ß-Cell Differentiation from a Human Pancreatic Cell Line in Vitro and in Vivo

Dominique Dufayet de la Tour, Tanya Halvorsen, Carla Demeterco, Björn Tyrberg, Pamela Itkin-Ansari, Mary Loy, Soon-Jib Yoo, Ergeng Hao, Stuart Bossie and Fred Levine

University of California San Diego Cancer Center La Jolla, California 92093-0912


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human ß-cells, inducibly transformed pancreatic ß-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of ß-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional ß-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human ß-cells is a major step toward developing a cell transplantation therapy for diabetes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The development of expanded populations of human pancreatic ß-cells that can be used for cell transplantation is a major goal of diabetes research (1). A number of alternative approaches are being pursued to achieve that goal, including using porcine tissue as a xenograft (2), expansion of primary human ß-cells with growth factors such as hepatocyte growth factor/scatter factor (HGF/SF) and extracellular matrix (ECM) (3), and generation of cell lines that exhibit glucose-responsive insulin secretion (4). Although there has been great interest in using porcine islets, they are difficult to manipulate in vitro, and concerns have been raised about endogenous and exogenous xenobiotic viruses being transmitted to graft recipients (5). With primary human ß-cells, entry into the cell cycle can be achieved using HGF/SF plus ECM (3, 6). However, this combination, while resulting in a 2–3 x 104-fold expansion in the number of cells, is limited by cellular senescence and loss of differentiated function, particularly pancreatic hormone expression (3, 7). Recently, the use of pancreatic or embryonic stem cells has been proposed as a source of cells for ß-cell replacement. Human duct cells have been shown to differentiate in vitro. However, the extent of expansion is limited so far (8). Murine embryonal stem cells were reported to exhibit glucose-responsive insulin secretion when selected with an insulin promoter driving a dominant selectable marker, but that system remains relatively uncharacterized and has not yet been applied to human cells (9).

The focus of our work has been on developing immortalized cell lines from the human endocrine pancreas (10, 11, 12). The cell lines are created by infecting primary cultures of cells from various sources, including adult islets, fetal islets, and purified ß-cells, with a retroviral vector expressing the potent dominant oncogenes SV40 T antigen and H-rasval12 (12, 13). The oncogenes are flanked by loxP DNA sequences, allowing for oncogene deletion by cre recombinase (7). The combined effect of the oncogenes is to trigger growth factor and ECM-independent entry into the cell cycle, as well as to prolong the lifespan of the cells from 10–15 population doublings for primary cells to approximately 150 doubling for the oncogeneexpressing cells (7). Further introduction of the gene encoding the hTERT component of telomerase results in immortalization, allowing the cells to be grown indefinitely (7).

Previously, we have shown that a cell line, TRM-6, derived from human fetal islets, was capable of differentiating along the pancreatic {delta}-cell lineage upon expression of the homeodomain transcription factor PDX-1 and promotion of cell-cell contact (11). Here, we show that ßlox5, a cell line derived from purified adult ß-cells, can be induced to exhibit glucoseresponsive insulin secretion in vitro and in vivo. The ability to grow an unlimited quantity of human ß-cells has implications for cell transplantation therapies for diabetes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pancreatic Hormones Are Not Expressed in ßlox5
ßlox5 is an immortalized cell line derived from a purified population of human ß-cells by infection with retroviral vectors expressing the SV40 T antigen, H- rasval12, and hTERT oncogenes (12). While low levels of insulin expression could be detected immediately after initial isolation of the cell line, insulin subsequently became undetectable (Fig. 1AGo, lane 9). Similar to other human pancreatic cell lines that we have developed (10, 11), somatostatin remained detectable in some samples at the limit of detection of the RT-PCR assay (Fig. 1BGo, lane 3). However, the strength of the somatostatin signal was so weak that no meaningful quantitation or comparison from one sample to another was possible.



