Summary
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, tyrosin hydroxylase or CA812. A two-step collagenase digestion protocol yielded 1.29 ± 0.17 x 105 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 < 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.
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Ziegler B, Witt S, Waterstradt B, Schlosser M, Ziegler M (1989) Heterogeneity of monoclonal antibodies against pancreatic Beta cells. Exp Clin Endoeripol 93:173–180
Witt S, Ziegler B, Waterstradt B, Besch W, Hehmke B, Ziegler M (1987) Generation and partial characterization of monoclonal antibodies reactive with islet cell antigens. Exp Clin Endocrinol 89:276–282
Vissing H, Papadopoulos G, Lernmark Å (1986) Monoclonal antibodies against pancreatic islet-cell-surface antigens selected by flow cytofluorometry. Scand J Immunol 23:425–433
Brogren C-H, Hirsch F, Wood P, Druet P, Poussier P (1986) Production and characterization of a monoclonal islet cell surface autoantibody from the BB rat. Diabetologia 29:330–333
Contreas G, Jorgensen J, Madsen OD (1990) Novel islet, duct, and acinar cell markers defined by monoclonal autoantibodies from prediabetic BB rats. Pancreas 5:540–547
Gerbitz KD, Boenke B, Paprotta A, Chemnitz U (1988) Human nesidioblastosis tissue as an immunogen for generation of islet cell specific monoclonal antibodies. J Clin Chem Clin Biochem 26:659–666
Warnock GL, Kneteman NM, Evans MG, Dabbs KD, Rajotte RV (1990) Comparison of automated and manual methods for islet isolation. Can J Surg 33:368–371
Van de Winkel M, Maes E, Pipeleers DG (1982) Islet cell analysis and purification by light scatter and autofluorescence. Biochem Biophys Res Comm 107:525–532
Like AA, Orci L (1972) Embryogenesis of the human pancreatic islets: a light and electron microscopic study. Diabetes 21 [Suppl 2]:511–534
Milstein C, Galfre G, Secher DS, Springer T (1979) Monoclonal antibodies and cell surface antigens. Cell Biol Int Rep 3:1–16
Nakane PK, Pierce GB (1967) Enzyme-labelled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol 33:307–318
Wicker R, Avrameas S (1969) Localization of virus antigens by enzyme-labelled antibodies. J Gen Virol 4:465–471
Keilacker H, Dietz H, Witt S, Woltanski KP, Berling R, Ziegler M (1986) Kinetic properties of monoclonal insulin antibodies. Biomed Biochem Acta 45:1093–1102
Rotman B, Papermaster BW (1966) Membrane properties of living mammalian cells as studies by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 55:134–141
Roth J, Bendayan M, Carlemalm E, Villiger W, Garavitto M (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29:663–671
Kalina M, Lukinius A, Grimelius L (1991) Ultrastructural localization of synaptophysin to the secretory granules of normal glucagon and insulin cells in human islets of Langerhans. Ultrastruct Pathol 15:215–219
Navone F, Jahn R, Di Giola G, Stukenbrok H, Greengard P, De Camilli P (1986) Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol 103:2511–2527
Hutton JC (1989) The insulin secretory granule. Diabetologia 32:271–281
Lukinius A, Wilander E, Westermark GT, Engström U, Westermark P (1989) Colocalization of islet amyloid polypeptide and insulin in the B-cell secretory granules of the human pancreatic islets. Diabetologia 32:240–244
Madsen OD, Blume N, Petersen JE, Kofod H, Dyrberg T (1990) Specific antisera to IAPP, proinsulin C-peptide 1, and 2: probes to study murine islet Beta-cell differentiation. Diabetologia 33 [Suppl]:A108 (Abstract)
Madsen OD, Nielsen JH, Michelsen B et al. (1991) Islet amyloid polypeptide and insulin expression are controlled differently in primary and transformed islet cells. Mol Endocrinol 5:143–148
Hegre OD, Marshall S, Schulte BA et al. (1983) Nonenzymic in vitro isolation of perinatal islets of Langerhans. In Vitro 19:611–620
Ono J, Takaki R, Fukuma M (1977) Preparation of single cells from pancreatic islets of adult rat by the use of dispase. Endocrinol Jpn 24:265–270
Pipeleers DG, In't Veld PA, Van de Winkel M, Maes E, Schuit FC, Gepts W (1985) A new in vitro model for the study of pancreatic A and B cells. Endocrinology 117:806–816
Kover K, Moore WV (1989) Development of a method for isolation of islets from human fetal pancreas. Diabetes 38:917–924
Hellerström C, Lewis NJ, Borg H, Johnson R, Freinkel N (1979) Method for large-scale isolation of pancreatic islets by tissue culture of fetal rat pancreas. Diabetes 28:769–776
Korsgren O, Sandler S, Schnell Landstrom A, Jansson L, Andersson A (1988) Large-scale production of fetal porcine pancreatic islet-like cell clusters. An experimental tool for studies of islet cell differentiation and xenotransplantation. Transplantation 45:509–514
Van de Winkel M, Maes E, Pipeleers DG (1982) Islet cell analysis and purification by light scatter and autofluorescence. Biochem Biophys Res Comm 107:525–532
MacDonald MJ (1981) Flavin content of intracellular compartments of pancreatic islets compared with acinar tissue and liver. Endocrinology 108:1899–1902
Rabinovitch A, Russell T, Shienvold F et al. (1982) Preparation of rat islet B-cell-enriched fractions by light-scatter flow cytometry. Diabetes 31:939–943
Nielsen DA, Lernmark ⫗A, Berelowitz M, Bloom GD, Steiner DF (1982) Sorting of pancreatic islet cell subpopulations by light scattering using a fluorescence activated cell sorter. Diabetes 31:299–306
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de Krijger, R.R., Aanstoot, H.J., Kranenburg, G. et al. Enrichment of Beta cells from the human fetal pancreas by fluorescence activated cell sorting with a new monoclonal antibody. Diabetologia 35, 436–443 (1992). https://doi.org/10.1007/BF02342440
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DOI: https://doi.org/10.1007/BF02342440