The Effect And Mechanism Of Translation Of Induced Pluripotent Stem Cells With Folic Acid-Derived Hydrogel For Mouse Myocardial Infarction Therapy

Background:Induced pluripotent stem(iPS)cells,capable of differentiating into variety of cell types,have the potential to generate cardiomyocytes for myocardial repair after ischemic damage,but their poor retention rate in ischemic myocardium significantly hinders the therapeutic success.In this study,we develop a supramolecular hydrogel of folic acid-modified peptide(FA hydrogel)formed by a biocompatiblie method(glutathione reduction),which is suitable for cell encapsulation and transplantation.This study for the first time reported a newly developed FA hydrogel that can improve the therapeutic capacity of iPS on MI,and the FA hydrogel based stem cell therapy can be a perspective strategy for treating MI.Objective:Investigatie the therapeutic capacity of iPS encapsulated in FA hydrogel on MI,and whether the FA hydrogel could be used as injectable scaffold for the treatment of MI.Methods and results:1.FA-modified peptide derivatives of FA-FFFRGDssEE was designed and synthesized by standard Fmoc-solid phase peptide synthesis.We then tested its self-assembling properties,and characterized the hydrogels of FA-FFFRGDssEE by transmission electron microscopy(TEM).The mechanical properties of the hydrogel was then characterized by rheology.Live/Dead assay and CCK-8 assay were used to test the biocompability of FA hydrogel.The results demonstrated the excellent self-assembling properties of FA-FFFRGDssEE.And the cells can proliferate and spread well in the FA hydrogel,which gradually degraded within 4 weeks in mice heart.2.iPS cells were cultured in a feeder-independent manner,and iPS was induced to differentiate into cardiomyocytes using EB(embryo body)method.The expressions of OCT3/4,MESP1,ISL-1,GATA4,MYH6,cTnT and vWF genes were detected by qPCR on the 0th,3rd,5th,7th,11th and 15th days of differentiation.Immunofluorescence stained MYH6/cTnT and vWF on day 15 of differentiation.The results inficated that the expression of all these markers changed with time,which was consistant with the development of embryo.MYH6/cTnT and vWF-positive cells can be seen in differentiated cells.Subsequently,the proliferation and viability of iPS cultured in FA hydrogel(1.0 wt%,0.75 wt%,0.5 wt%)was detected by CCK-8 and Live/Dead assay.The results showed that iPS cells could proliferate and survive well in all concentrations of FA hydrogel.So we selected the strongest one(1.0 wt%)for follow-up experiments.Differentiation of iPS cultured in 1.0 wt%FA hydrogel was detected by qPCR and immunofluorescence.The results showed that iPS could differentiate into cardiomyocytes and endothelial cells in 1.0 wt%FA hydrogel.We also used flow cytometry to examine the apoptosis of iPS in FA hydrogel under hypoxia stress.The results revealed that FA hydrogel significantly reduced the apoptotic rate of iPS in hypoxia stress.3.iPS encapsulated in FA hydrogel(1.0 wt%)was implanted into the infarcted hearts of C57BL/6 mice for treatment.The experiment is divided into 5 groups:Sham group,PBS group,FA hydrogel group,iPS group,FA hydrogel+iPS group.Transthoracic echocardiography was performed 28 days after transplantation.The results indicated that FA hydrogel+iPS group obviously improved contractile function of the infarcted heart compared with other groups.Masson staining showed that the FA hydrogel+iPS group had the minimal fibrosis.The results of immunofluorescence showed that the survival of iPS was higher in FA hydrogel+iPS group in vivo.MYH6 fluorescence staining showed that iPS differentiate towards cardiomyocyte like cells in mice heart.The vWF fluorescence staining showed that the density of blood vessels in the infarcted myocardium of the FA hydrogel+iPS group was significantly increased compared with the other three groups.Isolectin B4 staining showed that the capillary density of the infarct peripheral area was higher in the FA hydrogel + iPS group.WGA staining showed the minimal hypertrophy of myocardial cells in the peripheral area of myocardial infarction in the FA hydrogel + iPS group.Conclusion:1.FA-modified peptide derivatives of FA-FFFRGDssEE was designed and synthesized,which can form a stable and transparent hydrogel.It has good biocompatibility,tissue compatibility,and can be used as injectable scaffolds for myocardial tissue engineering.2.iPS was successfully induced to differentiate into cardiomyocytes in vitro.And iPS cultured in FA hydrogel can grow,propliferate and differentiate well.3.FA hydrogel increased survival and cardiac differentiation of transplanted iPS in vivo.And improved the therapeutic effects of iPS transplantation for myocardial infarction.