Abstract
BackgroundCell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking.Methodology/Principal FindingsUsing a 1H-Nuclear Magnetic Resonance (1H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the 1H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at −20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine.Conclusions/Significance 1H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.
Highlights
Human embryonic stem cells harbor the capacity to differentiate into all primary human cell types [1,2,3] and can be cultivated in vitro indefinitely under specified culture conditions [4]
The most abundant metabolites in the TeSR-1 media conditioned by passage 11 human foreskin fibroblasts (HFFs) (CMp11) are labelled and include energy substrates, amino acids and vitamins
To facilitate the rapid examination of global metabolic changes in the media following conditioning, 1H-Nuclear Magnetic Resonance (1H-NMR) spectral profiles from batches of unconditioned (UM) and conditioned CM prepared from passage 11 (CMp11) samples were analysed by Principal Components Analysis (PCA)
Summary
Human embryonic stem cells (hESCs) harbor the capacity to differentiate into all primary human cell types [1,2,3] and can be cultivated in vitro indefinitely under specified culture conditions [4]. Proteomic analysis of CM derived from human neonatal fibroblasts revealed a complex mixture of growth factors, extracellular matrix proteins and differentiation factors potentially involved in the derivation of hESCs [18] While such investigations have shed light on important protein components of CM, a paucity of information exists regarding the metabolite component of CM. Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking
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