Induced Pluripotent Stem Cell-and Nanofiber Scaffold-based Myocardial Tissue Engineering

BackgroundCardiovascular disease(C VD)is the leading cause of death all over the world.M In the past a few years,more than 30% of the total deaths each year were due to CVD,constituting both a heavy economic and social burden.Myocardial infarction(MI)is the most common type of CVD with high morbidity and mortality.MI frequently progresses to heart failure,eventually death,accompanied by cardiac remodeling with the permanent loss of up to billions of cardiomyocytes that are replaced by myofibroblasts to form scar tissue.With very limited self-regeneration ability,human cannot sufficiently regenerate the injured heart after MI.The current therapeutic approaches,such as medication,intervention and surgical bypass,can limit the disease developments,but they are incapable of restoring reduced cardiac function and reversing remodeling completely.Nowadays,whole heart transplantation is one of the most effective option to treat patients with severe MI.Over the past decade,great breakthroughs in stem cell biology have offered several potential strategies for heart regeneration.However,several drawbacks have been found during stem cell-based heart regeneration trials,such as implanted cells show poor survival and retention after transplantation,as well as unable to regenerated a fully organized and functional cardiac tissue within the host heart.Therefore,engineering a transplantable cardiac tissue patch in vitro has become an alternative option.ObjectiveThe present study was aimed to build a mature,mechanical and electrical coupled artificial cardiac tissue grafts with i PSCs and three-demensional nanofibrous biomimetic scaffolds.We tried to explore the feasibility of i PSC-and nanofibrous scaffold-based myocardial tissue engineeringMethods1)For the handing drop method,embryoid bodies(EBs)were formed in hanging drops consisting of a certain number of Oct4-GFP+ induced pluripotent stem cells(Oct4-GFP+ i PSCs).For the monolayer method,Oct4-GFP+ i PSCs were seeded in the Matrigel pre-treated culture plates.Spontaneous differentiation wit hout ascorbic acid or induced differentiation with ascorbic acid were carried out separately for 15 days and cardiomyocyte specific protein markers,such as cardiac troponin I(c Tn I)and myosin light chain-2a(MLC2a),were probed by immunocytochemistry.2)Ascorbic acid was used as an inducer during EBs differentiation and the proportion of beating EBs was calculated.3)Both of the three-demensional Poly-(?-caprolactone)(PC L)and PCL/Gelatin composite nanofibrous biomimetic scaffolds were produced using the electrospinning method.Induced cardiomyocyte differentiation of Oct4-GFP+ i PSCs on these scaffolds were performed,and the potential of future applications in cardiac tissue engineering was properly evaluated.4)The Oct4-GFP+ i PSCs were cultured on PCL and PCL/Gelatin composite nanofibrous biomimetic scaffolds to explore the possible influence of nanofibrous topography on i PSC differentiation and the possible mechanisms involved in.5)We employed monolayer method,scaffolds and a combination of several differentiation inducers to differentiate i PSCs into cardiomyoctes with a more mature phenotype.Flow cytometry was used to investigate the proportion of cardiac troponin T(c Tn T)positive cel s.6)Immunocytochemistry was adopted to reveal the difference of MLC2 v expressed in cells between scaffolds and tissue culture plates,and transmission electron microscopy was used to compare the difference of sarcomere structures formed on scaffold and in plates.7)Finally,we tried to engineer myocardial tissues de novo from the Oct4-GFP+ i PSCs with a spontaneous rhythm and synchronous contraction/relaxation activity by means of adding several inducers during the differentiation period on scaffolds,prolonging culture days from 15 days to 25 days and using the monolayer method as well.Results and Conclusions1)The EBs formed by the hanging drop method were of the same size,and could differentiate into beating EBs spontaneously.The addition of ascorbic acid increased the rate of beating EBs significantly.The monolayer method could also be used to induce i PSCs into cardiomyocytes.Immunocytochemistry results showed that some cells were c Tn I positive using both EB method and monolayer method,suggesting the Oct4-GFP+ i PSCs could be used to differentiate into cardiomycytes.2)PCL and PCL/Gelatin nanofibrous scaffold characterized by homogeneous nanofibers and a porous three-dimensional microstructure could be manufactured using the electrospinning method.The characteristic of these scaffolds were similar to native extracellular matrix,and could be used as substrates to differentiate i PSCs into c Tn T+ and/or MLC2a+ cells.RNA analysis revealed the dynamic gene expressions from pluripotent stem cells to mesenchymal cells,cardiac progenitor cells and finally to cardiomyocytes.Western b lot confirmed the existence of cardiac specific proteins such as c Tn T,?MHC and MLC2 Thus both the PC L and the PC L/Gelatin nanofibrous scaffold are good platforms for cardiomyocyte differentiation from iPSCs.3)Based on the results of Western blot,RT-PCR,q RT-PCR and flow cytometry,the PCL nanofibrous scaffold is more potent than tissue culture plates in directing i PSC differentiation towards cardiomyocytes.The topography and nanofibrous structure of the nanofibrous scaffold may responsible for this positive effect,and we found that the canonical Wnt signal may also participate in the progress.4)We achieved a high yield rate of cardiomyocytes,approximately more than 70% according to flow cytometric analysis,from i PSCs cultured on scaffolds by using a combination of ascorbic acid,CHIR99021 and Dickkopf-1(DKK-1).5)Cardiomyocytes differentiated from i PSCs on scaffolds were more mature than its counterparts in plates in terms of highly-organized sarcomere structures and more cells were cardiac specific proteins MLC2 v positive,suggesting that nanofibrous scaffolds can effectively promote the differentiation of mature cardiomyocytes6)By using several differentiation inducers,employing PC L/Gelatin nanofibrous scaffold and prolonging culture time,we obtained i PSC-derived cardiomyocytes in a more mature state with highly-organized sarcomeres observed by electron microscope.Immunocytochemistry also revealed a more clear arrangement of c Tn I proteins within these cardiomyocytes,which added evidence to support the idea of a more mature state.7)Finally,we successfully developed an engineer myocardial tissues de novo from the Oct4-GFP+ i PSCs with an independent rhythm and synchronous contraction/relaxation activity.In summary,our study demonstrated that the highly porous three-dimensional nanofibrous scaffold fabricated by e lectrospinning method is an ideal platform for cardiac lineage-specific differentiation from i PSCs.We also highlighted that an engineered myocardial syncytium with relatively mature cardiomyocytes,independent rhythm and synchronous systolic/diastolic functions was achievable by using this scaffold and some inducing factors.O ur research provided some practical experence for the construction of a transplantable tissue engineered myocardium.