Generation Of Induced Cardiomyocytes Derived From Somatic Cells Of Patients With Cardiovascular Diseases

Background:Cardiovascular disease has been a serious threat to human health,although with the progress of the study,epidemiological characteristics,clinical manifestations and some effective treatments have been found,but mechanism of most diseases is still unclear,especially some inherited heart diseases.One of the most important reasons is lack of disease-specific models or the models available cannot completely mimic the process of human diseases.In addition,as resident cardiomyocytes hardly have the capacity to regenerate after birth,the heart cannot fully play its role once injury,which may lead to heart failure or even cause death.Currently,major treatments to hypertension,coronary heart disease,arrhythmia,cardiomyopathy and congenital heart disease,except heart transplantation,are controlling symptoms,delaying progression,which mostly fail to achieve the purpose of completely curing the diseases.Moreover,even the heart transplant,it’s limited by donor shortages,immune response and many other unfavorable factors.However,with the advent of induced pluripotent stem cells(iPSCs)and induced cardiomyocytes(iCMs),the situation is expected to change.Objective:To generate patient-specific iPSCs and iPSCs-derived cardiomyocytes by collection and separation somatic cells of patents who are with cardiovascular disease,which can be applied to the study of mechanisms of disease and drug screening.Methods and Results:Urine and peripheral blood of patients who are suffering cardiovascular diseases were collected,from which somatic cells were isolated and cultured,and then episomal combinations coding Sox2,Klf4,Oct4,L-Myc,Lin28,mp53 DD,EBNA1 were electroporated into them.After induced pluripotent stem cells(iPSCs)were obtained,their pluripotency and stability were confirmed by standard procedures including prominent nucleoli structure,higher nucleo-cytoplasmic ratio,higher cell density of clone paving stone on the whole appearance and positive alkaline phosphatase staining observed under inverted microscope,and immunofluorescence staining and qPCR for expression of pluripotency markers including OCT4,SOX2,NANOG,SSEA-4 and TRA-1-60,which were totally similar to human ESC.Additionally,embryonic body in vitro and teratoma in vivo formation test demonstrated that iPSCs hold potential to differentiate into all three germ layers,which were also verified by immunofluorescence microscopy for the appropriate markers.Then,with the use of small molecules Rapamycin,CHIR99021,KY02111 and XAV939,we gained iCMs by method of monolayer and free growth factors.Beating cells were observed on the 10 th day,while cardiac-specific markers including cTnT,cTn I,MHY6,a-Actinin were also proved by immunofluorescence staining and qPCR.Conclusion:With the study of reprogramming and directional differentiation,we successfully established bank of urine cells and peripheral blood mononuclear cells,patient-derived and disease specific-iPSCs and iCMs,which can be widely used in the area of mechanism study and drug discovery,and may lay the foundation for personalized medicine and precision medicine.