The Establishment Of An Induced Pluripotent Stem Cell Model Of Dilated Cardiomyopathy

Dilated Cardiomyopathy (DCM) is a kind of idiopathic myocardial disease characterized by left ventricular or both ventriculars dilatation, systolic dysfunction, and progressive heart failure. DCM is the most common form of cardiomyopathy, the pathogenetic condition getting worse progressively and death can occur at any stage of the disease. DCM is the main cause of heart failure. The pathogenesis of DCM is still unknown, the onset of DCM are thought to include virus infection, autoimmunity, apoptosis, genetic factors. In recent years, clinical study revealed that some of the patients with DCM are familiar, and pedigree investigation and echocardiography of the family members of DCM confirm that25%-35%of DCM cases have obvious family history which known as familial dilated cardiomyopathy (FDCM). The inheritance modes of FDCM include autosomal dominant, autosomal recessive, X-linked inheritance and maternal inheritance. FDCM is an important resource for the genetics and pathogenesis study of DCM. However, due to the difficulty to get the pathological tissue of heart, we can not carry out the pathogenesis mechanism study in vivo.In2006, Japanese scientists Shinya Yamanaka first reported that forced expression of exogenous Oct4, Sox2, Klf4and c-Myc could reprogram mouse embryonic fibroblasts (MEF) into induced pluripotent stem cells (iPSCs). And then in2007, the Shinya Yamanaka group and Thomson group independently reported successful reprogramming of human cells. The morphology, whole genome expression patterns, epigenetics and differentiation potential of iPSCs are well resemble with that of embryonic stem cells (ESCs). Since the tissue for generating iPSCs can be easily obtained, and iPSCs showes same differentiation potential with ESCs, it is possible to generate the iPSCs using tissue from the patient, and differentiate them into functional cardiomyocytes, which may help us to investigate the pathogenesis of DCM in a patient originated cell model.The previously study in our laboratiary has collected a large DCM pedigree accompanied by conduction dysfunction. Genetic study suggested that TNIK c.153A>G heterozygous mutation co-segregated with the disease in the family. In order to study the possible pathogenesis mechanism of the TNIK mutation in cardiomyocytes, we obtained patient’s tissue by skin biopsy. The human dermal fibroblasts (HDF) were obtained through primary culture. Simultaneously, we used293T cells to package pseudovirus carrying Oct4, Sox2, Klf4and c-Myc, which were then used to infect the HDF. With the expression of Oct4, Sox2, Klf4and c-Myc, and addition of Vitamine C and valproate, the HDF were reprogrammed to ES-like clones eventually22days later. These clones can be expended by using ESCs’culture method, and maintain ESCs-like morphological characteristics. AP staining, Karyotype analysis, endogenous gDNA mutation detection, immunofluorescence staining, pluripotency genes expression analysis, and in vivo and in vitro differentiation were then carried out, the results revealed that these cells were fully reprogrammed iPSCs originated from HDF of the patient. Our study also used ESCs to establish a method for cardiac differentiation of pluripotent stem cell, we successful ontained the cardiomyocytes from ESCs but failed in iPSCs differention.