112. FETAL CALF SERUM AFFECTS hESC METABOLISM AND GENE EXPRESSION LEADING TO DIFFERENTIATION IN CULTURE

Abstract

Fetal calf serum (FCS) has conventionally been used to support the growth and maintenance of human embryonic stem cells (hESCs). FCS however, is an undefined complex mixture containing factors which potentially alter the functionality of hESCs. Inclusion of FCS during embryo culture negatively impacts embryo metabolism and viability but comparative studies on hESCs have been hindered by the lack of serum and feeder independent culture systems. Using a recently available defined culture system, the effects of FCS on hESC metabolism and pluripotentcy were investigated. Mel2 hESCs were grown at 37°C in 5% CO2 for 3 days on matrigel (hESC-qualified) coated tissue culture wells in mTeSR1 medium. hESCs were then cultured in mTeSR1 (control) or in mTeSR1 supplemented with 20% FCS from different manufacturers or knockout serum replacement (KOSR), for 96 hours. Media was renewed daily. At the end of the culture period, spent media was collected and cells were trypsinised and counted and/or collected for gene expression analysis. FCS decreased cell survival and altered hESC morphology from densely packed colonies with distinct borders into non-uniform heterogeneous populations comprising of hESC-like cells and fibroblastic-like cells with high cytoplasmic to nuclear ratios. Media analysis revealed altered cell metabolism with increased glucose consumption rates per cell (P < 0.01) with FCS supplementation, compared to cells cultured in mTeSR1 alone. Gene expression analysis revealed that FCS, regardless of its manufacturer, decreased the expression of some pluripotent markers and increased differentiation markers. A decrease in pluripotent gene expression was also observed in hESCs cultured with KOSR compared to mTeSR1 alone. Maintenance of homogeneity in hESC populations is crucial for the advancement of hESC clinical therapies. This study demonstrates that FCS promotes heterogeneity and impacts the metabolic function and gene expression of hESCs thereby supporting the need for serum-free culture systems as standard practice in hESC culture.