Study On The Differentiation Of Induced Pluripotent Stem (iPS) Cells Into Cardiomyocytes And Their Roles In Myocardium Reconstruction

Given the capacity of self-renewal and potential differentiation, embryonicstem cells (ESC) and induced pluripotent stem cell (iPSC) may be used forcardiac regenerative medicine. Different from ESC, iPSC generated from adultfibroblasts re-programming might provide a homologous source ofcardiomyocytes (CM) avoid ethical debate and immunological rejection aftertransplantation. Therefore, using the iPSC-derived cardiomyocytes instead ofprimary cardiomyocytes as the seed cells for cardiac tissue engineering will beimportant development trends in the cardiac regeneration field.ESC and iPSC have been confirmed that they can differentiate to functionalcardiomyocytes in vitro and in vivo. However, the low efficiency andheterogeneity of differentiated cells may lead to the risk of tumorigenesis andproarrhythmia, which limits their cardiac regeneration application. Although CMcan be efficiently derived from ESC, long-term maintaining structural andfunctional properties of these ESC-derived CM needs more research. The differentiation from stem cell to mature CM involves a complex network oftranscriptional regulators for cell differentiation. The interactions of cell-cell andcell-matrix may play an important role in the efficient differentiation of stem cells.Co-culture with other cell may contribute to the site-specific differentiationmicroenvironment. Here we combine Vc-induced and Co-culture to build up aCM differentiation model for ESC. By using the model, we investigate molecularmechanisms and signaling pathways leading to efficient differentiation from ESCin the co-culture system. We try to explore the available procedure to remove thecellular components from heart tissues and the role of ESC/iPSC in the cardiacregeneration field.1）Co-culture model for CM differentiationObjective: Observe the effects of MEF or NCM co-culture on ESC cardiacdifferentiation based on Vc-induced, to establish a CM differentiation model forESC.Methods: Mouse ESC were cultured in hanging drops to form EB and treatedwith0.1mmol/L ascorbic acid to induce the early-stage differentiation of ESCinto CM. In the indirect co-culture system, EBs were co-cultured with mouseembryonic fibroblasts (MEF) or neonatal CMs (NCM) by the hanging cell cultureinserts (PET membranes with1μm pores). The molecular expressions andfunctional properties of ESC-derived CM in prolonged culture course wereevaluated.Results: Beating EBs are first observed approximately8days after differentiation.The beating EBs can expressed cardiac-specific markers (GATA4、MLC-2V、CX43), and were stained positively for cTnI and CX43. During time course ofESC differentiation, the percentages of EBs with contracting areas in MEFs co-culture and NCMs co-culture were significantly higher than that withoutco-culture. The GATA4、MLC-2V、and CX43expressions were increased in theco-culture model, whereas GATA6expression were decreased. cTnI and CX43immunostaining indicates that the cardiac specific proteins were present indifferentiating EBs.Conclusions: These results indicate that MEFs as well as NCMs co-culturepromote ESC differentiation and co-culture with NCMs has a further effect oncell growth and differentiation.2) Study on the CM differentiation of iPSC in the co-culture modelObjective: Investigate the CM differentiation of iPSC in the co-culture modeliPSC, to reveal the mechanisms leading to efficient differentiation from iPSC inthe co-culture system.Methods: Mouse iPSC were cultured in hanging drops to form EB and treatedwith0.1mmol/L ascorbic acid to induce the early-stage differentiation of iPSCinto CM. In the indirect co-culture system, EBs were co-cultured with mouseembryonic fibroblasts (MEF) or neonatal CMs (NCM) by the hanging cell cultureinserts (PET membranes with1μm pores). The Oct-4、GATA4、Nkx2.5、ANF、CX43expressions were evaluated by RT-PCR and the GATA4、ANF expressionswere evaluated on4d、8d、12d、16d、20d、24d、28d、32d by real-time PCR.The functional properties of iPSC-derived CM during culture course wereevaluated by β-Adrenergic Stimulation. FCM and BrdU analysis were utilized todetect the proliferation of iPSC-derived CM. We detect the integrinα1andintegrinα2receptor expressions during differentiation and block the integrinreceptor to investigate their effect on αMHC、NKX2.5、Mef2C、GATA4expressions. Results: Just like ESC, Beating EBs were first observed approximately8daysafter differentiation. Oct-4is a mark of undifferentiated iPSC. Semi-quantitativeRT-PCR demonstrated that Oct-4expression was reduced over a time course. Thebeating EBs can expressed cardiac-specific markers (GATA4、Nkx2.5、ANF、CX43), and their expressions were increased in the co-culture model. Real timePCR analysis on GATA4and ANF showed that their expression was relativelymaintained by co-culture with NCMs in prolonged culture time course.Co-culture with MEF or NCM has an effect on the late-stage differentiation ofiPSC into CM. Compare with MEF, co-culture with NCM further improves theefficiency of iPSC. The results of FCM and BrdU analysis indicate that MEFsand NCMs co-cultures can promote the CM proliferation in late-stagedifferentiation. It is α1β1integrin, not α2β1integrin blocking did decrease theαMHC、NKX2.5、Mef2C、GATA4expressions.Conclusions: These results indicate that MEFs and NCMs co-cultures canpromote iPSC differentiate into CM involving the activation of α1β1integrinpathway to promote the CM proliferation in late-stage differentiation.3) Heart tissue acellularization and regenerationObjective: We try to explore the available procedure to remove the cellularcomponents from heart tissues and study the role of ESC/iPSC in the cardiactissue regenerationMethods: The heart was obtained from an adult rabbit and immediately washedin PBS. The cellular substances in myocardium tissue were removed by SDScleaning and trypsin digestion to obtain acellular myocardial tissue. Afterdocumented the acellularization by hematoxylin-eosin staining (HE staining), theacellular matrix was freeze-dried and treated with radiation for sterilization. The CM derived from ESC/iPSC in the co-culture group were trypsinized,re-suspended in culture medium, and then were seeded onto acellularmyocardium tissues with a density of approximately106~107cells per cm2. Thetissue regeneration was detected by HE staining and α-actininimmunocytochemistry.Results: SDS cleaning in myocardial tissue failed to remove the cellularsubstances completely. Treatment of the tissue with trypsin and NaOH convertedmyocardial tissue into a cell-free scaffold. After implantation, the acellular matrixwas incompletely covered by cells at1week, and the interstitium of acellularmatrix was found to be densely repopulated by cells at2weeks. The result ofα-actinin immunocytochemistry indicates that these repopulated cells are CMs.Conclusions: Treatment of the tissue with trypsin and NaOH convertedmyocardial tissue into a cell-free scaffold, which can be repopulated by cells. TheCM from iPSC can be used as seed cell for cardiac tissue engineering application.