Martin I. Mally

Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells.

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin, glucagon, and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM, respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak), whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets, the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development.

Regulation of Proliferation and Differentiation of Human Fetal Pancreatic Islet Cells by Extracellular Matrix, Hepatocyte Growth Factor, and Cell-Cell Contact

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.

Developmental gene expression in the human fetal pancreas.

ABSTRACTS: Differential developmental regulation of pancreas-specific genes has not been reported for the human fetal pancreas. We have therefore undertaken a systematic, quantitative analysis of the transcriptional levels of various genes in the human pancreas at different stages of fetal and postnatal development. Using sensitive ribonuclease protection assays, in situ hybridization, and the polymerase chain reaction, our results indicate the following: 7) Transcriptional levels of insulin and amylin remain lower in the fetal than in the adult pancreas, whereas glucagon and somatostatin mRNA levels are consistently greater after 14 wk gestation than postnatally. These results are in agreement with previous immunohistochemical studies of these gene products. 2) The reg gene exhibits a 20-fold increase in mRNA levels after 16 wk gestation. The gene is expressed exclusively in the acinar cells and does not colo-calize with insulin. This restricted exocrine expression does not indicate a direct role for the reg gene in islet development. 3) Glucose transporter 2 and glucokinase mRNA are detectable as early as 13 wk gestation and remain low throughout development. Glucose transporter 1 reaches adult transcriptional levels by 18 wk gestation. The early detection of glucose transporter 2 and glucokinase implies that lack of expression of these “glucose sensor” genes does not account for the known insensitivity of the fetal β-cells to glucose.

Ontogeny and Tissue Distribution of Human GAD Expression

One of the major β-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with α- and β-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.

Opposite Effects of β-cell Differentiation and Growth on Reg Expression in Human Fetal Pancreatic Cells

Reg is a gene associated with regeneration of pancreatic islets. We have previously shown that nicotinamide induces differentiation of human fetal β-cells in tissue culture and that hepatocyte growth factor/scatter factor (HGF/SF) is mitogenic for the fetal β-cells. We now tested whether these conditions, supporting either differentiation or growth, are associated with changes in reg gene expression in human fetal pancreatic cells. Culture for 7 days with 10 mM nicotinamide led to a fourfold decrease in reg mRNA levels (23 ± 12% of control, n = 5, P < 0.001). In contrast, HGF/SF increased reg expression threefold (302 ± 68% of control, n = 4, P < 0.05). Nicotinamide, which does not alter the differentiation level of adult β-cells, did not significantly affect reg expression in adult human islets (84 ± 4% of control, n = 2, NS). Thus, a higher level of endocrine differentiation is associated with a lower level of reg expression, and a higher rate of β-cell replication results in increased reg transcription. These results provide the first evidence of a molecular marker, the reg gene, to distinguish between proliferation and differentiation of human β-cells.

ICA69 is expressed equally in the human endocrine and exocrine pancreas.

SummaryIslet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.

Oncogene expression in murine splenic T cells and in murine T-cell neoplasms

The differential expression of a series of proto-oncogenes has been examined in a group of cultured murine T-cell lymphomas that were induced following virus inoculation into, or X irradiation of, C57BL/6 mice. Two classes of T lymphoma cell lines were studied: growth factor-dependent (autocrine) cells, and growth factor-independent T lymphoma cells. Cell lines that were established from X-irradiation-induced T lymphomas were growth factor dependent, whereas T lymphoma lines grown from virus-induced tumors were generally growth factor independent. Of 18 cellular proto-oncogenes studied, five (c-myc, c-myb, c-abl, c-rasHa, c-rasKi) were consistently expressed in all cell lines tested. Thirteen other proto-oncogenes (c-mos, c-sis, c-rel, c-yes, c-fes3, c-fes4, c-fos, c-fms, c-src, c-erbA, c-erbB, int-1, Pim-1) were not expressed in any of the T lymphoma cells tested. Concanavalin A-stimulated spleen cells, representative of replicating T cells, expressed c-myc, c-abl, and Pim-1, suggesting that the products of these proto-oncogenes are involved in T-cell proliferation. The results indicate no qualitative differences (albeit some quantitative differences) in proto-oncogene expression between the growth factor-dependent and growth factor-independent cells. This suggests that expression of the five proto-oncogenes is in itself not sufficient to induce the progression of the growth factor-dependent cells to their fully growth factor-independent counterparts. Changes in the regulation of one or more of the five active proto-oncogenes, i.e., from an inducible to a constitutive state, and/or additional changes in the expression of other cellular genes may be required to induce the transformation of neoplastic T cells from growth factor dependence to growth factor independence.

IDENTIFICATION OF A MARKER TO ENRICH POPULATIONS OF ENDOCRINE CELL PRECURSORS FROM THE HUMAN FETAL PANCREAS

Isolation of the endocrine cell precursors from the human fetal pancreas is important for the understanding of islet cytodifierentiation. The progenitor cells, from which all four islet endocrine cell types arise, are present within the epithelium ol the fetal pancreatic duct. Following enzymatic digestion and culture of the fetal pancreas WG obtained cell clusters resembling islets but with a high content of undifferentiated cells. Histochemical staining revealed very high acid β-galaclosidase activity in most cells within the clusters. After transplantation into athymic nude mice, the clusters gave rise to tissue rich in differentiated endocrine cells. The histochemical finding of high acid β-galactosidase activity was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid β-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic β-galactosidase substrate, we were able to enrich for a subpopulation of cells high in acid β-galactosidase activity with flow cytometry. Evidence identifying these cells as potential islet cell precursors included, besides the transplantation experiments, the colocalization in vitro of lyrosine hydroxylase, a known marker of embryonic islet cells. Thus, our results indicate that high acid β-galactosidase activity serves as a marker to enrich populations of endocrine cell precursors.

FIBROBLAST GROWTH FACTORS 1 AND 2 ACT ON THE IMMATURE HUMAN FETAL ISLET CELL BUT NOT ON THE MATURE β-CELL

Fibrpblast growth factors (FGFs) play an important role in the embryogenesis of many organs. In order to elucidate the regulation of pancreatic islet development, we have used tissue culture of human fetal pancreas to study the effects of FGFs. Our method is based on the formation of islet-like cell clusters (ICCs), consisting of 87% undifferentiated cells and 13% islet hormone-positive cells. Supplementation of the culture medium with FGF-2 (basic FGF) more than doubled the formation of ICCs, stimulated their DNA synthesis, inhibited the insulin release (ED50-1 ng/ml), but did not affect the insulin content. This activity was shared by FGF-1 (acidic FGF) but not by FGF-6 or FGF-7 (KGF). When ICCs were treated for 72h with bFGF-Saporin (F-SAP), a mitotoxin targeted against FGF-receptor expressing cells, a distinct population (45%) of cells within the ICCs were killed, whereas some strongly hormone-positive (mature) islet cells were left intact. F-SAP treatment also blocked the endocrine differentiation induced by nicotinamide. Similar experiments with isolated adult human islets showed no activity of FGF-2 or F-SAP, supporting a role for FGF in the early development of human islet cells.