CRISPR/Cas9-based Study Of Dynamics And Function Of Transplanted Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

Acute myocardial infarction(AMI)is a myocardial ischemic necrosis caused by a sharp decrease or interruption of coronary blood flow on the basis of coronary artery disease.It is also the disease with the highest mortality rate among cardiovascular diseases.Severe arrhythmia,heart failure,and shock after AMI can further increase the infarct size.From the last century till now,the treatment of AMI has experienced the development of landmark methods and technologies such as heart transplantation,drug therapy,surgical recanalization,and cell therapy-based heart regeneration.However,methods such as drugs and surgery methods cannot regenerate necrotic myocardium.In recent years,stem cell therapy has become a research hotspot.Among them,human induced pluripotent stem cells derived cardiomyocytes(hi PSC-CMs)have been considered a promising tool for regenerating injured heart tissue.Despite of the rapid development and promising application prospects of stem cell therapy,the mechanisms of survival,proliferation,differentiation,migration as well as safety issue of stem cell transplantation in vivo still cause confusion.The inadequate in vivo research and the disconnection of translational medicine have severely restricted the true promotion of stem cell therapy to clinical applications.Therefore,it is critical to perform in vivo longitudinal imaging to evaluate the biological behavior and efficacy of transplanted cells.Molecular imaging is a cutting-edge cross field combining molecular biology and modern medical imaging.It can reflect the biological processes of stem cells in live subjects non-invasively at the molecular level and can perform qualitative and quantitative analysis.Currently,the commonly used molecular imaging techniques include optical imaging,positron emission tomography(PET),magnetic resonance imaging(MRI),and ultrasound imaging.These imaging technologies have their own advantages and disadvantages,but none of them can meet the high sensitivity and high resolution requirements at the same time.Research and innovation of multi-modal imaging methods are necessarily needed to further solve the problems related to the clinical transformation of stem cell therapy.For this purpose,the study uses a new generation of clustered regularly short palindromic repeats/CRISPR-associated nuclease 9(CRISPR/Cas9)gene editing technology to integrate a plasmid carrying the triple fusion reporter gene TF(triple fusion)into human urine-derived induced pluripotent stem cells(h Ui PSCs),and thus to construct TF-h Ui PSCs line.TF-h Ui PSCs was further differentiated into cardiomyocytes(TF-h Ui CMs).After CRISPR/Cas9 gene editing,TF-h Ui PSCs and TF-h Ui CMs maintain the characteristics of stem cells and cardiomyocytes,respectively.Also,they show good bioluminescence and PET molecular imaging capabilities,which confirmed by in vitro experiments.Further,in vivo experiments have confirmed that the reporter gene imaging system successfully monitored the biological behaviors including survival,proliferation,and migration of transplanted TF-h Ui CMs.Transplanted cells showed cardioprotective effect by improving left ventricular contractile function,microvascular formation and glucose metabolism.This study combines the latest genetic engineering technology with in vitro and in vivo imaging applications of stem cells to realize the clinical transformation potential of hi PSC-CMs therapy.Part 1: The construction of stem cell lines carrying multiple reporter genes by CRISPR/Cas9 technology In this study,for the first time,we obtained TF-h Ui PSCs that carry reporter genes and verified the pluripotency of TF-h Ui PSCs from multiple levels.The reporter gene plasmids of monomeric red fluorescent protein(mrfp),firefly luciferase(fluc)and herpes simplex virus thymidine kinase(hsvtk)were integrated into the AAVS1 gene locus of TF-h Ui PSCs by using CRISPR/Cas9 technology.The mrfp contained in the reporter gene cell line can be used to evaluate the expression ratio of positive cells during the transfection process,and fluc and hsvtk can achieve bioluminescence imaging(BLI)and PET imaging in vivo,respectively.We also proved that the exogenous reporter gene has no negative impact on the vitality and proliferation capacity of stem cells,which laid the foundation for further in vitro and in vivo experiments.Part 2: In vitro imaging capabilities of reporter gene cell lines and in vivo fate tracking of transplanted TF-h Ui CMs This part conducts imaging studies on TF-h Ui PSCs and TF-h Ui CMs and realizes the multi-modal imaging capabilities of TF-h Ui CMs both in vitro and in vivo.We monitor the longitudinal cell fate of TF-h Ui CMs after transplantation in a real-time.To address the problem of low survival rate after cell transplantation,we pre-treated TF-h Ui CMs with a pro-survival cocktail before transplantation.Multimodal imaging showed that the signal intensity of TF-h Ui CMs gradually increased within 5 weeks after transplantation,indicating the proliferation of transplanted cells in the body.In vivo imaging tracked a series of biological behaviors of transplanted cells,including survival,engraftment,proliferation and migration.Part 3: Study on therapeutic effect and potential mechanism of TF-h Ui CMs treatment on acute myocardial infarction In this part,the improvement of the heart function was explored after cell tranplantation.Transplanted cells promote the cardiac function by improving the microvessel density in the infarct border zone and regulating glucose metabolism,which is manifested by the improvement of left ventricular ejection fraction and left ventricular fractional shortening.Compared with the remote myocardium,TF-h Ui CMs still showed an immature phenotype 5 weeks after transplantation,and the sarcomere structure was similar to fetal cardiomyocytes.Genomic RNA sequencing showed that the myocardial glucose metabolism pathway was up-regulated and the VEGF pathway was up-regulated in the ischemic microenvironment,which are consistent with the corresponding in vivo and ex vivo experimental results.It is helpful to understand the underlying mechanism of TF-h Ui CMs cardioprotection.This study is expected to provide ideas for in vivo fate tracking and functional research after stem cell transplantation.