Detecting de novo insulin synthesis in embryonic stem cell-derived populations.
Several studies in recent years have described protocols, both genetic- and culture-based, that induce the differentiation of embryonic stem (ES) cells towards a pancreatic beta-cell type. The success of previous protocols in generating insulin-producing beta-cells has been questioned due in part to uncertainty regarding cell lineage but also due to the controversy regarding the source of any insulin detected in these cells. In an attempt to address the latter, we designed a novel assay that can identify de novo insulin synthesis. The method is based on metabolic labeling combined with a modified radio-immunoassay and will routinely detect less than 5 pg/microl of de novo insulin synthesis in lysates from the insulinoma cell line MIN6. This assay failed to detect any newly translated insulin in an ES cell-derived population generated using an adapted version of a previously published, 5-stage differentiation protocol. In combination with other techniques, including immunofluorescent staining and western blot analysis to detect and quantify C-peptide, we conclude that the majority of the insulin found in these differentiated ES cell cultures is medium-derived.
Mc Kiernan
Eadaoin
E
National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland.
Barron
Niall W
NW
O'Sullivan
Finbarr
F
Barham
Paul
P
Clynes
Martin
M
O'Driscoll
Lorraine
L
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002096:C-Peptide / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D019070:Cell Lineage; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D007328:Insulin / Q000096:biosynthesis* / Q000378:metabolism; D051379:Mice; D046509:Tissue Culture Techniques
D015415:Biomarkers; D002096:C-Peptide; D007328:Insulin