Human Beta Cell Proliferation Research Articles

Serum from pregnant donors induces human beta cell proliferation

ABSTRACT Pancreatic beta cells are among the slowest replicating cells in the human body and have not been observed to increase in number except during the fetal and neonatal period, in cases of obesity, during puberty, as well as during pregnancy. Pregnancy is associated with increased beta cell mass to meet heightened insulin demands. This phenomenon raises the intriguing possibility that factors present in the serum of pregnant individuals may stimulate beta cell proliferation and offer insights into expansion of the beta cell mass for treatment and prevention of diabetes. The primary objective of this study was to test the hypothesis that serum from pregnant donors contains bioactive factors capable of inducing human beta cell proliferation. An immortalized human beta cell line with protracted replication (EndoC-βH1) was cultured in media supplemented with serum from pregnant and non-pregnant female and male donors and assessed for differences in proliferation. This experiment was followed by assessment of proliferation of primary human beta cells. Sera from five out of six pregnant donors induced a significant increase in the proliferation rate of EndoC-βH1 cells. Pooled serum from the cohort of pregnant donors also increased the rate of proliferation in primary human beta cells. This study demonstrates that serum from pregnant donors stimulates human beta cell proliferation. These findings suggest the existence of pregnancy-associated factors that can offer novel avenues for beta cell regeneration and diabetes prevention strategies. Further research is warranted to elucidate the specific factors responsible for this effect.

349-OR: Selected DYRK1A Inhibitors Simultaneously Drive Both Human Beta-Cell Proliferation and Differentiation

Small molecule DYRK1A inhibitors induce adult human beta cell proliferation and expand human beta cell mass. Harmine also enhances differentiation and function in human beta cells in vitro and in vivo, increasing expression of key beta cell genes. It is unknown whether this “pro-differentiation effect” is a feature of all DYRK1A inhibitors. We compared harmine, 2-2c, 5-Iodotubericidin (5-IT), GNF4877, INDY, CC-401 and Leucettine-41 at doses that maximally drive human beta cell proliferation. As anticipated, harmine, 2-2c and 5-IT all increased expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC30A8, and ENTPD3. These effects were observed in normal human islets as well as in islets from donors with T2D. Surprisingly, in simultaneous experiments, the DYRK1A inhibitors GNF4877, INDY, CC-401 and Leucettine failed to induce these beta cell differentiation markers. The pro-differentiation effect of harmine was independent of DYRK1A inhibition: silencing DYRK1A in human islets induced beta cell proliferation, but had no effect on differentiation; harmine treatment in DYRK1A-silenced islets restored beta cell pro-differentiation effects. The pro-differentiation effect was also independent of NFAT signaling and DREAM Complex effects, since NFAT overexpression, or p130, p107, E2F4 and E2F5 silencing had no effect on beta cell differentiation. Similarly, silencing other known harmine targets (DYRK2, 3, 4, the CLK and HIPK family) had no effect on either human beta cell differentiation nor proliferation. In summary, harmine, 2-2c and 5-IT are unique among the DYRK1A inhibitor family in their simultaneous ability to enhance both beta cell proliferation and differentiation. While the beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor drug development. Disclosure P. Wang: None. K. Kumar: None. H. Liu: None. O. Wood: None. E. Karakose: None. L. Choleva: None. L. Lambertini: None. A. Garcia-Ocana: Consultant; Sun Pharmaceutical Industries Ltd. R.J. DeVita: None. A.F. Stewart: None.

Mitogen Synergy: An Emerging Route to Boosting Human Beta Cell Proliferation.

Decreased number and function of beta cells are a key aspect of diabetes mellitus (diabetes), a disease that remains an onerous global health problem. Means of restoring beta cell mass are urgently being sought as a potential cure for diabetes. Several strategies, such as de novo beta cell derivation via pluripotent stem cell differentiation or mature somatic cell transdifferentiation, have yielded promising results. Beta cell expansion is another promising strategy, rendered challenging by the very low proliferative capacity of beta cells. Many effective mitogens have been identified in rodents, but the vast majority do not have similar mitogenic effects in human beta cells. Extensive research has led to the identification of several human beta cell mitogens, but their efficacy and specificity remain insufficient. An approach based on the simultaneous application of several mitogens has recently emerged and can yield human beta cell proliferation rates of up to 8%. Here, we discuss recent advances in restoration of the beta cell population, focusing on mitogen synergy, and the contribution of RNA-sequencing (RNA-seq) to accelerating the elucidation of signaling pathways in proliferating beta cells and the discovery of novel mitogens. Together, these approaches have taken beta cell research up a level, bringing us closer to a cure for diabetes.