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Figure 1. RT-PCR Analysis of Pancreatic Hormone Gene Expression in ßlox5

The conditions tested were ßlox5 grown in monolayer culture, ßlox5 cells infected with a retroviral vector expressing PDX-1 (ßlox5/PDX-1), ßlox5 grown as three-dimensional aggregates, and ßlox5 treated with exendin-4. A, Insulin; B, other pancreatic hormones: PP, glucagon, somatostatin, and IAPP. Human adult islet cDNA was used as positive control. In this figure as well as in Figs. 2Go and 4BGo, the lower band on the gel represents PCR primers.

 
A Subset of Insulin Transcription Factors Are Expressed in ßlox5
To begin to address the reason for the loss of hormone expression, the expression of some of the transcription factors that are important in establishing and maintaining ß-cell differentiated function was examined (Fig. 2Go). Most significantly, PDX-1, a homeodomain transcription factor that is required for ß-cell development and function, is not expressed in ßlox5 by RT-PCR (not shown) or electrophoretic mobility shift assay (EMSA) (Fig. 2AGo, lane 2). This differs from growth-stimulated primary cells, where PDX-1 continues to be expressed (3). BETA2 (NeuroD1), a basic helix-loop-helix (bHLH) factor important in neurogenesis as well as pancreatic endocrine cell development and function, is not expressed in ßlox5 (not shown and Fig. 2CGo). CREB (cAMP-response element-binding protein), which plays a critical role in cAMP responsiveness in ß-cells, is expressed at a low level, being variably detectable at the limit of sensitivity of the Western blot (Fig. 2DGo). Pax6, which is expressed in all endocrine cells, was expressed at high levels in ßlox5, consistent with an origin from primary endocrine cells (Fig. 2BGo, lane 5). RIPE3b, a yet uncloned factor that plays an important role in insulin gene expression by binding to the human insulin promoter C1 element (14, 15), is present as detected by EMSA (Fig. 2EGo). Overall, it is clear that some, but not all, of the transcription factors that play important roles in establishing and maintaining ß-cell function are expressed in ßlox5 cells.



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Figure 2. Analysis of Transcription Factors Expressed in ßlox5 Cells

A, EMSA for PDX-1; B, RT-PCR analysis of Pax6. cDNAs from Jurkat, a T cell line that does not express Pax6 and from total pancreas were used as negative and positive controls, respectively. C, RT-PCR analysis of BETA2. Human adult islet cDNA was used as a positive control; D, Western blot analysis of CREB. E, EMSA for RIPE3b. Nuclear extracts from the TRM6 cell line that does not express RIPE3b was as a negative control.

 
PDX-1, Cell-Cell Contact, and Glucagon-Like Peptide 1 Receptor (GLP-1R) Activation Work Synergistically to Activate Insulin Gene Transcription
Previously, we showed that introduction of PDX-1 into a cell line, TRM-6, derived from human fetal islets, resulted in an increase in somatostatin gene expression (11). Furthermore, PDX-1 acted synergistically with cell-cell contact to further increase the level of somatostatin expression to 1,000-fold above that of cells not expressing PDX-1, to a level similar to that found in normal human islets (11). To determine whether ßlox5 could be induced to express pancreatic hormones, PDX-1 was expressed in ßlox5 using a retroviral vector (11). ßlox5 did not respond to PDX-1 and cell-cell contact with an increase in hormone expression (Fig. 1Go, A and B, lane 3), despite the fact that PDX-1 expression in ßlox5 resulted in functional protein as evidenced by its ability to bind DNA in EMSA (Fig. 2AGo, lane 3). Therefore, other factors were examined for their ability to induce hormone expression in ßlox5 cells expressing PDX-1. Activin has been reported to be able to induce endocrine differentiation in cell lines and primary cells (16, 17, 18, 19). However, it had no effect on hormone expression in ßlox5 (data not shown). Similarly, nicotinamide and HGF, which induce endocrine differentiation of human fetal pancreatic cells (20, 21), also had no effect.

