Dissociated Islet Cells Research Articles

TRNA-derived fragments in T lymphocyte-beta cell crosstalk and in type 1 diabetes pathogenesis in NOD mice.

tRNAs play a central role in protein synthesis. Besides this canonical function, they were recently found to generate non-coding RNA fragments (tRFs) regulating different cellular activities. The aim of this study was to assess the involvement of tRFs in the crosstalk between immune cells and beta cells and to investigate their contribution to the development of type 1 diabetes. Global profiling of the tRFs present in pancreatic islets of 4- and 8-week-old NOD mice and in extracellular vesicles released by activated CD4+ T lymphocytes was performed by small RNA-seq. Changes in the level of specific fragments were confirmed by quantitative PCR. The transfer of tRFs from immune cells to beta cells occurring during insulitis was assessed using an RNA-tagging approach. The functional role of tRFs increasing in beta cells during the initial phases of type 1 diabetes was determined by overexpressing them in dissociated islet cells and by determining the impact on gene expression and beta cell apoptosis. We found that the tRF pool was altered in the islets of NOD mice during the initial phases of type 1 diabetes. Part of these changes were triggered by prolonged exposure of beta cells to proinflammatory cytokines (IL-1β, TNF-α and IFN-γ) while others resulted from the delivery of tRFs produced by CD4+ T lymphocytes infiltrating the islets. Indeed, we identified several tRFs that were enriched in extracellular vesicles from CD4+/CD25- T cells and were transferred to beta cells upon adoptive transfer of these immune cells in NOD.SCID mice. The tRFs delivered to beta cells during the autoimmune reaction triggered gene expression changes that affected the immune regulatory capacity of insulin-secreting cells and rendered the cells more prone to apoptosis. Our data point to tRFs as novel players in the crosstalk between the immune system and insulin-secreting cells and suggest a potential involvement of this novel class of non-coding RNAs in type 1 diabetes pathogenesis. Sequences are available from the Gene Expression Omnibus (GEO) with accession numbers GSE242568 and GSE256343.

Automated high-throughput, high-content 3D imaging of intact pancreatic islets.

Diabetes poses a global health crisis affecting individuals across age groups and backgrounds, with a prevalence estimate of 700 million people worldwide by 2045. Current therapeutic strategies primarily rely on insulin therapy or hypoglycemic agents, which fail to address the root cause of the disease - the loss of pancreatic insulin-producing beta-cells. Therefore, bioassays that recapitulate intact islets are needed to enable drug discovery for beta-cell replenishment, protection from beta-cell loss, and islet-cell interactions. Standard cancer insulinoma beta-cell lines MIN6 and INS-1 have been used to interrogate beta-cell metabolic pathways and function but are not suitable for studying proliferative effects. Screening using primary human/rodent intact islets offers a higher level of physiological relevance to enhance diabetes drug discovery and development. However, the 3-dimensionality of intact islets have presented challenges in developing robust, high-throughput assays to detect beta-cell proliferative effects. Established methods rely on either dissociated islet cells plated in 2D monolayer cultures for imaging or reconstituted pseudo-islets formed in round bottom plates to achieve homogeneity. These approaches have significant limitations due to the islet cell dispersion process. To address these limitations, we have developed a robust, intact ex vivo pancreatic islet bioassay in 384-well format that is capable of detecting diabetes-relevant endpoints including beta-cell proliferation, chemoprotection, and islet spatial morphometrics.

92-OR: The Survival and Function of Whole or Dissociated Islet Transplants Are Improved by Immunomodulatory Microporous Annealed Particle (MAP) Gel

