Enrichment of beta cells from the human fetal pancreas by fluorescence activated cell sorting with a new monoclonal antibody.
The aim of this study was to produce an antibody reactive to the surface of endocrine pancreatic cells and use this antibody for the purification of endocrine cells from the human fetal pancreas by fluorescence activated cell sorting. We describe such an antibody, called N1, reacting with the surface and cytoplasm of endocrine cells in the adult and fetal human pancreas (12 to 18 weeks gestational age). While unreactive to exocrine and mesenchymal cells, it was not specific for endocrine cells, as evidenced by its staining pattern in tissues other than pancreas. Almost 40% of the N1-positive pancreatic cells contained either insulin, glucagon or somatostatin. Conversely, more than 90% of each of the hormone-containing cells was N1 positive. An additional 40% of N1-positive cells, not containing other pancreatic hormones, was shown to contain islet amyloid polypeptide, synaptophysin, chromogranin, tyrosine hydroxylase or CA812. A two-step collagenase digestion protocol yielded 1.29 +/- 0.17 x 10(5) cells per mg pancreatic tissue. After Percoll gradient centrifugation, the suspension contained 15.6 +/- 5.7% (n = 25, mean +/- SD) cells reactive with N1. By fluorescence activated cell sorting using the antibody N1, the single-cell suspension was enriched from 3.0 +/- 1.4% to 16.2 +/- 4.8% (n = 10, p less than 0.01) Beta cells. Alpha and Delta cells were also enriched significantly by this procedure. The percentage of N1-positive cells increased from 17 +/- 4% to 83 +/- 6%. This preparation enriched for endocrine cells allows future studies on possible endocrine precursor cells.
de Krijger
R R
RR
Department of Pediatrics, Erasmus University Rotterdam, The Netherlands.
Aanstoot
H J
HJ
Kranenburg
G
G
Verkerk
A
A
Jongkind
J F
JF
van Strik
R
R
Lafferty
K J
KJ
Bruining
G J
GJ
D000022:Abortion, Spontaneous; D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D005260:Female; D005333:Fetus; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D000882:Haplorhini; D006801:Humans; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000166:cytology* / Q000196:embryology*; D051379:Mice; D008815:Mice, Inbred Strains / Q000276:immunology; D010179:Pancreas / Q000166:cytology* / Q000196:embryology; D011247:Pregnancy; D051381:Rats
D000911:Antibodies, Monoclonal; D016650:Fluorescein-5-isothiocyanate