Title
A specific amino acid metabolic state of human ES/iPS cells and its significance

Nobuaki Shiraki1,#, Yasuko Shiraki3,#, Genta Nagae4, Tomonori Tsuyama1, Hiroyuki Aburatani4, Kazuhiko Kume1, Fumio Endo2,3, and Shoen Kume1, 2,

1 Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, 2 G.COE, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan 3 Department of Pediatrics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan 4 Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan

Abstract:
Embryonic stem (ES) cells possess a characteristic high rate of proliferation and a short G1 phase; they have an unlimited ability to replicate as well as the potential to differentiate into most cell types in an organism, including hepatic lineage. Recent studies showed that mouse ES cells are in a high-flux metabolic state, showing a high dependence on threonine catabolism, in particular (Wang et al., 2009). Marked differences in the differentiation propensity exist among human ES (hES) cell lines and form an obstacle for the directed differentiation of hES/iPS cells in vitro .
Here, we identified a specific dependence of the pluripotent human ES/iPS cells on a particular amino acid for their growth. We have established a differentiation procedure for human ES/iPS cells to differentiate into the definitive endoderm derivatives of the pancreas and liver (Shiraki et al., 2008, 2011). To establish a more endoderm selective differentiation method, we focused on the differences of amino acid metabolisms between the undifferentiated and differentiated definitive endoderm cells. When cultured in media deprived of the particular amino acid, undifferentiated human ES/iPS cells rapidly ceased to proliferate and apoptosis occurred, which resulted in an increased proportion of Sox17-positive definitive endoderm cells. Further differentiation into hepatic lineages was potentiated, to yield a 3-fold increase in Albumin secretion. Moreover, the elimination of undifferentiated cells using the particular amino acid deprived media was applicable to several different human ES/iPS cell lines.
In this study, a novel human ES/iPS cell differentiation system, based on the difference of a specific amino acid metabolism between undifferentiated cells and differentiated cells, is established. By using the media deprived of a particular amino acid, undifferentiated cells can be specifically eliminated during endoderm differentiation. This method is useful for directing human ES/iPS cell lines that are resistant to differentiation to achieve a higher differentiation efficiency.