Glucagon-like peptide-1 (GLP-1), a small peptide cleaved from the proglucagon molecule, is an insulin secretagogue (22). A homolog of GLP-1 derived from Gila lizards, exendin-4, has recently been found to stimulate rodent ß-cell differentiation in vitro and in vivo (23, 24). Therefore, exendin-4 was tested for its ability to stimulate ß-cell differentiation in ßlox5. By itself, exendin-4 had no effect on hormone expression in ßlox5 (Fig. 1AGo, lane 8 and not shown). However, when ßlox5 cells expressing PDX-1 were grown in suspension culture as three-dimensional aggregates in the presence of exendin-4, a substantial amount of insulin mRNA was detectable (Fig. 1AGo, lane 2). The synergistic nature of the interaction between exendin-4, cell-cell contact, and PDX-1 is evident from the fact that no insulin mRNA was detectable with any one or two of the three inducing factors (Fig. 1AGo). Quantitative RT-PCR revealed that the level of insulin mRNA in induced ßlox5 cells ranges from one to one-tenth that of adult islets, depending on the quality of the islet preparation (data not shown). Islet amyloid polypeptide (IAPP) mRNA, a hormone that is cosecreted with insulin, was also expressed in induced ßlox5 cells (Fig. 1BGo, lane 2). However, the other major pancreatic hormones, glucagon, somatostatin, and pancreatic polypeptide (PP), were not induced (Fig. 1BGo).

Induction of ß-Cell Differentiation in ßlox5 Correlates with Up-Regulation of BETA2/NeuroD1 and CREB Expression
The expression of transcription factors important in ß-cell development was examined to determine whether they had been induced by PDX-1, cell-cell contact, and exendin-4. RIPE3b binding was not altered by PDX-1, cell-cell contact, or exendin-4 (Fig. 2EGo). BETA2 was induced only by the combination of all three inducing factors (Fig. 2CGo, lane 3). In addition to acting as an insulin secretagogue (25), GLP-1 has been reported to stimulate insulin gene promoter activity by a protein kinase A-independent mechanism mediated by a bZIP transcription factor related in structure to CREB (26). Therefore, it was interesting that CREB, which plays a critical role in the cAMP response of ß-cells, is increased in cells exposed to exendin-4, independently of cell-cell contact and PDX-1 (Fig. 2DGo). This is the first report that activation of the GLP-1 receptor can induce CREB expression, suggesting that GLP-1 may act at multiple levels in promoting cAMP signaling.

Induced ßLox5 Cells Exhibit Glucose-Responsive Insulin Secretion in Vitro
A hallmark of functional ß-cells is the ability to secrete insulin in response to blood glucose. Primary ß-cells store large quantities of insulin and secrete it in response to a variety of physiological stimuli, most significantly extracellular glucose. Induced ßlox5 cells have an insulin content between 6 and 21 pmol/µg DNA, compared with adult islet preparations, which in our hands have insulin contents between 50 and 250 pmol/µg DNA.

Intracellular insulin is detectable by immunohistochemistry in the induced ßlox5 cells (Fig. 3BGo), whereas no cells were stained before aggregation (Fig. 3AGo). In parallel, very few secretory granules were found in the uninduced cells (Fig. 3CGo). Those granules were pale stained, with the appearance of somatostatin granules, consistent with the known presence of a very low level of somatostatin mRNA in the uninduced cells (Fig. 1BGo, lane 3). In contrast, the morphology of the granules in the induced cells (Fig. 3DGo) varied from electron opaque granules with a white halo typical for primary ß-cells, to pale staining granules that might be immature insulin granules (Fig. 3DGo, inset). In both conditions, electron opaque structures at about the size of small mitochondria, filled with spiral membranes, were found. These structures are easily mistaken for large secretory granules in the overview pictures (Fig. 3Go, C and D), but have a morphology consistent with remnants of hyperproliferating endoplasmic reticulum.



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Figure 3. Insulin Immunohistochemical and Ultrastructural Analysis of Uninduced (A and C) and Induced (B and D) ßlox5 Cells

A and B, Insulin immunohistochemistry (105x magnification); C and D, electron micrographs (2,900x magnification, inset 22,000x).

