title: Adaptation of a commonly used, chemically defined medium for human embryonic stem cells to stable isotope labeling with amino acids in cell culture.
abstract: Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151.
authors.affiliation: Weill Cornell Medical College in Qatar, Doha, Qatar.
authors.forename: Albert R
authors.initials: AR
authors.lastname: Liberski
authors.forename: Muna N
authors.initials: MN
authors.lastname: Al-Noubi
authors.forename: Zahra H
authors.initials: ZH
authors.lastname: Rahman
authors.forename: Najeeb M
authors.initials: NM
authors.lastname: Halabi
authors.forename: Shaima S
authors.initials: SS
authors.lastname: Dib
authors.forename: Rasha
authors.initials: R
authors.lastname: Al-Mismar
authors.forename: Anja M
authors.initials: AM
authors.lastname: Billing
authors.forename: Roopesh
authors.initials: R
authors.lastname: Krishnankutty
authors.forename: Faizzan S
authors.initials: FS
authors.lastname: Ahmad
authors.forename: Christophe M
authors.initials: CM
authors.lastname: Raynaud
authors.forename: Arash
authors.initials: A
authors.lastname: Rafii
authors.forename: Kasper
authors.initials: K
authors.lastname: Engholm-Keller
authors.forename: Johannes
authors.initials: J
authors.lastname: Graumann
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D003470:Culture Media / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D051379:Mice; D040901:Proteomics / Q000379:methods*
chemical_list: D000596:Amino Acids; D003470:Culture Media