IGFBP-4-induced Differentiation Of IPSCs Into Cardiomyocytes And The Defense Mechanism Of PML-NBs Protein On Rep Protein-mediated AAV Plasmid Integration System

Part 1 Insulin-like growth factor binding protein-4 enhancing cardiomyocyte induction in mouse induced pluripotent stem cellsThe establishment of induced pluripotent stem cells(iPSCs) potentially provide a unique resource for generating patient specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, the efficiency and yield of cardiomyocytes currently limits the application of this technology.Given the critical role of Wnt/β-catenin signaling plays in embryogenesis, IGFBP-4, a recently discovered inhibitor of Wnt/p-catenin signaling is a valuable protein to dissect cardiomyocyte differentiation in iPSCs.In the present study, we first identified IGFBP-4 treatment protocol by counting the contract embryoid bodies (EBs) under different time window and various dose of IGFBP-4. The expression of cardiac-specific genes and proteins were significantly enhanced by addition of IGFBP-4(day6-8). Using immunocytochemical staining, those induced cardiomyocytes showed typical cardiac markers. Early cardiac transcription factors but not mesodermal differentiation was promoted by IGFBP-4. Finally, IGFBP-4 mediated differentiation was not cardiomyocyte specific and it did not affect iPSCs maintenance or early differentiation. Thus, cardiomyocyte differentiation was enhanced in overexpressed IGFBP-4-iPSCs as compared with control iPSCs. IGFBP-4, addition acturally inhibited the expression of β-catenin protein in Wnt/p-catenin signaling pathway.Our study established a strong foundation and rationale to pursue future research using IGFBP-4 in the ongoing development of iPSCs transplantation therapy for the treatment of heart failure.Part 2 Role and fate of SP100 protein in Reponse to Rep-dependent nonviral integration systemPreviously, we studied an AAVS1 site specific non-viral integration system with a Rep-donor plasmid (pRC) and a plasmid containing adeno-associated virus (AAV) integration element. Earlier study mostly focused on the plasmid vector itself, but the cellular response to the system was still unknown. SP100 is a member of the PML nuclear bodies (PML-NBs). It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. In this study, we revealed that SP100 inhibited the Rep-dependent nonviral integration. Conversely, transient expression of Rep78 increased the degradation of SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome. SP100 and Rep78 are both located in the nucleolus, which provides the spatial possibility for their interaction. Rep78 was co-immunoprecipitated with the EGFP-SP100 fusion protein but not EGFP, which verified the interaction between Rep78 and SP100. These results have enriched our knowledge about the cellular protein SP100 and Rep-dependent nonviral integration. It may lead to an improvement in the application of Rep-related transgene integration method and in the selection of target cells.