 
Consistent with the presence of morphologically mature insulin granules, media from induced ßlox5 cells contain processed insulin that comigrates with bovine insulin (Fig. 4AGo). Interestingly, out of six duplicate wells of induced ßlox5 cells, one did not exhibit any insulin secretion (Fig. 4AGo, lane 4) or mRNA by RT-PCR (not shown). We have found repeatedly that occasional cultures of exendin-4-treated ßlox5/PDX-1 aggregates fail to differentiate. The reason for this is under investigation but seems to be due in large part to loss of PDX-1 expression over time. Previously, we have found that PDX-1 causes a decrease in the growth rate of the human pancreatic endocrine cell line TRM-6 (11). ßLox5 cells exhibit an even more pronounced growth arrest in response to PDX-1 expression, leading to strong selection for loss of PDX-1 over time in culture.



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Figure 4. Analysis of Glucose-Responsive Insulin Secretion

A, Insulin Western blot analysis of conditioned medium from induced ßlox5 cells. Bovine insulin was used as a positive control. Fresh tissue culture media were used as a negative control; B, RT-PCR for glucokinase. cDNA from human total pancreas was used as a positive control; C, Assay for insulin secreted from induced ßlox5 cells grown in culture medium containing increasing concentrations of glucose. Two independent cultures of induced ßlox5 cells were studied (open boxes) and compared with a single preparation of adult human islets (closed triangle).

 
A major component of the glucose-sensing apparatus is glucokinase (27). Induced ßlox5 cells express glucokinase mRNA, while uninduced cells do not (Fig. 4BGo). The extent to which ßlox5 cells exhibit glucose-responsive insulin secretion was tested by culturing two independent cultures of induced ßlox5 cells in different concentrations of glucose and measuring insulin release in the culture medium by ELISA (Fig. 4CGo). Consistent with the lower insulin content of the induced ßlox5 cells, the amount of insulin secreted in response to glucose is 3- to 4-fold lower than adult islets. Induced ßlox5 cells exposed to high concentrations of glucose for prolonged periods become depleted of insulin more rapidly than primary islets (not shown).

Oncogene-Deleted ßlox5 Cells Exhibit Glucose-Responsive Insulin Secretion in Vivo
To test the function of ßlox5 cells in vivo, cells were transplanted under the kidney capsule of nude mice. To reduce the risk of tumorigenicity after transplantation, the LTPRRTNllox retroviral vector incorporates lox sites in the long terminal repeats (LTRs). This allows the oncogenes to be deleted by expression of cre recombinase. To express cre, ßlox5 cells were infected with an adenoviral vector (Ad-cre) (28) at a multiplicity of infection of 50. While this resulted in a large reduction in SV40 T antigen expression, a low level of expression persisted, reflecting inefficiency of infection and/or cre-mediated recombination (Fig. 5Go). The following conditions were studied: PDX-1 expression, oncogene deletion with Ad-cre, and exposure of the cells to exendin-4 in vitro and/or in vivo. Animals were monitored for tumor formation and circulating human C-peptide levels up to a maximum of 3 months after transplantation, when a final C-peptide determination was made (Fig. 6Go). Substantial levels of circulating human C-peptide were present only in animals grafted with cells expressing PDX-1. Furthermore, oncogene deletion with Ad-cre and/or exendin-4 treatment in vitro was also required for circulating C-peptide to be present. The incidence of tumors was much lower with oncogene-deleted cells (1 of 10 animals) compared with oncogene-expressing cells (16 of 23 animals). The level of C-peptide secreted by the grafted cells is comparable to the amount secreted by primary islet cell grafts that have been shown to cure diabetes in streptozotocin-treated nude mice (29). Interestingly, animals injected with exendin-4 in vivo did not have any detectable C-peptide, suggesting that continued administration is deleterious to graft function. Significantly, C-peptide levels in fasting animals before and after intraperitoneal glucose stimulation demonstrated that the C-peptide secretion was glucose responsive.



