GATA-4 promotes the differentiation of P19 cells into cardiac myocytes.
ABSTRACT The aim of this study was to investigate the effects of GATA-4 on the differentiation of P19 cells into cardiomyocytes and to examine the relationship between GATA-4 and cardiomyocytes. We constructed vectors to overexpress and silence GATA-4. These vectors, as well as empty ones were transfected into P19 cells. Subsequently, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were performed. The morphology of P19 cells during differentiation was observed using an inverted microscope. Total RNA was extracted from P19 cells. We used real-time PCR to evaluate the expression levels of 6 genes: GATA-4, GATA-6, transthyretin (TTR), alpha-fetoprotein (AFP), Nkx2.5, and alpha-myosin heavy chain (alpha-MHC). The gene expression pattern of these 6 genes is graphically shown for each group. The GATA-4 mRNA level in cells overexpressing GATA-4 was notably higher than that in the controls, whereas the levels in the controls were notably higher than those in the GATA-4-silenced P19 cells. The cell lines overexpressing GATA-4 expressed higher levels of Nkx2.5 and alpha-MHC than the controls. However, the controls expressed higher levels of AFP, GATA-6 and TTR than the cells overexpressing GATA-4. The RNAi group expressed lower levels of TTR, Nkx2.5, and alpha-MHC than the controls, but there were no differences in the RNAi group and the controls with regard to the expression levels of AFP and GATA-6. The gene expression pattern in the cells overexpressing GATA-4 was biased toward the Nkx2.5 and alpha-MHC. On the other hand, the gene expression pattern in GATA-4-silenced cells and the controls was biased toward the TTR and AFP. The overexpression of GATA-4 enhances the differentiation of P19 cells into cardiac myocytes, whereas its down-regulation suppresses this trend.
Article: Directing cardiomyogenic differentiation of human pluripotent stem cells by plasmid-based transient overexpression of cardiac transcription factors.[show abstract] [hide abstract]
ABSTRACT: Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) possess a high potential for regenerative medicine. Previous publications suggested that viral transduction of a defined set of transcription factors (TFs) known to play pivotal roles in heart development also increase cardiomyogenesis in vitro upon (over)expression in mouse or human ES cells. To circumvent issues associated with viral approaches such as insertional mutagenesis, we have established a transient transfection system for straightforward testing of TF-combinations. Applying this method, the transfection efficiency and the temporal pattern of transgene expression was extensively assessed in hPSCs by qRT-PCR, TF-specific immunofluorescence analysis and flow cytometry. Testing TF-combinations in our approach revealed that BAF60C, GATA4 and MESP1 (BGM) were most effective for cardiac forward programming in human induced pluripotent stem cell (hiPSC) lines and human ES cells as well. Removal of BAF60C slightly diminished formation of CM-like cells while depletion of GATA4 or MESP1 abolished cardiomyogenesis. Each of these TFs alone had no inductive effect. In addition, we have noted sensitivity of CM formation to cell density effects which highlights the necessity for cautious analysis when interpreting TF-directed lineage induction. In summary, this is the first report on TF-induced cardiomyogenesis of hPSCs applying a transient, non-integrating method of cell transfection.Stem cells and development 11/2012; · 4.15 Impact Factor