Electrospun Nanofibrous Scaffold Induces Cardiomyocyte Differentiation Of I Ps Cell And Its Mechanism

Background Nowadays, ischemic cardiovascular disease is becoming the leading cause of patient death and disability worldwide. Among them are three most frequent causes of death: heart failure, chronic ischemic heart disease and acute myocardial infarction(MI). Until now, there is no definitive or radical therapy for endstage heart failure which is also the final stage of many kinds of ischemic heart diseases, other than heart transplantation. However, several drawbacks such as the shortage of donor hearts and immunological rejection associated with this approach have long been a concern for clinicians and scientists. New approaches for treatment of cardiovascular disorders are clearly and urgently needed. Recent advances in stem cell biology indicate that regenerative medicine and tissue engineering could play a pivatol role in the future of cardiovascular diseases. Myocardial regeneration requires stem cells to be capable of producing billions of homologous cardiomyocytes(CMs). Among a variety of stem cells, induced pluripotentstem cell(i PSC) is considered as the ideal cell source. Biomaterial scaffold is another critical aspect of myocardial regeneration because it is in the scaffold that the stem cells live, proliferate and later differentiate into billions of homologous target cells. In heart, CMs are surrounded by interconnected porous extracellular matrix(ECM) scaffold, and an electrospinning nanofibrous scaffold can be highly similar to the natural heart ECM in its structural and fibrous characteristics. Thus, the electrospinning nanofibrous scaffold is capable to provide appropriate physical microenvironment for stem cell growth and differentiation in vitro. Most of the existing researches have focused on the adminstration of various cytokines to induce stem cells to efficiently differentiate into CMs. Application of artificial biomimetic scaffold in myocardial regeneration has attracted the growing attention of scientists ever since. Among the few reports of mayocardial reconstruction in vitro, most researches used differentiated CMs combining with scaffolds, some other studies blended ECM ingredients into material scaffold or simply fabricated ECM ingredients into bioscaffolds to culture with CMs. However, the roles of the physical microenvironment of a biomimetic nanofibrous scaffold on CM differentiation of i PSC are still poorly understand. Since there are intimate interactions between stem cells and scaffolds, it is necessary to figure out the possible influences of scaffolds on CM differentiation of i PSCs for future myocardium regeneration.Objective The present study was aimed to investigate the potential effects and mechanisms of the structure and nano-topography characteristics of a three-demensional nanofibrous biomimetic scaffold on CM differentiation of i PSCs, and to explore the feasibility of this bioscaffold for application in future myocardial tissue engineering.Methods 1) Poly-(ε-caprolactone)(PCL) and PCL/gelatin composite nano fibers were produced using the electrospinning method, and the three-demensional PCL and PCL/gelatin nanofibrous biomimetic scaffold were prepared. 2) The physical and chemical properties, as well as cytocompatibility of scaffolds werecharacterized. 3) The mi PSCs were cultured without mouse embryo fibroblasts(MEFs) feeder layer to evaluate the feasibility of the PCL scaffolds applied for mi PSCs proliferation. 4) To explore the influence of biophysical signals originating from the nanofibrous topography and porous structure on mi PSC cardiac myocyte commitment, the Oct4-GFP+ mi PSCs were maintained on the PCL nanofibrous scaffold and in TCPs using the monolayer culture method for CM spontaneous differentiation without supplementary cytokines or small moleculars. 5) To explore the possible factors that influence the CM differentiation of mi PSCs, using western blot to detect and compare the expression levels of those important protein markers, such as c Tn T and α-actinin, of samples with or without scaffolds. Using q PCR to test the m RNA levels of MESP1, GATA4, NKX2.5 and c TNNT2. Applying immunocytochemistry to examine MLC2 a and c Tn T expression and localization. 6) Modulating Wnt/β-catenin signaling with selective activator or inhibitor to investigate the underlying mechanism.Results and Conclusions 1) PCL nanofibrous scaffold characterized by homogeneous nanofibers and a porous three-dimensional microstructure could be manufactured using the electrospinning method, and gelatin coating significantly improved the hydrophilicity of the PCL scaffold without changing its surface morphology. PCL/gelatin composite nanofibrous scaffold could also be fabricated but with larger viriaties in fiber diameters. 2) Gelatin coating significantly elevated the hydrophily of PCL nanofibrous scaffold, leading to improved cell adhesion and proliferation. Thus this PCL scaffold are suitable for application in bioengineering. 3) The mi PSCs could adhere to and proliferate on PCL nanofibrous scaffolds over time, which means the PCL nanofibrous scaffold could be used as substrate for i PSC long time culture. 4) The mi PSCs cultured on PCL nanofibrous scaffolds using the monolayer methodcould spontaneously differentiate into CMs, which was evidenced by cardiac marker protein and m RNA expression. Furthermore, our results indicate that PCL nanofibrous scaffolds are a suitable tissue engineering candidate for potentiating the CM differentiation of mi PSCs. 5) The results of western blot detection and m RNA expression confirmed that the PCL nanofibrous scaffolds are superior to tissue culture plates in directing mi PSC differentiation towards CM. The topographic and structural properties of PCL nanofibrous scaffolds should be responsible for the differences. 6) The Wnt/β-catenin signaling participates in the CM differentiation of mi PSCs on PCL nanofibrous scaffolds. The dual roles of Wnt/β-catenin signaling during cardiomyocyte differentiation of mi PSCs on PCL nanofibrous scaffolds were also observed.In summary, our study demonstrated that a highly porous three-dimensional PCL nanofibrous scaffold fabricated using the electrospinning method was an ideal platform both for i PSC cultivation and cardiac lineage-specific differentiation. We also revealed that PCL nanofibrous scaffolds can direct CM differentiation, which may be mediated by the activation of canonical Wnt/β-catenin signaling at an early stage through interactions between the mi PSCs and the PCL nanofibrous scaffold. Our findings highlighted the intimate relationship between extracellular biophysical signals and intracellular biochemical pathways regulated by cell/ECM adhesion, and the important roles of these interactions in the cardiac myocyte commitment of mi PSCs during monolayer differentiation. These findings will help researchers devise innovative biomimetic scaffolds to efficiently direct the cardiac fate of i PSCs for future myocardial-tissue engineering and regenerative medicine.