The aim was to investigate if MAP gel can support the function of whole islets or dissociated islet cells, and improve transplantation under kidney capsule or within the epididymal fat pad. A) Whole or dissociated BL/6 islets were transplanted into syngeneic diabetic recipients in the presence or absence of MAP gel. 100 islets were transplanted under the kidney capsule (n=6/group). Mice receiving transplant within the fat pad were divided into 6 groups (n=2/group): 300 whole islets; 400 dissociated islets; MAP gel alone; and 200, 300 or 400 dissociated islets with MAP gel. Blood glucose (BG) was measured daily. B) MAP gel or NP gel was transplanted within the fat pad of NOD mice (n=4/group) to analyze the immune response. Graft-containing fat pad was excised 6 weeks post-transplantation and analyzed for cytokines by flow cytometry. Our results indicated that transplantation of dissociated islets and MAP gel under the kidney capsule permanently restored normoglycemia within 18 days. In contrast, uncoated dissociated islet cells failed to reverse diabetes. Treatment efficacy continued 40 days post-transplantation, the entire period of observation. At Day 30, average BG was 163±38mg/dL. Diabetic mice transplanted with dissociated islet cells without MAP gel remained hyperglycemic. Transplantation of 400 dissociated islet cells with MAP gel within the fat pad also achieved normal BG levels that continued 44 days post-transplantation, the period of observation. In contrast, transplantation of MAP gel, or 300 whole islets, or dissociated islets alone into the fat pad, failed to reverse diabetes. An upregulation of IL-10 and downregulation of IL-6 was observed only in the graft microenvironment of the MAP gel group, validating the immunomodulating capacity of MAP gel. To conclude, MAP gel can promote transplantation outcomes under the kidney capsule or within the fat pad by improving the survival and function of both, whole and dissociated islets. Disclosure M.Ma: None. C.A.Roosa: None. D.R.Griffin: Board Member; Tempo Therapeutics. K.L.Brayman: None. P.Chhabra: None. Funding LaunchPad for Diabetes Innovation Fund (GF004946); LaunchPad for Diabetes Ignite Program (169098)

P12.11: Immunomodulatory Microporous Annealed Particle (MAP) Gel Protects Delivery of Islets to Promote Islet Transplantation Outcomes

Introduction: To investigate if coating dissociated or whole islets with microporous annealed particle (MAP) gel could support cell function and viability and improve islet transplantation outcomes. Methods: a) C57BL/6 mouse islets were mixed with MAP gel and cultured in 24 well plates at 37°C. The viability of islets was scored after Propidium Iodide-Fluorescein Diacetate (PI/FDA) staining and Glucose stimulated insulin secretion (GSIS) assay performed on Day 1, 3, 5, and 7 post-culture. The experiment was done thrice. b) Single cells from C57BL/6 mouse dissociated islets were mixed with MAP gel, and cultured in 24 well plates at 37°C. Viability of cells was scored after PI/FDA staining and GSIS assay performed 1, 24, 48, and 72 hours post-culture. c) 100 C57BL/6 mouse islets were dissociated into single cells by Trypsin, mixed with MAP gel, and transplanted into syngeneic diabetic recipients under the kidney capsule. Blood glucose (BG) was measured daily. Results: Mouse islets protected with MAP gel displayed similar GSIS and viability scores when compared to uncoated islets. Both groups retained above 95% viability, with the stimulation index (SI) of MAP gel-protected islets 3.2 and that of uncoated islets is 2.4. Viability of dissociated islets protected with MAP gel was 84% with a SI=8.5 at 72 hours post culture. In contrast, uncoated dissociated islets were nearly dead with only 35% viability and an SI=0.38 at 72 hours post culturing. In MAP scaffold, dissociated islet cell viability was density dependent − cell viability in the 50 and 12.5 dissociated islet conditions was on average >78%, while dissociated cells from 2.5 islets demonstrated 56% viability after 72 hours. In in vivo studies, no significant difference was observed in the ability to return Streptozotocin-induced diabetic mice to normoglycemia when either MAP gel coated or uncoated whole islets (75-100 islets) were transplanted under the mouse kidney capsule. C57BL/6 mouse islets (100 islets) that were dissociated to single cells and co-transplanted with MAP gel under the kidney capsule, permanently returned transplanted mice to normoglycemia, with glucose levels below 200 mg/dL within 18 days post-transplantation (n=6). In contrast, uncoated dissociated islets failed to restore normoglycemia in tranplanted mice. Treatment efficacy continued 40 days post-transplantation (time mice were monitored). The average blood glucose measured at Day 30 was 163 ± 38.2 mg/dL. Recipient mice transplanted with cells from dissociated islets without MAP gel remained hyperglycemic (n=6/group). Conclusion: The MAP scaffold protects delivery of dissociated islet cells, and helps promote islet transplantation outcomes. Moreover, MAP scaffold provides a possible platform for delivering stem cell-derived insulin-secreting cells. LaunchPad Ignite. NIH grant: NIBIB R21 Trailblazer [1R21EB028971-01A1].

