiffness and mitochondrial maturation.Methods: Mitotracker green staining to show mitochondrial morphology.Immunofluorescence staining of mitochondrial marker Tom20 combined with ?-Actinin to show mitochondrial cellular orgnization.The ultrastrucuture of mitochondria on stiffness matrix can be observed by EM.Mitochondrial structure proteins and genes expression to be detected by WB and HTA2.0.The mitochondrial membrane potential of mitochondria in hiPSC-CMs on stiffness can be detected by TMRE and MTG staining.ATP lite Luminescence Assay System(ATP lite)detected the cellular ATP production of hiPSC-CMs on stiffness matrix.WB and HTA2.0 detected the proteins and genes expression of oxidative phosphorylation complexes(OXPHOS).CM-H2 DCFDA and MTDR staining showed the reactive oxygen species(ROS).Mitochondrial calcium can be detected by Rhod-2 and MTG staining.LTDR and MTG staining show the lysosome contents.Immunofluorescence staining of Cytochrome C(Cyto C)and Tom20 and HTA2.0 to show mitophagy.Results: MTG staining show the mitochondria on Soft PDMS had more elongated and network morphology compared with the stiffer groups.Mitochondria on stiffer matrix were frequently clustered around the nucleus and had little association with the immature contractile apparatus,while the mitochondria on soft matrix were arranged more in a linear pattern spinning the length of the cell and between the developing contractile filaments.Mitochondria on Soft PDMS had tubular cristae,and increased in mitochondrial length and area and the cristae length.On Soft PDMS,cellular ATP production increased by ATP lite.And related proteins and genes in OXPHOS were increased.Mitochondrial membrane potential increased on Soft PDMS,as well as Oxygen Consumption Rate.Soft PDMS decreased the accumulation of ROS and calcium and lysosome.There were no Cyto C released from mitochondria,and HTA2.0 showed apoptosis was inhibited on Soft PDMS.Conclusions: The stiffness improved the mitochondrial maturation of hiPSC-CMs on elongated mitochondrial morphology with tubular cristae,increased oxidative phosphorylation and decreased the accumulation of toxic substances.Part 4 The mechanism of Soft PDMS improved the hiPSC-CMs maturationAim: Detected the expression of YAP/TAZ,the mechanical transducer,in hiPSC-CMs,to explore the possibility of substrate stiffnes modulate the maturation of hiPSC-CMs by YAP / TAZ.Examined YAP/TAZ expression and morphology and function of mitochondria by activating / inhibiting Wnt signaling pathway,to investigate the relationship among Wnt signaling pathway,YAP / TAZ and mitochondrial dynamics in hiPSC-CMs on stiffness substrate,to illustrate Soft PDMS enhanced the maturation of hiPSC-CMs.Methods: WB and HTA2.0 to detect the genes and proteins expression of Mfn2,Opa1,Drp1 and YAP/TAZ.Immunofluorescence staining to show YAP localization in cell.HTA2.0 showed the gene expression in Wnt pathway,Hippo pathway and significance genes.By adding CHIR99021 and IWR-1 to TCPs and Soft PDMS,to detect the mitochondrial morphology by MTG staining and mitochondrial membrane potential of mitochondria by TMRE and MTG staining,and using WB to detect the expression of Mfn2,Drp1 and YAP/TAZ.Results: On Soft PDMS,Mfn2 and Opa1 increased,Drp1 decreased.There was no relocalization of YAP in cell by immunofluorescence staining.HTA2.0 showed YAP decreased while TAZ increased comfirmed by WB.On TCPs by adding CHIR,mitochondria morphology had more elongated and network,increased Mfn2 expression and mitochondrial membrane potential,and decreased YAP/TAZ expression while on Soft PDMS by adding IWR-1 had decreased network mitochondrial morphology,Mfn2 expression,mitochondrial membrane potential and increased YAP/TAZ expression.Conclusions: Soft PDMS enhanced mitochondrial fusion and decreased fission.The stiffness did not affect the nuclear localization of YAP/TAZ.TAZ,independent of YAP,significantly increased expression on Soft PDMS.There was a crosstalking between Wnt pathway,mitochondrial dynamics and YAP/TAZ which inhibition of wnt pathway can decreased mitochondrial fusion and increased YAP/TAZ expression.In summary,the crosstalk among Wnt pathway,mitochondrial dynamics and YAP/TAZ enhanced hiPSC-CMs on Soft PDMS with the organized sarcomere,increased calcium transient,enlongated mitochondrial morphology and enhanced mitochondrial function on.