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Figure 5. Western Blot of SV40LT Protein in ßlox5 before and after Infection with Ad-cre

Whole cell extract from 293T cells was used as a positive control and from 293 GFP as a negative control.

 


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Figure 6. Human C-Peptide Levels in Mice Grafted with ßlox5 Cells before and after Glucose Stimulation

Black bars represent basal C-peptide levels before glucose stimulation. Open bars represent glucose-stimulated Cpeptide levels. The presence (+) or absence (–) of a tumor in each animal is indicated immediately beneath the C-peptide data from that animal. Seven groups were transplanted with ßlox5 cells: uninduced cells, Ad-Cre infected cells, ßlox5/Pdx-1 cells, Ad-Cre infected ßlox5/Pdx-1 cells, ßlox5/Pdx-1 cells exposed to exendin-4 in vitro (induced cells), ßlox5/Pdx-1 cells exposed to exendin-4 in vivo, and ßlox5/Pdx-1 cells exposed to exendin-4 in vitro and in vivo. Animals were scored as having a tumor if the size of the graft was significantly greater than the 10 µl volume of cells initially grafted.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Beginning with a cell line that exhibited few ß-cell characteristics despite having been originally derived from human ß-cells, we have successfully induced ß-cell function in vitro and in vivo. Induction of ß-cell differentiation in ßlox5 is complex, requiring synergistic interactions between at least three inducing factors, PDX-1, cell-cell contact, and activation of the GLP-1 receptor. In addition, it is likely that those three inducing factors interact with gene products expressed in ßlox5 to induce ß-cell differentiation, since another cell line, TRM-6, responds to the PDX-1 and cell-cell contact by differentiating along the {delta}-cell lineage (11). Cell lines such as TRM-6 ({delta}-cell lineage) and ßlox5 (ß-cell lineage) are powerful tools that should allow identification of the full complement of genes that are required for endocrine cell development and function. Furthermore, the requirement for multiple interacting inducing factors provides an opportunity to study how different signal transduction pathways interact with one another to control a complex differentiation program.

A problem with our current technology is that PDX-1 expression leads to a substantial decrease in the growth rate of the cells. Unlike TRM-6, where we have been able to maintain stable populations of cells expressing PDX-1 indefinitely, it has not been possible to maintain PDX-1 expression in ßlox5. Expressing PDX-1 under inducible control may solve this problem and such studies are underway. The level of insulin stored and secreted by induced ßlox5 cells, while substantial compared with rodent insulinoma cell lines, remains substantially below that of normal human islets. Whether this level of insulin is sufficient to cure diabetes remains to be tested. It is possible that higher levels of insulin production could be engineered into the cells, either directly by overexpressing the proinsulin gene or indirectly by further manipulation of insulin gene transactivators. It is encouraging that the shape of the glucose response curve is quite similar to that of normal human islets. Most other insulinoma cell lines exhibit maximal insulin secretion at a much lower glucose concentration, which is thought to be due to an imbalance in the glucokinase/hexokinase ratio (30).

The availability of an unlimited source of functional human ß-cells has important implications for diabetes. One straightforward application is in exploring aspects of ß-cell biology that would benefit from an unlimited, homogeneous source of cells. High-throughput screening for new diabetes drugs is one such application. Ultimately, we hope to be able to use cells such as those presented here in a cell transplantation therapy for diabetes. However, a number of issues must be dealt with before an immortalized cell line can be used in humans. One problem has to do with effects of the oncogenes on the biological properties of the cells. Cell lines from the human endocrine pancreas developed using SV40 T antigen plus H-rasval12 are tumorigenic (10). Although this does not prevent them from differentiating into fully functional ß-cells in vitro, tumorigenic cells cannot be transplanted into humans unless they are safely encapsulated. The vector used to create the cell lines incorporates lox sites so that the oncogenes can be deleted by expression of the cre recombinase in the cells after expansion (12), so it may be possible to avoid encapsulation by oncogene deletion before transplantation. The development of improved vectors is necessary to allow for complete oncogene deletion. Such studies are in progress. Immune rejection of grafted cells has been a major obstacle to successful islet transplantation, but recent advances in therapy for allograft rejection may make this less of a concern (31, 32). A potential advantage of using an immortalized cell line as a source of transplantable cells is that they can be engineered to exhibit desirable qualities, including avoidance or suppression of host immune responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
All the cells were maintained in DMEM with 5.5 mM glucose and 10% FBS. Aggregation of cells into three-dimensional clusters and the retroviral vector expressing PDX-1 have been described previously (11). Infection with the PDX-1 virus was done at a multiplicity of infection of 25. Exendin-4 (Sigma, St Louis, MO) was used at a concentration of 10 nM in PBS.