Delivery of Dissociated Islets Cells within Microporous Annealed Particle Scaffold to Treat Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune loss of insulin-producing beta cells in the pancreas. The only clinical approach to patient management of blood glucose that doesn't require exogenous insulin is pancreas or islet transplantation. Unfortunately, donor islets are scarce and there is substantial islet loss immediately after transplantation due, in part, to the local inflammatory response. The delivery of stem cell-derived beta cells (e.g., from induced pluripotent stem cells) and dissociated islet cells hold promise as a treatment for T1D; however, these cells typically require re-aggregation in vitro prior to implantation. Microporous scaffolds have shown high potential to serve as a vehicle for organization, survival, and function of insulin-producing cells. In this study, we investigated the use of microporous annealed particle (MAP) scaffold for delivery of enzymatically dissociated islet cells, a model beta cell source, within the scaffold's interconnected pores. We found that MAP-based cell delivery enables survival and function of dissociated islets cells both in vitro and in an in vivo mouse model of T1D.

Cryopreservation and post-thaw characterization of dissociated human islet cells.

The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to –40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+ and Ca2+ channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.

Gsα-dependent signaling is required for postnatal establishment of a functional β-cell mass

ObjectiveEarly postnatal life is a critical period for the establishment of the functional β-cell mass that will sustain whole-body glucose homeostasis during the lifetime. β cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult β cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal β-cell development. MethodsTo study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from β-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. β-cell mass was studied using histomorphometric analysis and optical projection tomography. β-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted β cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. ResultsElimination of Gsα in β cells led to reduced β-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key β-cell identity and maturation genes in postnatal β-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. ConclusionWe conclude that Gsα is required for β-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal β cells, which may have implications for using cAMP-raising agents to promote β-cell regeneration and maturation in diabetes.

Microelectrode Array based Functional Testing of Pancreatic Islet Cells.

Electrophysiological techniques to characterize the functionality of islets of Langerhans have been limited to short-term, one-time recordings such as a patch clamp recording. We describe the use of microelectrode arrays (MEAs) to better understand the electrophysiology of dissociated islet cells in response to glucose in a real-time, non-invasive method over prolonged culture periods. Human islets were dissociated into singular cells and seeded onto MEA, which were cultured for up to 7 days. Immunofluorescent imaging revealed that several cellular subtypes of islets; β, δ, and γ cells were present after dissociation. At days 1, 3, 5, and 7 of culture, MEA recordings captured higher electrical activities of islet cells under 16.7 mM glucose (high glucose) than 1.1 mM glucose (low glucose) conditions. The fraction of the plateau phase (FOPP), which is the fraction of time with spiking activity recorded using the MEA, consistently showed distinguishably greater percentages of spiking activity with high glucose compared to the low glucose for all culture days. In parallel, glucose stimulated insulin secretion was measured revealing a diminished insulin response after day 3 of culture. Additionally, MEA spiking profiles were similar to the time course of insulin response when glucose concentration is switched from 1.1 to 16.7 mM. Our analyses suggest that extracellular recordings of dissociated islet cells using MEA is an effective approach to rapidly assess islet functionality, and could supplement standard assays such as glucose stimulate insulin response.

