Abstract
Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 μM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography–mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.
Highlights
Single cell cultures in suspension of hPSCs undergo apoptosis despite the use of culture conditions conducive to stem cell maintenance[9,10]
Existing metabolic switches on hESCs and hiPSCs were similar on both cell types up to 48 h of exposure to Y-27632, with 24 h exposure being identified to be critical as a turning point in metabolism
The above indicate a dynamic process of adaptation of the cells to the altered environment, which is mostly projected to the metabolic level
Summary
Single cell cultures in suspension of hPSCs undergo apoptosis despite the use of culture conditions conducive to stem cell maintenance[9,10] This problem has been addressed with the inhibition of ROCK (Rho-associated protein kinase), a serine-threonine kinase that phosphorylates and activates the myosin II pathway[11,12,13,14,15,16] resulting in the maintenance of the differentiation potential for up to 72 hours, which is considered to be primarily mediated via the inhibition of an E-cadherin-dependent apoptotic pathway[17,18,19,20]. Existing metabolic switches on hESCs and hiPSCs were similar on both cell types up to 48 h of exposure to Y-27632, with 24 h exposure being identified to be critical as a turning point in metabolism Correlating with these metabolic changes, a differential expression of the metabolic regulators, p5328,29 and mTORC14,30, was observed. The above indicate a dynamic process of adaptation of the cells to the altered environment, which is mostly projected to the metabolic level
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