Global gene expression profiling and histochemical analysis of the developing human fetal pancreas.

AIMS/HYPOTHESIS: An immunohistochemical and genomic analysis of human pancreatic development from 9-23 weeks of fetal age was undertaken to provide a comparative analysis of human and murine islet development. METHODS: Human fetal pancreases obtained at gestational ages 9-23 weeks were processed in parallel for immunohistochemistry and gene expression profiling by Affymetrix microarrays. RESULTS: By 9-11 weeks, the pancreas was made up principally of mesenchymal tissue infiltrated by branched epithelial structures containing scattered hormone-negative neurogenin3-positive endocrine cells. Protoacinar structures emerged by 15-19 weeks, along with clusters of endocrine cells producing either glucagon or insulin. By 20-23 weeks, vascularised islet-like structures appeared. More than 70% of endocrine cells produced a single hormone at any age. Analysis of Ki67 immunoreactivity showed that the replicative rate of endocrine cells was low and suggested that the endocrine expansion was derived from hormone-negative precursors. Insulin, glucagon, somatostatin, ghrelin and pancreatic polypeptide transcripts were present at 9-10 weeks and increased progressively, commensurate with the expansion of endocrine cell volume. The human equivalent of a mouse endocrine secondary transition was not evident, neither in terms of morphology nor in dramatic changes in endocrine-specific transcriptional regulators. By contrast, exocrine genes showed a marked transition at around 11 weeks, associated with a greater than sixfold increase in exocrine gene transcripts. CONCLUSIONS/INTERPRETATION: The observed extension of terminal differentiation of human endocrine tissue into late gestation is in contrast with findings in the mouse. It indicates that the human fetal pancreas could provide an abundant islet precursor cell population that could be expanded ex vivo for therapeutic transplantation.

Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells.

AIMS AND HYPOTHESIS: Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family with a high degree of specificity for epithelial cells in vitro and in vivo. Our aim was to study the effect of KGF on beta-cell growth and differentiation on islet-like cell clusters derived from human fetal pancreas. METHODS: We investigated the effects of KGF, in vitro, on beta-cell differentiation from undifferentiated pancreatic precursor cells and in vivo after transplantating human fetal pancreatic cells into athymic rats treated with KGF. RESULTS: Treatment of islet-like cell clusters with KGF in vitro did not change the number of insulin producing cells, as measured by the measurement of insulin content or DNA. The in vivo treatment of recipient rats with KGF increased the number of beta cells within the grafts 8 weeks after transplantation. At this time, glucose-stimulated insulin secretion was evaluated by glucose stimulation tests in rats bearing the transplants. Measurements of human C-peptide concentrations after glucose challenge showed that the newly differentiated beta cells in the KGF-treated group were functionally competent as opposed to the control group, where the graft failed to release insulin appropriately. CONCLUSION/INTERPRETATION: These findings suggest that in vivo, KGF is capable of inducing human fetal beta-cell expansion. The growth promoting effect of KGF on beta cells occurred mainly through the activation of ductal cell proliferation and their subsequent differentiation into beta cells.

A role for activin A and betacellulin in human fetal pancreatic cell differentiation and growth.

Activin A (Act.A), a member of the transforming growth factor beta family of secreted proteins, has been implicated in the regulation of growth and differentiation of various cell types. Betacellulin (BTC), a member of the epidermal growth factor family, converts exocrine AR42J cells to insulin-expressing cells when combined with Act.A. We have used primary cultures of human fetal pancreatic tissue to identify the effects of Act.A and/or BTC on islet development and growth. Exposure to Act.A resulted in a 1.5-fold increase in insulin content (P < 0.005) and a 2-fold increase in the number of cells immunopositive for insulin (P < 0.005). The formation of islet-like cell clusters, containing mainly epithelial cells, during a 5-day culture, was stimulated 1.4-fold by BTC (P < 0.05). BTC alone caused a 2.6-fold increase in DNA synthesis (P < 0.005). These data suggest that Act.A induces endocrine differentiation, whereas BTC has a mitogenic effect on human undifferentiated pancreatic epithelial cells.

Protection from cell death in cultured human fetal pancreatic cells.

Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote beta-cell growth.

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.

PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line.

Cell lines from the fetal and adult pancreas that were developed by retroviral transfer of the SV40T and ras(val12) oncogenes lose insulin expression but retain extremely low levels of somatostatin and glucagon mRNA. In contrast to expanded populations of primary human islet cells, none of them express the homeodomain transcription factor PDX-1. When that factor was expressed in the cell lines by retroviral-mediated gene transfer, one of the cell lines, TRM-6, derived from human fetal islets, exhibited a 10- to 100-fold increase in somatostatin gene expression. This is the first report of induction of the endogenous somatostatin gene by PDX-1. Promotion of cell-cell contact by aggregation of TRM-6/PDX-1 into islet-like clusters produced a further 10- to 100-fold increase in somatostatin mRNA, to a level similar to that of freshly isolated islets, which resulted in production of somatostatin protein. Thus, we demonstrate here that signals induced by cell-cell contact act in synergy with PDX-1 to up-regulate the endogenous somatostatin promoter in an immortalized cell line from human fetal islets. This system provides a powerful model for studying human islet cell development and, particularly, the role of cell-cell contact in the differentiation process.

Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro.

Widespread application of beta-cell replacement strategies for diabetes is dependent upon the availability of an unlimited supply of cells exhibiting appropriate glucose-responsive insulin secretion. Therefore, a great deal of effort has been focused on understanding the factors that control beta-cell growth. Previously, we found that human beta-cell-enriched islet cultures can be stimulated to proliferate, but expansion was limited by growth arrest after 10-15 cell divisions. Here, we have investigated the mechanism behind the growth arrest. Our studies, including analyses of the expression of senescence-associated beta-galactosidase, p16(INK4a) levels, and telomere lengths, indicate that cellular senescence is responsible for limiting the number of cell divisions that human beta-cells can undergo. The senescent phenotype was not prevented by retroviral transduction of the hTERT gene, although telomerase activity was induced. These results have implications for the use of primary human islet cells in cell transplantation therapies for diabetes.

Sustained proliferation of PDX-1+ cells derived from human islets.

Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined.

Gene transfer to human pancreatic endocrine cells using viral vectors.

We have studied the factors that influence the efficiency of infection of human fetal and adult pancreatic endocrine cells with adenovirus, murine retrovirus, and lentivirus vectors all expressing the green fluorescent protein (Ad-GFP, MLV-GFP, and Lenti-GFP, respectively). Adenoviral but not retroviral vectors efficiently infected intact pancreatic islets and fetal islet-like cell clusters (ICCs) in suspension. When islets and ICCs were plated in monolayer culture, infection efficiency with all three viral vectors increased. Ad-GFP infected 90-95% of the cells, whereas infection with MLV-GFP and Lenti-GFP increased only slightly. Both exposure to hepatocyte growth factor/scatter factor (HGF/SF) and dispersion of the cells by removal from the culture dish and replating had substantial positive effects on the efficiency of infection with retroviral vectors. Studies of virus entry and cell replication revealed that cell dispersion and stimulation by HGF/SF may be acting through both mechanisms to increase the efficiency of retrovirus-mediated gene transfer. Although HGF/SF and cell dispersion increased the efficiency of infection with MLV-GFP, only rare cells with weak staining for insulin were infected, whereas approximately 25% of beta-cells were infected with Lenti-GFP. We conclude that adenovirus is the most potent vector for ex vivo overexpression of foreign genes in adult endocrine pancreatic cells and is the best vector for applications where high-level but transient expression is desired. Under the optimal conditions of cell dispersion plus HGF/SF, infection with MLV and lentiviral vectors is reasonably efficient and stable, but only lentiviral vectors efficiently infect pancreatic beta-cells.

Transplantation of human fetal pancreas: fresh vs. cultured fetal islets or ICCS.

The paucity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreas a potential alternative. Fetal pancreatic endocrine cells grow and differentiate over time when fresh explants or cultured islet-like cell clusters (ICCs) are transplanted under the kidney capsule in athymic nude mice. We have recently developed a procedure to isolate fetal islets, which differ from ICCs in their beta-cell content. This study was undertaken to compare the maturation and growth of grafts from purified fetal islets, containing mostly beta-cells, to grafts of mostly undifferentiated endocrine cell precursors, cultured as ICCs, and fresh, uncultured tissue. Total insulin content was highest in the fresh tissue pre-transplant while insulin levels fell precipitously during culture as either fetal islets or ICCs. Although 500 fetal islets contained more insulin than 500 ICCS before transplantation, the insulin content of the resulting grafts was the same 3 months post-transplantation. The degree of stimulation following glucose challenge was comparable, as was the histological appearance. However 70 mg of fresh tissue was needed to generate the fetal islets while only 30 mg was needed for the ICCs. Grafts of 30 mg fresh tissue also had similar total insulin contents and stimulation following glucose challenge, but, when normalized to DNA there was a significantly higher concentration of insulin in the grafts from ICCs or fetal islets. Moreover there were distinct morphological differences, with fibrous and ductal elements prominent in the grafts from fresh tissue, which were also much larger and more diffuse, with cystic elements evident macroscopically. Quantitative immunohistochemical analysis showed that grafts from cultured tissue were 48.3+/-5% positive for immunoreactive insulin compared with grafts from fresh tissue which were only 13.3+/-1.4% positive for insulin. In conclusion cultured ICCs, a heterogeneous mixture of hormone-containing and undifferentiated endocrine cells, are a preferable source for transplantation than either purified fetal islets or uncultured tissue.

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Microfabricated immunoisolating biocapsules.

A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes.

Experimental transplantation of human fetal and adult pancreatic islets.

