Influence of culture pH on proliferation and cardiac differentiation of murine embryonic stem cells

• Embryonic stem cells are sensitive to pH during cardiac differentiation.
• pH of 6.8 affected proliferation, viability and differentiation of mESCs.
• Optimal culture performance for E14TG2a mESCs was at pH 7.1–7.4.

Embryonic stem cells (ESCs) possess great potential in many tissue engineering applications such as cardiac regeneration. Culture pH is important in stem cell cultures as they could impact vitality of the culture and cell fate decisions. In this study, we demonstrated how sensitive ESCs are to pH, utilizing murine ESCs as a cell model and experiments conducted at three different pH conditions (pH 6.8, 7.1 and 7.4). Maintenance of culture pH was achieved via a perfused rotary bioreactor while murine ESCs were encapsulated in alginate hydrogels, which served as a three-dimensional (3D) platform and matrix support for the ESC culture. Our results showed that at pH 6.8, ESC viability was inferior to those at pH 7.1 and 7.4. Lower cardiac gene expressions and percentage of cardiac troponin-I positive cells at pH 6.8 indicated that cardiac differentiation of ESCs was significantly compromised. Concurrently, residual pluripotency of ESCs was better conserved at pH 6.8 as compared to higher pH conditions. Finally increased levels of MAPK14 and HIF-2α suggest an impact of pH on kinase and HIF regulated pathways. This study highlights how a small change in pH could significantly affect the growth and differentiation of ESCs toward cardiomyocytes. Therefore, there is an important need for good control of culture parameters such as pH in ESC cultures, so as to obtain the optimal and desired cell output.