Author + information
- Wanwen Lin and
- Chaoquan Peng
human pluripotent stem cells (hPSCs) is generally including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These cells are multipotential differentiation and are a potentially inexhaustible supply of human cells, including cardiomyocytes. Because the convenient accesses, hPSCs offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. In the domain of inducing hPSCs into myocardial cells, there is a problem about the choose of 5azacytidine concerntrations and how to increase the differntiation.
Enriched hPSCs approximately 6*105 cells per milliliter were seeded in poly- L-lysine-coated eight-well slide chambers in a differentiation medium (IMDM supplemented with 10% fetal calf serum, 100 U/mL penicillin G, L-glutamine DMEM-Ham F-12 mix containing 2% B27, 0.1 mmol/L 2- mercaptoethanol, 10 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 40 nmol/L cardiotrophin-1, 40 nmol/L thrombin, L-glutamine). After 2 days the medium was replaced with fresh growth medium. Once confluent, the hPSCs were treated with 5uM or 10uM or 20uM 5-azacytidine, then each group were incubated in media alone in normoxic conditions (untreated controls) or 10 ng/mL recombinant mouse TNF and/or exposed to hypoxia for 6 hours. Then the RNA of each group was isolated and quantitative real-time PCR was performed. In about 4 weeks of differentiation, cardiac-specific transcription factors-myosin heavy chain (MHC) and alpha sarcomeric actin (α-actin)was measured by immunofluorescence stain.
Cultured hPSCs with 10uM 5aza and TNF, hypoxia presented the highest number of α-actin and MHC-positive/1×105 cells compared to the other group(**P<0.01), and Real time quantitative reverse transcriptase polymerase chain reaction(Q-PCR) show the fold rise in expression of α-actin and MHC after treatment(**P<0.01). The α-actin and MHC expression of the group treated with 5uM 5aza is obvious less than the 10uM group. While the 20uM group undergoes a significant of cell apoptosis.
5-azacytidine is a pronouncing inducer of cardiomyocytes differentiation. 10uM of 5-azacytidine is the most appropriate concentration of inducing hPSCs into cardiomyocytes. What's more, TNF, hypoxia intervention will increase the 5aza's induction differentiation.