Metabolic Impact of Culture Media on Pluripotent Stem Cell Growth and Differentiation

Since the discovery of culture conditions that allow for the maintenance and proliferation of human pluripotent stem cells (hPSCs) researchers have attempted to develop chemically defined media which can be used to safely expand hPSCs.  These efforts have included the engineering of functional matrices that mimic or replace undefined substrates (e.g. Matrigel) and the addition of signaling molecules which maintain hPSCs in a highly proliferative state.  Early studies focused on the design of culture media that does not require conditioning by embryonic fibroblasts, and more recently researchers have successfully developed chemically defined liquid media containing as few as 8 components.  These latter efforts have been motivated, in part, by experiences in the bioprocess industry, as undefined components added to clinical biologics processes can be a source of contamination by adventitious agents.  Furthermore, such additives may also cause lot-to-lot variability which complicates production processes and/or and interpretation of experimental outcomes.  These concerns are well-founded and of critical importance as we attempt to scale-up pluripotent stem cell and adult progenitor cell cultures for clinical applications in regenerative medicine.  However, unlike common industrial bioprocesses that produce therapeutic protein products in Chinese Hamster Ovary (CHO) cells, cell-based therapies are themselves the clinical product and must therefore match or replace the function of tissues in the body.  Importantly, these functions almost always involve highly regulated metabolic activities.  Researchers must therefore pay increasingly close attention to the metabolic performance of stem cells and their derivatives as we attempt to develop and scale-up cell-based therapies for more functionalized tissues.

     To better characterize the metabolic behavior of hPSCs we have applied 13C metabolic flux analysis (MFA) to human embryonic stem cells and their derivatives.  These studies involve application of stable isotope-labeled substrates such as [13C]glucose or [13C]glutamine, mass spectrometry-based metabolomics, and systems-level analysis of isotope enrichment in metabolic networks to estimate intracellular fluxes.  Our investigations of metabolic performance in standard (conditioned) versus several commonly used defined media formulations identified profound differences in the metabolism of hESCs, though all media maintained OCT4 expression for over 20 passages.  Defined media induce hESCs to increase glycolytic flux while decreasing oxidative TCA metabolism, with a concomitant decrease in oxygen consumption.  Steady state metabolomics analysis indicated that TCA intermediates were more abundant in defined culture conditions, while lipogenic metabolites such as glycerol-3-phosphate (G3P) and citrate became depleted.  Reductive flux of glutamine carbon in the TCA cycle was increased to support lipid biosynthesis.  In fact, while cells in standard hESC medium exhibit minimal de novo lipogenesis, hESCs in defined media synthesize significant amounts of fatty acids and cholesterol.  To support these processes hESCs increase NADPH production in the oxidative pentose phosphate pathway but also exhibit increases in total reactive oxygen species (ROS).  These changes are further manifested by the activation of upstream signaling pathways that are commonly upregulated in transformed cells.  By supplementing these defined media with specific additives we can normalize the metabolic behavior of hESCs.  Intriguingly, the nutritional microenvironment also influences differentiation efficiency.  Current studies are now addressing the extent that these culture conditions affect the metabolic performance of hPSC-derivatives, including cardiomyocytes which have significant bioenergetic needs.  These results suggest that caution should be used in employing minimal media for the maintenance of hPSCs and highlight the importance of metabolic function in characterizing cell function.