Identification of proteins from feeder conditioned medium that support human embryonic stem cells.
The maintenance of undifferentiated human embryonic stem cells (hESC) requires feeder cells, either in co-culture or feeder-free with conditioned medium (CM) from the feeders. In this study, we compared the CM of a supporting primary mouse embryonic feeder (MEF) and an isogenic but non-supporting MEF line (DeltaE-MEF) in order to gain an insight to the differential expression profile of secreted factors. Using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight (MALDI) tandem mass spectrometry, 13 protein identities were found to be downregulated in DeltaE-MEF compared to MEF, of which 4 were found to be soluble factors and 3 proteins were membrane-associated or related to the extracellular matrix. In addition, four other proteins were identified to be differentially expressed in MEF-CM using high pressure liquid chromatography (HPLC) and cytokine arrays. In functional experiments where CM was replaced with six of the factors identified, hESC were able to proliferate for five continuous passages whilst maintaining 68-82% and 74-98% expression of pluripotent markers, Oct-4 and Tra-1-60, respectively. Using proteomic tools, important proteins from CM that supports hESC culture have been identified, which when replaced with recombinant proteins, continue to support undifferentiated hESC growth in a feeder-free culture platform.
Chin
Angela Chui Ping
AC
Bioprocessing Technology Institute, Biomedical Sciences Institutes, Singapore.
Fong
Wey Jia
WJ
Goh
Lin-Tang
LT
Philp
Robin
R
Oh
Steve Kah Weng
SK
Choo
Andre Boon Hwa
AB
D000818:Animals; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D050810:Octamer Transcription Factors / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016207:Cytokines; D050810:Octamer Transcription Factors; D011506:Proteins; D011994:Recombinant Proteins