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

899 Effect of maternal serum on proliferation of the EndoC-ßH1 human beta cell line

During pregnancy, the placenta secretes hormones that increase insulin resistance and raise blood glucose to support the growing fetus. To prevent maternal hyperglycemia, pregnancy is also associated with an increase in pancreatic beta cell mass. If molecular mechanisms that trigger maternal beta cell expansion can be determined, such pathways could be targeted for therapeutic beta cell regeneration. Exosomes, a type of extracellular vesicle, contain signaling molecules and are released by placental cells. Exosomes may be a source of inter-organ communication that signals beta cells to proliferate during pregnancy. Our purpose is to determine if exposure to maternal serum containing placental-derived exosomes triggers human beta cell proliferation. The genetically engineered human beta cell line EndoC-βH1 was used for this study because its phenotype and function are similar to primary human beta cells, and it proliferates reliably. EndoC-βH1 cells were cultured for one week in the presence of third trimester and postpartum serum drawn within 48 hours of delivery. We compared the maternal serum to serum from a male donor, fetal bovine serum, and serum-free conditions. Cell proliferation was measured using an EdU(5-ethynyl-2-deoxyuridine) assay. Six third trimester and postpartum donors were studied. Serum from three of six maternal donors induced a substantial (20-30%) increase in beta cell proliferation relative to serum-free conditions (Figure 1A). Of the three donors, five of six samples showed significant proliferation (p<0.05). (Figure 1A). Male human serum and fetal bovine serum only increased beta cell proliferation moderately (7-9%). We found no difference in the degree of beta-cell expansion induced when comparing serum samples collected during the third trimester and postpartum (Figure 1B). The results suggest a factor present in serum from 50% of pregnant donors triggers beta cells to proliferate. In future studies, we will deplete maternal serum of exosomes to determine whether the increased proliferation is attributable to maternal exosomes.

2141-P: Nrf2 Mediates Glucose-Stimulated Beta-Cell Proliferation

Glucose catabolism drives adaptive beta cell mass expansion by promoting proliferation under conditions of increased insulin demand. Previously, we showed that induction of the antioxidant regulator, Nrf2, is required for glucose-stimulated β-cell proliferation, and that overexpression of Nrf2 stimulates human beta cell proliferation. Here we manipulate the Nrf2 pathway using pharmacological and genetic approaches to explore the link between glucose metabolism, Nrf2 activation and beta cell proliferation. Treatment of isolated mouse islets with either a Nrf2 inhibitor (brusatol) or Cre-recombinase deleted Nrf2, resulted in complete inhibition of glucose-stimulated proliferation (both n=3, p &amp;lt; 0.01). Conversely, stimulation of Nrf2 by CDDO-me or genetic gain of function (Cre-mediated depletion of Nrf2 inhibitor, Keap1) increased beta cell proliferation of beta cells from isolated mouse islets cultured in 5 mM glucose (4.9 ± 0.2 fold and 6.9 ± 1.4 fold, respectively; n=3, p &amp;lt; 0.05). Inhibition or depletion of Nrf2 was associated with a remarkable decrease in insulin content, suggesting that beta cell identity requires basal levels of Nrf2 expression. Importantly, isolated human beta cells displayed similar responses to Nrf2 loss and gain of function. Activation and nuclear translocation of Nrf2 requires dissociation from its inhibitor, Keap1, and phosphorylation of Ser-40 on Nrf2. Five minutes of 20 mM glucose treatment profoundly increased Ser-40 phosphorylation and translocation of Nrf2, while the same effect by CDDO-Me treatment took 50 minutes in INS1 cells. The glucose-mediated activation and translocation of Nrf2 was blocked by the antioxidant N-acetylcysteine (NAC), which also attenuated glucose-stimulated proliferation of isolated mouse beta cells. In addition, the activation and phosphorylation of Nrf2 was prevented by the pan-PKC inhibitor, calphostin C. We conclude that manipulation of the Nrf2 pathway is an exciting novel target for regulation of beta cell proliferation. Disclosure S. Baumel-Alterzon: None. L.S. Katz: None. G. Brill: None. A. Garcia-Ocaña: None. D. Scott: None. Funding American Diabetes Association (1-17-IBS-116 to D.S.); National Institutes of Health

Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells

Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFβSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFβ inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both invitro and invivo.