RT-PCR
RT-PCRs for somatostatin, glucagon, IAPP, and glucokinase have been described previously (11, 33). Quantitative insulin RT-PCR was done by interpolation from a standard curve constructed using a plasmid containing the human insulin cDNA (3). RT-PCR for BETA2 and Pax-6 were performed using the following primers: BETA2 Forward-cagaaccaggacatgccc, BETA2 Reverse-atcaaaggaagggctggtg, Pax-6 Forward-gccttttattgtgagagtgg, Pax-6 Reverse-ctcacacatccgttggacac. PCR amplification conditions were 5 min at 94 C followed by 40 cycles of 94 C for 45 sec, 60 C for 45 sec, 72 C for 45 sec. All cDNA preparations were checked for the quality and concentration of cDNA by RT-PCR for the housekeeping gene porphobilinogen deaminase (11).

Protein Expression
EMSA for Pdx-1 and RIPE3b were performed using probes derived from the human insulin promoter A5 and C1 elements, respectively (11, 14). Western blot for CREB was performed using a polyclonal antibody (a kind gift from Dr. Marc Montminy) at a dilution of 1:200 on 10 µg of nuclear extracts. Western blot for insulin was done using a monoclonal antibody (Insulin 2D11-H5, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a dilution of 1:500 on 40 µl conditioned media. Anti-SV40 T antigen antibody (PharMingen, La Jolla, CA) was used at a concentration of 1 µg/ml. Induced ßlox5 cells were cultured in DMEM containing a single concentration of added glucose for 1 h. Medium was harvested and assayed for insulin by RIA (Diagnostic Products, Los Angeles, CA) or enzyme-linked immunosorbent assay (ELISA) (Ultrasensitive human insulin ELISA; Mercodia, Uppsala, Sweden).

Immunohistochemistry
ßlox5 aggregates or single cells were fixed in 4% paraformaldehyde for 3 h, embedded in 2% agarose, fixed for another 21 h, processed for paraffin embedding, and sectioned in 5 µm slices. Specimens were stained for insulin with an antihuman insulin antibody (Santa Cruz Biotechnology, Inc.) and a peroxidase/diaminobenzidine detection system providing a brown color precipitate (EnVision+, DAKO Corp., Glostrup, Denmark).

Ultrastructural Analysis
ßlox5 aggregates or single cells were washed in 0.1 M cacodylate buffer and fixed in 2% glutaraldehyde in cacodylate buffer for 1 h. Specimens were then rinsed in cacodylate buffer and dH2O, postfixed in 1% OsO4 in dH2O for 1 h, rinsed in dH2O and 70% ethanol, stained en bloc with 2% uranyl acetate in 70% ethanol, dehydrated in a graded series of ethanol, embedded in Epon 812 (34), sectioned in 80-nm slices, and contrasted with 2% lead citrate and 2% uranyl acetate. The specimens were randomly photographed in a transmission electron microscope (Hitachi-600; Hitachi Scientific Instruments, Inc., Tokyo, Japan) at an acceleration voltage of 75 kV.

Transplantation Studies
Human cell transplants were performed by inserting 10 µl of packed aggregated cells under the kidney capsule of 7-week-old NIH Swiss homozygous athymic nude mice as previously described (35).