We examined morphology and function following transplantation of human fetal islet-like clusters (ICCs) in nude mice and compared the functional efficiency of human adult islets and fetal ICCs after transplantation. To assess the optimal site we first transplanted ICCs under the kidney capsule, pancreas, lung, and liver in nude mice. Grafts to the kidney and pancreas matured functionally and morphologically, as evidenced by a 4-fold increase in C peptide after glucose stimulation and the presence of insulin in the grafts of all animals. Grafts to the lung, liver, and spleen did poorly; C peptide was only measurable in two of eight, two of five, or three of five of mice grafted to the lung, liver, or spleen, respectively. Using chemically diabetic nude rats as recipients, we were able to restore normoglycemia using 15,000 ICCs/kg. Lastly, when transplanted under the kidney capsule of normal nude mice, ICCs had significantly higher insulin contents and C peptide release than equivalent grafts of adult islets. In summary, ICCs are an efficient source of insulin-producing cells of potential use in clinical transplantation. In nude mice, both the kidney and the pancreas provide suitable environments for the growth and maturation of undifferentiated human beta-cells.

Phosphatidylinositol 3-kinase is a negative regulator of cellular differentiation.

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor-induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.

Ex vivo expansion of human pancreatic endocrine cells.

Cell transplantation as a therapy for type 1 diabetes is facilitated by ex vivo cell expansion of pancreatic beta-cells without loss of differentiative characteristics. The aim of this study was to determine the optimal conditions for in vitro growth of functional human pancreatic endocrine tissue. We examined the mitogenicity of matrixes from a variety of cell lines; proliferation was greater in cells growing on matrixes from bladder carcinoma cell lines, especially in monolayers grown on matrix from the human cell line HTB-9. After 14-day culture, there was a more than 100-fold proliferative increase, which was augmented to a more than 200-fold when hepatocyte growth factor/scatter factor was added; however, hepatocyte growth factor/scatter factor induced a rapid decrease in insulin content. Without the growth factor, fetal cell monolayers expanded 4-fold with no insulin loss; however, after 12-fold expansion, the insulin levels decreased to 40% of those in unexpanded cells. Adult islet cells expanded 3-fold without insulin loss. After 5-fold expansion, insulin levels decreased by 25% compared to those in free floating islets while retaining a normal response to secretagogues. Together, these results indicate that HTB-9 matrix provides the best stimulatory effect on replication of human endocrine cells, with little loss of in vitro function.

Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.

The scarcity of available tissue for transplantation in diabetes and the need for multiple donors make it mandatory to use an optimal cryopreservation method that allows maximal recovery and preservation of beta-cell function. We have developed a method to cryopreserve islets with excellent survival of endocrine cells. Current methods use DMSO as cryoprotectant. Our method involves introducing both DMSO and the disaccharide trehalose into the cells during cooling. Uptake and release of trehalose occurred during the thermotropic lipid-phase transition measured in pancreatic endocrine cells between 5 degrees and 9 degrees C, using [14C]trehalose. Recovery of adult islets after cryopreservation with 300 mmol/l trehalose was 92 vs. 58% using DMSO alone. In vitro function, in terms of insulin content and release in response to secretagogues, was indistinguishable from fresh islets. Grafts from islets cryopreserved with trehalose contained 14-fold more insulin than grafts from islets cryopreserved without trehalose. Results with human fetal islet-like cell clusters (ICCs) were more pronounced: recovery from cryopreservation was 94%, compared with 42% without trehalose. Complete functionality of fetal cells was also restored; tritiated thymidine incorporation and insulin content and release were similar to fresh tissue. After transplantation in nude mice, there was a 15-fold increase in insulin content of grafts from ICCs cryopreserved with trehalose compared with ICCs cryopreserved without trehalose. Thus, the addition of trehalose to cryopreservation protocols leads to previously unobtainable survival rates of human pancreatic endocrine tissue.

Functional beta-cell mass after transplantation of human fetal pancreatic cells: differentiation or proliferation?

The scarcity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreatic cells desirable. Human fetal pancreatic cells grow and differentiate after transplantation in nude mice. It is unclear whether proliferation of preexisting endocrine cells or differentiation of precursor cells is mainly responsible for the increased islet mass and if beta-cell enrichment before transplantation enhances the functional outcome of the graft. To answer these questions, we transplanted purified human fetal islets, islet-like cell clusters (ICCs), and fresh tissue under the kidney capsule of nude mice. Insulin content was highest in the fresh tissue but fell rapidly during culture as either fetal islets or ICCs. Although fetal islets contained fourfold more insulin than ICCs before transplantation, the insulin content of the resulting grafts was the same after 3 months in vivo. The degree of stimulation after glucose challenge was comparable; however, more tissue was needed to generate the fetal islets. Grafts of fresh tissue also had similar total insulin contents, but when normalized to DNA, insulin concentration was significantly higher in the grafts from cultured tissue. Moreover, there were distinct morphological differences; the grafts from fresh tissue were more fibrous, with prominent ductal and cystic elements. Grafts from cultured tissue were two- to threefold enriched in endocrine tissue when compared with grafts originating from fresh tissue. These results suggest that islet cells identified in the grafted ICCs are mainly derived through differentiation of endocrine precursors and that cultured ICCs are more preferable than either fetal islets or uncultured tissue for 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.