Alpha1-antitrypsin ameliorates islet amyloid-induced glucose intolerance and β-cell dysfunction

ObjectivePancreatic β-cell failure is central to the development and progression of type 2 diabetes (T2D). The aggregation of human islet amyloid polypeptide (hIAPP) has been associated with pancreatic islet inflammation and dysfunction in T2D. Alpha1-antitrypsin (AAT) is a circulating protease inhibitor with anti-inflammatory properties. Here, we sought to investigate the potential therapeutic effect of AAT treatment in a mouse model characterized by hIAPP overexpression in pancreatic β-cells. MethodsMice overexpressing hIAPP (hIAPP-Tg) in pancreatic β-cells were used as a model of amyloid-induced β-cell dysfunction. Glucose homeostasis was evaluated by glucose tolerance tests and insulin secretion assays. Apoptosis and amyloid formation was assessed in hIAPP-Tg mouse islets cultured at high glucose levels. Dissociated islet cells were cocultured with macrophages obtained from the peritoneal cavity. ResultsNontreated hIAPP-Tg mice were glucose intolerant and exhibited impaired insulin secretion. Interestingly, AAT treatment improved glucose tolerance and restored the insulin secretory response to glucose in hIAPP-Tg mice. Moreover, AAT administration normalized the expression of the essential β-cell genes MafA and Pdx1, which were downregulated in pancreatic islets from hIAPP-Tg mice. AAT prevented the formation of amyloid deposits and apoptosis in hIAPP-Tg islets cultured at high glucose concentrations. Since islet macrophages mediate hIAPP-induced β-cell dysfunction, we investigated the effect of AAT in cocultures of macrophages and islet cells. AAT prevented hIAPP-induced β-cell apoptosis in these cocultures without reducing the hIAPP-induced secretion of IL-1β by macrophages. Remarkably, AAT protected β-cells against the cytotoxic effects of conditioned medium from hIAPP-treated macrophages. Similarly, AAT also abrogated the cytotoxic effects of exogenous proinflammatory cytokines on pancreatic β-cells. ConclusionsThese results demonstrate that treatment with AAT improves glucose homeostasis in mice overexpressing hIAPP and protects pancreatic β-cells from the cytotoxic actions of hIAPP mediated by macrophages. These results support the use of AAT-based therapies to recover pancreatic β-cell function for the treatment of T2D.

Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.

Translational assessment of a genetic engineering methodology to improve islet function for transplantation.

BackgroundThe functional quality of insulin-secreting islet beta cells is a major factor determining the outcome of clinical transplantations for diabetes. It is therefore of importance to develop methodological strategies aiming at optimizing islet cell function prior to transplantation. In this study we propose a synthetic biology approach to genetically engineer cellular signalling pathways in islet cells.MethodsWe established a novel procedure to modify islet beta cell function by combining adenovirus-mediated transduction with reaggregation of islet cells into pseudoislets. As a proof-of-concept for the genetic engineering of islets prior to transplantation, this methodology was applied to increase the expression of the V1b receptor specifically in insulin-secreting beta cells. The functional outcomes were assessed in vitro and in vivo following transplantation into the anterior chamber of the eye.FindingsPseudoislets produced from mouse dissociated islet cells displayed basic functions similar to intact native islets in terms of glucose induced intracellular signalling and insulin release, and after transplantation were properly vascularized and contributed to blood glucose homeostasis. The synthetic amplification of the V1b receptor signalling in beta cells successfully modulated pseudoislet function in vitro. Finally, in vivo responses of these pseudoislet grafts to vasopressin allowed evaluation of the potential benefits of this approach in regenerative medicine.InterpretationThese results are promising first steps towards the generation of high-quality islets and suggest synthetic biology as an important tool in future clinical islet transplantations. Moreover, the presented methodology might serve as a useful research strategy to dissect cellular signalling mechanisms of relevance for optimal islet function.

