Characterization of L‐Threonine transport in mouse embryonic stem cells

Abstract

Stem cells are at the forefront of current regenerative and biomedical research. There exists an imperative need to understand the mechanisms that drive stem cell function in order to exploit their use as a therapeutic tool. Amino acids are potent inducers of signaling cascades that drive stem cell proliferation and differentiation. With a focus on mouse embryonic stem (mES) cells, L‐threonine is the only amino acid required in culture medium for mES cell proliferation. Current research associates this need for L‐threonine with threonine dehydrogenase (TDH), which catabolizes L‐threonine to glycine and acetyl‐CoA in mES cells. While this may be true for mES cells, human embryonic stem (hES) cells do not express a functional tdh gene. Additionally, the concentration of 3‐hydroxynorvaline (3‐HNV), a potent inhibitor of TDH, needed to inhibit mES cell proliferation is more than an order of magnitude less than the apparent Ki for TDH inhibition. Such findings suggest a different mechanism responsible for the relationship between L‐threonine and ES cell proliferation, which may be explained by Thr transport. To explore this novel idea, we characterized the L‐threonine transport systems in mES cells by conducting radiolabelling transport studies. We found that there is a Na+‐dependent and Na+‐independent component of transport, with the Na+‐dependent component predominating. We also found that Thr, Ser, Cys, Ala, 4‐OH‐Pro, Asn, 3‐HNV, and Met were the strongest inhibitors of the Na+‐dependent component of transport. Such findings are consistent with characteristics of the ASC transport system, suggesting that this system is responsible for majority of Thr transport in mES cells. We confirmed the presence of the ASC system transporters, ASCT1 and ASCT2, in mES cells using RT‐PCR. Project supported by Midwestern University.