Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.
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.
Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK.
Spyros I
SI
Vernardis
Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK.
Konstantinos
K
Terzoudis
Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK.|Department of Haematology, Imperial College, London, UK.
Nicki
N
Panoskaltsis
Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, UK.
Athanasios
A
Mantalaris
D000577:Amides / Q000494:pharmacology*; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1 / Q000235:genetics / Q000378:metabolism; D055442:Metabolome; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011725:Pyridines / Q000494:pharmacology*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D000577:Amides; D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; D015415:Biomarkers; D004791:Enzyme Inhibitors; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011725:Pyridines; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D055746:Stage-Specific Embryonic Antigens; C495901:TP53 protein, human; C519803:TRA-1-81 antigen, human; D016159:Tumor Suppressor Protein p53; C052924:stage-specific embryonic antigen-3; C108830:Y 27632; D000076222:Mechanistic Target of Rapamycin Complex 1; D054460:rho-Associated Kinases