    ACKNOWLEDGMENTS
 
We thank the Juvenile Diabetes Foundation Islet Isolation Program and particularly the Islet Transplant Centers at the University of Minnesota and University of Miami for providing human islets for these studies.


    FOOTNOTES
 
Address requests for reprints to: Dr. Fred Levine, Center for Molecular Genetics, University of California San Diego School of Medicine, La Jolla, California 92093-0634. E-mail: flevine{at}ucsd.edu

This work was supported by grants to Fred Levine from the NIDDK (DK-55065 and DK-55283), the Juvenile Diabetes Foundation (197035), and the Stern Foundation. Bjorn Tyrberg was supported by the Swedish Academy of Pharmaceutical Sciences Fellowship. Soon-Jib Yoo was supported by a research scholarship from the Catholic University of Korea.

Received for publication September 19, 2000. Revision received October 26, 2000. Accepted for publication November 21, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Diabetes Research Working Group 1999 Conquering Diabetes: A Strategic Plan For the 21st Century. National Institutes of Health, Bethesda, MD, pp 67–69
  2. Groth CG, Tibell A, Wennberg L, Korsgren O 1999 Xenoislet transplantation: experimental and clinical aspects. J Mol Med 77:153–154[CrossRef][Medline]
  3. Beattie GM, Itkin-Ansari P, Cirulli V, Leibowitz G, Lopez AD, Bossie S, Mally MI, Levine F, Hayek A 1999 Sustained proliferation of PDX-1+ cells derived from human islets. Diabetes 48:1013–1019[Abstract]
  4. Levine F 1997 Gene therapy for diabetes: strategies for ß-cell modification and replacement. Diabetes Metab Rev 13:209–46[CrossRef][Medline]
  5. Weiss RA 1998 Transgenic pigs and virus adaptation. Nature 391:327–8[CrossRef][Medline]
  6. Hayek A, Beattie GM, Cirulli V, Lopez AD, Ricordi C, Rubin JS 1995 Growth factor/matrix-induced proliferation of human adult ß-cells. Diabetes 44:1458–1460[Abstract]
  7. Halvorsen TL, Beattie GM, Lopez AD, Hayek A, Levine F 2000 Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro. J Endocrinol 166:103–109[Abstract/Free Full Text]
  8. Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, O’Neil JJ 2000 In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA 97:7999–8004[Abstract/Free Full Text]
  9. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F 2000 Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49:157–162[Abstract]
  10. Wang S, Beattie GM, Mally MI, Cirulli V, Itkin-Ansari P, Lopez AD, Hayek A, Levine F 1997 Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas. Cell Transplant 6:59–67[CrossRef][Medline]
  11. Itkin-Ansari P, Demeterco C, Bossie S, de la Tour DD, Beattie GM, Movassat J, Mally MI, Hayek A, Levine F 2000 PDX-1 and cell-cell contact act in synergy to promote {delta}-cell development in a human pancreatic endocrine precursor cell line. Mol Endocrinol 14:814–822[Abstract/Free Full Text]
  12. Halvorsen TL, Leibowitz G, Levine F 1999 Telomerase activity is sufficient to allow transformed cells to escape from crisis. Mol Cell Biol 19:1864–70[Abstract/Free Full Text]
  13. Wang S, Beattie GM, Hayek A, Levine F 1996 Development of a VSV-G protein pseudotyped retroviral vector system expressing dominant oncogenes from a lacO-modified inducible LTR promoter. Gene 182:145–50[CrossRef][Medline]
  14. German M, Ashcroft S, Docherty K, Edlund H, Edlund T, Goodison S, Imura H, Kennedy G, Madsen O, Melloul D, Moss L, Olson K, Permutt MA, Philippe J, Robertson RP, Rutter WJ, Serup P, Stein R, Steiner D, Tsai M-J, Walker MD 1995 The insulin gene promoter; a simplified nomenclature. Diabetes 44:1002–1004[Medline]
  15. Sharma A, Fuscodemane D, Henderson E, Efrat S, Stein R 1995 The role of the insulin control element and Ripe3b1 activators in glucose-stimulated transcription of the insulin gene. Mol Endocrinol 9:1468–1476[Abstract]
