Stem Cell Biology

Abstract

Differentiation of human embryonic stem cells (hESCs) into cardiomyocytes in culture may offer unique opportunities for modeling genetic disorders, screening potentially cardiotoxic pharmaceutical agents or replacing cells of the diseased heart. However, while more than 200 hESC lines have been derived worldwide, cardiac differentiation has been documented in less than 4% of lines. This is likely a consequence of the laborintensive nature of maintaining multiple lines that apparently require different culture media, passaging methods, culture substrates and feeder cell types / densities. Since these varying culture parameters may influence differentiation capacity, it is challenging to extrapolate the broad applicability of specific differentiation protocols and thus each must be tested empirically. Thus, our laboratory is standardizing culture conditions between the independently-derived hESC lines to facilitate interline comparison and produce generic cardiac differentiation protocols. Maintenance of the BG01 (derived by BresaGen, Georgia) and HUES-7 (derived at Harvard University) has routinely used different media and employed manualand trypsin-passaging, respectively, on mouse embryonic fibroblast (MEF) feeder layers. We find that proliferating, undifferentiated BG01 and HUES-7 can be adapted to trypsin-passaging on MEF feeder layers or on Matrigel in feeder-free conditions in standardized medium. These cultures were induced to differentiate by forming embryoid body formation (EB) in suspension culture with MEF conditioned medium. Subsequent culture in fetal bovine serum-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 ± 2.3 and 41.1 ± 3.1 for BG01-EBs and HUES-7-EBs, respectively. Beating EBs responded appropriately to isoprenaline and propranolol, generated calcium waves, and expressed relevant ion channels and cardiac-specific genes. Thus these standardized culture and differentiation conditions function between two independently-derived hESC lines and will serve to provide a platform for examining the generic efficacy of cardiac inducers.