CD99 as surface anchor for human islet endocrine cell purification.
Rat and human beta cell proteomes were quantified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), searching for cell surface markers. In human beta cells, CD99 (cluster of differentiation 99) was ranked among the plasma membrane proteins that combine a high molar abundance with a relative degree of selectivity for the endocrine cells of the islets of Langerhans. Therefore, the applicability of CD99 as anchor for islet endocrine cell purification was investigated. The CD99 gene and protein expression were studied using microarray, LC-MS/MS, western blotting, flow cytometry and immunofluorescence, and a protocol was developed for magnetic bead-mediated beta cell enrichment from human pancreas digests using available anti-CD99 antibodies. In human, but not in rat, CD99 protein and mRNA were abundantly expressed by islet endocrine cells but undetectable in exocrine pancreas. The extracellular CD99 epitopes appeared to be trypsin-resistant, enabling the binding of anti-CD99 antibodies to an insulin+/TSQ+ cell subset and efficient coupling of magnetic beads for positive selection of CD99+ cells. A MACS-CD99 purification of human pancreas fractions with low endocrine purity consistently yielded a fourfold enrichment of insulin+/TSQ+ cells and formation of viable and functional endocrine aggregates after 24 h of culture. It is concluded that CD99 is a human beta cell surface marker that, by virtue of its high molar abundance and resistance to tryptic digestion, can be used as anchor for upscalable magnetic bead-mediated islet endocrine cell purification. Copyright © 2016 John Wiley & Sons, Ltd.
Centre for Beta Cell Therapy in Diabetes, Vrije Universiteit Brussel, Brussels, Belgium.
Geert A
0000-0003-1208-6289
GA
Martens
Centre for Beta Cell Therapy in Diabetes, Vrije Universiteit Brussel, Brussels, Belgium.
Veerle
V
De Punt
Centre for Beta Cell Therapy in Diabetes, Vrije Universiteit Brussel, Brussels, Belgium.
Geert
G
Stangé
D000071443:12E7 Antigen / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D000072417:Protein Domains; D051381:Rats
D000071443:12E7 Antigen; C061828:CD99 protein, human
biological markers; cell separation; diabetes mellitus type 1; in vitro techniques; insulin-producing cells; islets of Langerhans transplantation; membrane proteins