Emerging Roles for Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) in Pancreatic Beta Cells and Diabetes.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was originally identified as a secreted trophic factor for dopamine neurons in vitro. It protects and restores damaged cells in rodent models of Parkinson’s disease, brain and heart ischemia, spinocerebellar ataxia and retina in vivo. However, its exact mechanism of action is not known. MANF is widely expressed in most human and mouse organs with high levels in secretory tissues. Intracellularly, MANF localizes to the endoplasmic reticulum (ER) and ER stress increases it’s expression in cells and tissues. Furthermore, increased MANF levels has been detected in the sera of young children with newly diagnosed Type 1 (T1D) diabetes and Type 2 (T2D) diabetic patients. ER stress is caused by the accumulation of misfolded and aggregated proteins in the ER. It activates a cellular defense mechanism, the unfolded protein response (UPR), a signaling cascade trying to restore ER homeostasis. However, if prolonged, unresolved ER stress leads to apoptosis. Unresolved ER stress contributes to the progressive death of pancreatic insulin-producing beta cells in both T1D and T2D. Diabetes mellitus is characterized by hyperglycemia, caused by the inability of the beta cells to maintain sufficient levels of circulating insulin. The current medications, insulin and antidiabetic drugs, alleviate diabetic symptoms but cannot reconstitute physiological insulin secretion which increases the risk of devastating vascular complications of the disease. Thus, one of the main strategies in improving current diabetes therapy is to define and validate novel approaches to protect beta cells from stress as well as activate their regeneration. Embryonic deletion of the Manf gene in mice led to gradual postnatal development of insulin-deficient diabetes caused by reduced beta cell proliferation and increased beta cell death due to increased and sustained ER stress. In vitro, recombinant MANF partly protected mouse and human beta cells from ER stress-induced beta cell death and potentiated mouse and human beta cell proliferation. Importantly, in vivo overexpression of MANF in the pancreas of T1D mice led to increased beta cell proliferation and decreased beta cell death, suggesting that MANF could be a new therapeutic candidate for beta cell protection and regeneration in diabetes.

Increasing beta cell mass to treat diabetes mellitus.

Finding a radical cure for diabetes has reached paramount importance in medicine due to the widespread prevalence of the disease. A substantial reduction in insulin-secreting beta cells is evident in diabetes. The failure of cyclin-dependent kinases (CDKs) and cyclins to access the nucleus is responsible for quiescence or senescence in human and rodent beta cells. The augmentation of beta cell proliferation is supposed to reverse diabetes. This concept has inspired the discovery of newer drugs that encourage the proliferation of beta cells. Although it is a rational step towards a cure for diabetes, the differences in biochemical pathways in rodents and human beta cells pose difficulty in promoting the proliferation of human beta cells. Primarily, it is mandatory to clearly understand the intracellular pathways involved in the proliferation of beta cells so as to pave the way for therapeutic interventions. There are several intrinsic factors that trigger the proliferation of beta cells. Furthermore, it is also obvious that the early death of beta cells due to oxidative stress-related upregulation of pro-apoptotic genes also predisposes individuals to diabetes mellitus. Polyphenols, exendin 4, histone deacetylase inhibitors, glucagon-like peptide 1, phenyl pyruvic acid glucoside, and several flavonoids reduce the early apoptosis of beta cells partly through their role in the reduction of oxidative stress. A better understanding of intracellular pathways, the identification of specific mitogens, the induction of beta cell proliferation, and the inhibition of apoptosis may help us treat diabetes mellitus through an increase in beta cell mass.

Advances in drug discovery for human beta cell regeneration.

The numbers of insulin-secreting pancreatic beta cells are reduced in people with type 1 and type 2 diabetes. Driving beta cell regeneration in the pancreases of people with diabetes would be an attractive approach to reversing diabetes. While adult human beta cells have long been believed to be terminally differentiated and, therefore, irreversibly quiescent, it has become clear over recent years that this is not true. More specifically, both candidate and unbiased high-throughput screen approaches have revealed several classes of molecules that are clearly able to induce human beta cell proliferation. Here, we review recent approaches and accomplishments in human beta cell regenerative drug discovery. We also list the challenges that this rapidly moving field must confront to translate beta cell regenerative therapy from the laboratory to the clinic.

Cell-Based Methods to Identify Inducers of Human Pancreatic Beta-Cell Proliferation.

