Human Extended Pluripotent Stem Cell-derived Cardiomyocytes Promote Functional Recovery Of Myocardial Infarction In Rats

Due to the limited ability of heart regeneration in adults,it is impossible to fundamentally improve myocardial necrosis and ventricular remodeling after cardiac disease.Induced pluripotent stem cell(i PSC)is a type of stem cells with pluri-directional differentiation potential.Its derived functional human cardiomyocytes are widely used for cardiac disease modeling,pathogenic mechanisms,drug screening and cell therapy.However,because of i PSC heterogeneity,the poor survival rate in a single cell,and differentiation bias,limiting differentiated cardiomyocytes of application for low maturity and unstable efficiency.Extended pluripotent stem cell(EPSC)is a new type of stem cells with different pluripotency states and molecular characteristics.Due to its outstanding bidirectional developmental potential of embryonic and extraembryonic,superior interspecies chimeric ability and longterm stability of culture,it will become a new candidate seed for differentiation functional cells in vitro.It was proved that EPSC-derived hepatocytes showed improved function and a more similar transcriptome to human primary hepatocytes than i PSC-derived hepatocytes.However,whether EPSCs can efficiently generate other lineages such as cardiomyocytes and how the EPSC-derived cardiomyocyte(EPSC-CM)function compared with ESC/i PSC-derived cardiomyocyte(ESC/i PSC-CM)have not been studied yet.This research mainly conducts the following three parts:1)To explore whether EPSC can be directed to differentiate into mesoderm lineage cardiomyocytes,as well as the efficiency and stability of differentiation.First,two cell lines,i PSC and ESC,were transitionally transformed into EPSC.Then,attempt different transition media,initial cell confluence,small molecules concentrations and differentiation duration times to discover the optimal CM differentiation scheme of EPSC.Next compared the efficiency and stability of markers at different stages,as well as transcriptome analysis the differentiation tendency of different PSCs.Last,in an attempt to simplify the components of preconditioning system for EPSC differentiation.The result are as follows: We successfully obtained two different EPSC lines: EPSWTC and EPSH9 can be stably cultivated in vitro.But cardiomyocytes differentiation efficiency is low if initiate from EPSC directly.The optimal differentiation conditions are treated EPSC with m Te SR?1 for 2days combined with WNT signaling small molecule regulators CHIR,i WR-1,IWP2.Moreover,EPSCs generated a higher percentage of 88% CTNT+ cardiomyocytes more robustly and the efficiency was still very stable for culture over 100 passages.Besides,we found that treatment with FGF2,TGFβ,and insulin instead of m Te SR?1 for preconditioning significantly increased the CM generation efficiency.MTe SR?1 treated extended pluripotent stem cell(EPMC)has enhanced pluripotency potential than i PSC,also were in a state more prone to differentiate.2)To explore what’s the specific characteristics and functions of EPSC-CM,and whether the CMs are superior to the maturity of i PSC-CM?The characteristics and functions of EPSC-differentiated cardiomyocytes were comprehensively evaluated from following aspects: basic contractile unit and connection structure,mitochondrial morphology and function,calcium handling capacity,cell proliferation rate and myocardial electrophysiology through immunofluorescence,flow cytometry,seahorse,MEA,patch clamp,force testing system,transmission electron microscope and optical mapping technologies.In addition,through two three-dimensional forms of engineered heart tissue(EHT)strips and sheets were constructed.the microscopic arrangement structure,mechanical contractility and calcium conduction were evaluated in micro-tissue level.The results are as follows: EPSC-CM has significantly increased mitochondrial content and higher ATP generation from oxidative metabolism.Calcium transient in EPSC-CM has higher amplitude and shorter duration,faster depolarization speed.Besides,two groups proliferation rate was the same in both groups,but EPSC-CM single cell APD30 was shorter and beating faster.Transcriptome analysis also enriched the high expression of genes related to calcium cycling,contraction,mitochondrial function,cell survival and oxidative metabolism in EPSC-CM.What’s more,EHT-Bundle in EPSC-CM group showed construction of T tubules,M lines and cell junctions distribution polarization and contractile properties of high absolute force,positive pacing response,strong drug sensitivity,good frequency-force relationship(FFR)in microtissue level.EHT-Patch calcium handling in EPSC-CM group have improved uniform conduction direction,higher maximum amplitude,faster depolarization rate and shorter 80%duration than i PSC-CM group.3)To explore whether EPSC-CM transplantation could survive in the myocardium and be used as a good cell source to potentially improve myocardial ischemia heart injury?First,EPSC-CM mixed with Matrigel,small chemical compounds was directly injected into the myocardium of nude rats.After 4 weeks transplantation,the cell resident status of EPSCCM and cardiac function were evaluated.Next,we performed myocardial infarction(MI)nude rat model and transplant cell compounds again to detect the transplantation safety,cell retention,proliferation,apoptosis and rat cardiac function,fibrosis,infarct size for comparing the function recovery of EPSC-CM and i PSC-CM.The results are as follows: After injecting EPSC-CM into normal nude rat hearts for 4 weeks,we confirmed the cell engraftments were largely survival with relatively mature sarcomere structure,formed basic cell connections,and vascularized regeneration in transplantation area and surrounding areas.Before and two days after MI modeling and EPSC/i PSC-CM transplantation,there was no significant difference in cell proliferation,residency,and fibrosis and cardiac function of two groups.Six weeks after cell transplantation in MI rats,EPSC derived grafts were significantly better improved 10% of heart LVEF,less infarcted size,fibrotic replacement and lower apoptosis rate than i PSC derived grafts while the two cell types formed comparable sizes of graft implants.In conclusion,this study was the first to explore a way of human EPSC for differentiation of the cardiac lineage.Cardiomyocytes derived from EPSC showed high efficiency and robustness and exhibited improved mitochondrial function,calcium handling,and contractility properties at monolayer and microtissue levels in contrast to ESC/i PSC-CM.Most importantly,EPSCCMs restored cardiac function and performed better than i PSC-CM in the nude rat MI model,thus being a better candidate cell source for cardiomyocyte maturation and regenerative therapy.This work will contribute to a broader understanding of future expectations for human-induced cardiomyocyte clinical study.