Published online Jul 26, 2013. doi: 10.4252/wjsc.v5.i3.86
Revised: June 3, 2013
Accepted: June 8, 2013
Published online: July 26, 2013
AIM: To develop an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells.
METHODS: Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcription-polymerase chain reaction.
RESULTS: hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar.
CONCLUSION: The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.
Core tip: Nutrient supplements were screened for improving cell survival during the cardiomyocyte differentiation process of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) in a serum-free medium based on the inhibition of p38 MAPK (SB media). Soy hydrolysate and bovine serum albumin supplementation was found to improve cell viability and therefore increased the yield of HES-3 cardiomyocytes by 2.6-fold over non-supplemented SB medium. The enhancing effect of this medium was demonstrated in an additional hESC line (H1) and Siu1-hiPSC cell line (15 and 9 fold respectively). The cardiomyocytes formed expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like.