Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers

Abstract

We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells

“…Day 0-5 EBand d3 monolayer-induced cells were stained with anti-CXCR4-phycoerythrin (BD; 1:100), anti-CD31-phycoerythrin (BD; 1:10), anti-CD117-allophycocyanin (Invitrogen; 1:100) and anti-KDRallophycocyanin (R&D Systems; 1:10). Day 20-25 cells were stained with HPi3 (1:20), HPa2 (1:20), HPx1 (1:20), HPd1 (1:20) (Dorrell et al, 2008) and anti-mouse IgG-phycoerythrin (Jackson ImmunoResearch;1:200). Intracellular antigens were detected by staining with goat anti-human SOX17 (R&D Systems; 1:40), goat anti-FOXA2 (clone M20, Santa Cruz; 1:50), rat anti-human C-peptide (AB1921, Beta Cell Biology Consortium; 1:300), mouse anti-GCG (Sigma; 1:500), donkey anti-goat IgG-Alexa 488 (Invitrogen; 1:400), donkey anti-rat IgG-Alexa 488 (Invitrogen; 1:400), goat anti-mouse allophycocyanin (BD; 1:200).…”

“…To gain a better understanding of the end-stage population we stained cells with a panel of antibodies recognizing different cellular subsets of the adult pancreas (alpha, ductal, exocrine and pan-islet) (Dorrell et al, 2008). Approximately 30% (34±16%, n3) of the day 22 population stained with the pan-islet (HPi3) antibody, whereas 43% (43±4%, n3) stained with the panexocrine antibody (HPx1) (Fig.…”

Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

“…Cell surface markers or marker combinations for beta-cells exist, but they will also label immature insulinpositive cells early in development [117][118][119]. At the moment, there are no cell surface markers that specifically label mature beta-cells.…”

Flow Cytometric Sorting of Cells from Solid Tissue – Reagent Development and Application

“…trypsin) to disperse tightlyassociated cells. Specific protocols successfully used for tissues such as mouse liver (Dorrell et al, 2008b), and human pancreas (Dorrell et al, 2008a) are described in the next section.…”

“…Subsequent treatment with cyclophosphamide kills the lymphocytes which have responded to any of these antigens, so that after subsequent immunizations with the tissue/cells of interest, only the new and relevant antigens cause an immune response. This approach was used successfully to raise antibodies against human pancreatic islet cells after pre-immunization with trypsin and calf serum, which might otherwise have been confounding antigens required for islet dispersal and cell storage (Dorrell et al, 2008a). Subtractive immunization is compatible with any of the immunogen sources described above.…”