title: Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells.
abstract: OBJECTIVE
  To development of 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Sherwin Ting, Marti Lecina, Shaul Reuveny, Steve KW Oh, Bioprocessing Technology Institute, ASTAR (Agency for Science, Technology and Research), Singapore 138668, Singapore.
authors.forename: Sherwin
authors.initials: S
authors.lastname: Ting
authors.forename: Marti
authors.initials: M
authors.lastname: Lecina
authors.forename: Yau-Chi
authors.initials: YC
authors.lastname: Chan
authors.forename: Hung Fat
authors.initials: HF
authors.lastname: Tse
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.forename: Steve Kw
authors.initials: SK
authors.lastname: Oh
keywords: Bovine serum albumin; Cardiomyocyte; Differentiation; Human embryonic stem cells; Human induced pluripotent stem cells; Soy hydrolysate
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