Diabetes is the result of the insufficiency or dysfunction of pancreatic beta cells alone or in combination with insulin resistance. The replacement or regeneration of beta cells can effectively reverse diabetes in humans and rodents. Therefore, the identification of novel small molecules that promote pancreatic beta-cell proliferation is an attractive approach for diabetic therapy. While numerous hormones, small molecules, and growth factors are able to drive rodent beta cells to replicate, only a few small molecules have demonstrated the ability to stimulate human beta-cell proliferation. Hence, there is an urgent need for therapeutic agents that induce regeneration and expansion of adult human beta cells. Here, we describe a detailed protocol for coating chamber slides, culturing primary islets, performing islet cell disassociation, seeding cells on chamber slides, treating islet cells with compounds or infecting them with adenovirus, immunostaining of proliferation markers and imaging, and data analysis.

Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue

IntroductionThe low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. MethodsTo identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. ResultsThe frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (p<0.0001), Cyclin C (p<0.0001), and p57 (p=0.018) expression levels were lower. The abundance of Cyclin D3 (p=0.62) and p27 (p=0.68) was not different between the groups. The reduced expression of p16 (p<0.0001) and p57 (p=0.012) in insulinomas and the unchanged expression of Cyclin D3 (p=0.77) and p27 (p=0.55) were confirmed using qRT-PCR. ConclusionsThe expression of certain cell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration.

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.

Osteoprotegerin and Denosumab Stimulate Human Beta Cell Proliferation through Inhibition of the Receptor Activator of NF-κB Ligand Pathway

Diabetes results from a reduction of pancreatic β-cells. Stimulating replication could normalize β-cell mass. However, adult human β-cells are recalcitrant to proliferation. We identified osteoprotegerin, a bone-related decoy receptor, as a β-cell mitogen. Osteoprotegerin was induced by and required for lactogen-mediated rodent β-cell replication. Osteoprotegerin enhanced β-cell proliferation in young, aged, and diabetic mice. This resulted in increased β-cell mass in young mice and significantly delayed hyperglycemia in diabetic mice. Osteoprotegerin stimulated replication of adult human β-cells, without causing dedifferentiation. Mechanistically, osteoprotegerin induced human and rodent β-cell replication by modulating CREB and GSK3 pathways, through binding Receptor Activator of NF-κB (RANK) Ligand (RANKL), a brake in β-cell proliferation. Denosumab, an FDA-approved osteoporosis drug, and RANKL-specific antibody induced human β-cell proliferation in vitro, and in vivo, in humanized mice. Thus, osteoprotegerin and Denosumab prevent RANKL/RANK interaction to stimulate β-cell replication, highlighting the potential for repurposing an osteoporosis drug to treat diabetes.

A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges.

WS6 induces both alpha and beta cell proliferation without affecting differentiation or viability.

Agents that stimulate human pancreatic beta cell proliferation are needed to improve diabetes mellitus treatment. Recently, a small molecule, WS6, was observed to stimulate human beta cell proliferation. However, little is known about its other effects on human islets. To better understand the role of WS6 as a possible beta cell regenerative therapy, we carried out in-depth phenotypic analysis of WS6-treated human islets, exploring its effects on non-beta cell proliferation, beta cell differentiation, and islet cell viability. WS6 not only stimulated beta cell proliferation in cultured human islets (in agreement with previous reports), but also human alpha cell proliferation, indicating that WS6 is not a beta cell-specific mitogen. WS6 did not change the proportion of insulin-positive beta cells or the expression of beta cell-specific transcription factors, suggesting that WS6 does not alter beta cell differentiation, and WS6 had no effect on human islet cell apoptosis or viability. In conclusion, WS6 stimulates proliferation of both human beta and alpha cells while maintaining cellular viability and the beta cell differentiated phenotype. These findings expand the literature on WS6 and support the suggestion that WS6 may help increase human islet mass needed for successful treatment of diabetes.

Functional Genomic HTS to Study Primary Human Beta-Cell Proliferation

s / Can J Diabetes 37 (2013) S13eS84 S58 processes and that at high glucose islets convert a significant portion of the sugar into glycerol and FFA, which are exported outside the b-cell. The identification of fuel surfeit detoxification mechanisms by a targeted metabolomic approach should reveal additional pathways of b-cell fuel detoxification. Targeting fuel detoxification processes may provide a novel approach to preserve b-cell mass and function.

Effect of osteocalcin on human beta cell proliferation and function and its role on insulin secretion

Effect of osteocalcin on human beta cell proliferation and function and its role on insulin secretion

In Vitro Proliferation of Adult Human Beta-Cells

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched (“beta-cells”) population were plated on extracellular matrix from rat (804G) and human bladder carcinoma cells (HTB9) or bovine corneal endothelial ECM (BCEC). Cells were maintained in culture+/−liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted) or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted), independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05).These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

A Human Islet Cell Culture System for High-Throughput Screening

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D1, known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.