Functional proteomics screen enables enrichment of distinct cell types from human pancreatic islets.

The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+), glucagon-producing alpha cells (CD9-/CD56+) and trypsin-producing acinar cells (CD9-/CD56-). This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.

Proteomics-based dissection of human endoderm progenitors by differential cell capture on antibody array.

Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomics platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated differential cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. We used this platform to fractionate the hitherto unresolved early endoderm compartment of CXCR4+ cells and identify several endoderm (CD61+ and CD63+) and non-endoderm (CD271+, CD49F+, CD44+ and B2M+) sub-populations. We provide evidence that one of these sub-populations, CD61+, is directly derived from CXCR4+ cells, displays characteristic kinetics of emergence, and exhibits a distinct gene expression profile. The results demonstrate the potential of the cell-capture antibody array as a powerful proteomics tool for detailed dissection of heterogeneous cellular systems.

Regulation of the GPR40 locus: towards a molecular understanding.

GPR40 {FFAR1 [non-esterified ('free') fatty acid receptor 1]} is a G-protein-coupled receptor expressed preferentially in pancreatic beta-cells. GPR40 functions as a receptor for medium and long-chain fatty acids, and has been implicated in mediating both physiological and pathological effects of fatty acids on beta-cells. The GPR40 gene is encoded at an interesting chromosomal locus that contains several genes: at the 5'-end of the locus, located approximately 4 kb upstream of GPR40, is CD22, a gene encoding a receptor expressed selectively in lymphocytes and involved in B-lymphocyte maturation and function. At the 3'-end of the locus are the GPR41 (FFAR3) and GPR43 (FFAR2) genes encoding receptors activated by short-chain fatty acids. The intergenic region between CD22 and GPR40 contains several evolutionarily conserved sequence blocks, among them HR2 and HR3. beta-Cell-specific expression of GPR40 is controlled at the transcriptional level through HR2, a potent beta-cell-specific enhancer. The mechanisms controlling cell-specific expression of the remaining genes in the cluster are unknown. Given the divergent modes of expression of the genes within the locus and their demonstrated physiological significance, it is important to analyse further the locus with a view to fully understanding the basis for transcriptional regulation of the encoded genes.