  16. Demeterco C, Beattie GM, Dib SA, Lopez AD, Hayek A 2000 A role for activin A and beta cellulin in human fetal pancreatic cell differentiation and growth. J Clin Endocrinol Metab 85:3892–3897[Abstract/Free Full Text]
  17. Huotari MA, Palgi J, Otonkoski T 1998 Growth factor-mediated proliferation and differentiation of insulinproducing INS-1 and RINm5F cells: identification of betacellulin as a novel ß-cell mitogen. Endocrinology 139:1494–1499[Abstract/Free Full Text]
  18. Mashima H, Ohnishi H, Wakabayashi K, Mine T, Miyagawa J, Hanafusa T, Seno M, Yamada H, Kojima I 1996 Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest 97:1647–54[Abstract/Free Full Text]
  19. Yamaoka T, Idehara C, Yano M, Matsushita T, Yamada T, Ii S, Moritani M, Hata J, Sugino H, Noji S, Itakura M 1998 Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants. J Clin Invest 102:294–301[Abstract/Free Full Text]
  20. Otonkoski T, Beattie GM, Mally MI, Ricordi C, Hayek A 1993 Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J Clin Invest 92:1459–1466[Medline]
  21. Otonkoski T, Beattie GM, Rubin JS, Lopez AD, Baird A, Hayek A 1994 Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells. Diabetes 43:947–953[Abstract]
  22. Drucker DJ 1998 Glucagon-like peptides. Diabetes 47:159–69[Abstract]
  23. Xu G, Stoffers DA, Habener JF, Bonner-Weir S 1999 Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased ß-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48:2270–2276[Abstract]
  24. Zhou J, Wang X, Pineyro MA, Egan JM 1999 Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 48:2358–2366[Abstract]
  25. Serre V, Dolci W, Schaerer E, Scrocchi L, Drucker D, Efrat S, Thorens B 1998 Exendin-(9–39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and ß-cell glucose competence. Endocrinology 139:4448–4454[Abstract/Free Full Text]
  26. Skoglund G, Hussain M, Holz G 2000 Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activatino of the rat insulin I gene cAMP response element. Diabetes 49:1156–1164[Abstract]
  27. Matschinsky FM, Glaser B, Magnuson MA 1998 Pancreatic ß-cell glucokinase: closing the gap between theoretical concepts and experimental realities. Diabetes 47:307–315[Abstract]
  28. Wang Y, Krushel LA, Edelman GM 1996 Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. Proc Natl Acad Sci USA 93:3932–3936[Abstract/Free Full Text]
  29. Hayek A, Beattie GM 1997 Experimental transplantation of human fetal and adult pancreatic islets. J Clin Endocrinol Metab 82:2471–2475[Abstract/Free Full Text]
  30. Clark SA, Quaade C, Constandy H, Hansen P, Halban P, Ferber S, Newgard CB, Normington K 1997 Novel insulinoma cell lines produced by iterative engineering of GLUT2, glucokinase, and human insulin expression. Diabetes 46:958–967[Abstract]
  31. Kenyon NS, Chatzipetrou M, Masetti M, Ranuncoli A, Oliveira M, Wagner JL, Kirk AD, Harlan DM, Burkly LC, RicordiC 1999 Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci USA 96:8132–8137[Abstract/Free Full Text]
  32. Shapiro A, Lakey J, Ryan E, Korbutt G, Toth E, Warnock G, Kneteman N, Rajotte R 2000 Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238[Abstract/Free Full Text]
  33. Mally MI, Otonkoski T, Lopez AD, Hayek A 1994 Developmental gene expression in the human fetal pancreas. Pediatr Res 36:537–544[Abstract]
  34. Luft J 1961 Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409–414[Abstract/Free Full Text]
  35. Beattie G, M, Levine F, Mally MI, Otonkoski T, O’Brien JS, Salomon DR, Hayek A 1994 Acid ß-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas. J Clin Endocrinol Metab 78:1232–1240[Abstract]