2137-P: Cholecystokinin Protects Mouse Pancreatic Beta Cells against Cytokine Insult through the Cholecystokinin A Receptor

Cholecystokinin (CCK) is an incretin-like hormone that is also produced by pancreatic β-cells under conditions of stress and obesity. In the past, we have established a role for CCK in protection from β-cell apoptosis. However, the specific CCK receptor responsible for this protective effect was unknown. Both Cckar and Cckbr mRNA are expressed in mouse islet, although Cckbr expression is very low. Incubation of WT mouse pancreatic islets with CCK-8 (100nM) increases the transcript levels of Cckar but does not impact Cckbr expression. Conversely, 24-hour treatment with proinflammatory cytokine cocktail decreases CCKAR transcript levels but again does not modulate Cckbr expression. Therefore, we hypothesized that the CCKA receptor was the most likely mediator of CCK signaling in the β-cell. We used isolated islets from mice with germline knockout of CCK receptors to determine which receptor was required for the pro-survival effects of CCK on the β-cell. Using imaging flow cytometry of dissociated islet cells stained for annexin V and propidium iodide (PI) we find that islet cells from CCKBR knockout mice have a 19.3% (p<0.005) reduction in cytokine-mediated cell death when treated with CCK-8 (100 nM), similar to the protection seen in wild type islets. This protective effect is lost in the CCKAR and double knockout mouse islets. We also confirm this finding using insulin and TUNEL immunohistochemistry to specifically identify β-cell apoptosis. In CCKBR knockout mouse islets, CCK-8 (100 nM) reduces the % TUNEL positive β-cells from 12.98% to 4.39% (p<0.05) after cytokine exposure, similar to protection seen in WT mouse islets (10.47% to 4.48%). Again, CCK-8 does not protect β-cells from cytokine-induced apoptosis in CCKAR knockout or double CCKR null mouse islets. Taken together, CCKAR, but not CCKBR, is required for CCK-mediated protection of pancreatic β-cells from cytokine-induced stress. Disclosure H. Kim: None. S. Sacotte: None. R.A. Williams: None. A.H. de Souza: None. J. Han: None. J.T. Bartosiak: None. G.H. Yang: None. D.B. Davis: None. Funding National Institutes of Health

Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice

Aims/hypothesisIslet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.MethodsHuman islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1−/− mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.ResultsCFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).Conclusions/interpretationThe ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.

In Vitro Proliferation of Porcine Pancreatic Islet Cells for β-Cell Therapy Applications.

β-Cell replacement through transplantation is the only curative treatment to establish a long-term stable euglycemia in diabetic patients. Owing to the shortage of donor tissue, attempts are being made to develop alternative sources of insulin-secreting cells. Stem cells differentiation and reprograming as well as isolating pancreatic progenitors from different sources are some examples; however, no approach has yet yielded a clinically relevant solution. Dissociated islet cells that are cultured in cell numbers by in vitro proliferation provide a promising platform for redifferentiation towards β-cells phenotype. In this study, we cultured islet-derived cells in vitro and examined the expression of β-cell genes during the proliferation. Islets were isolated from porcine pancreases and enzymatically digested to dissociate the component cells. The cells proliferated well in tissue culture plates and were subcultured for no more than 5 passages. Only 10% of insulin expression, as measured by PCR, was preserved in each passage. High glucose media enhanced insulin expression by about 4–18 fold, suggesting a glucose-dependent effect in the proliferated islet-derived cells. The islet-derived cells also expressed other pancreatic genes such as Pdx1, NeuroD, glucagon, and somatostatin. Taken together, these results indicate that pancreatic islet-derived cells, proliferated in vitro, retained the expression capacity for key pancreatic genes, thus suggesting that the cells may be redifferentiated into insulin-secreting β-like cells.

Maternal protein restriction leads to pancreatic failure in offspring: role of misexpressed microRNA-375.

The intrauterine environment of the fetus is a preeminent actor in long-term health. Indeed, mounting evidence shows that maternal malnutrition increases the risk of type 2 diabetes (T2D) in progeny. Although the consequences of a disturbed prenatal environment on the development of the pancreas are known, the underlying mechanisms are poorly defined. In rats, restriction of protein during gestation alters the development of the endocrine pancreas and favors the occurrence of T2D later in life. Here we evaluate the potential role of perturbed microRNA (miRNA) expression in the decreased β-cell mass and insulin secretion characterizing progeny of pregnant dams fed a low-protein (LP) diet. miRNA profiling shows increased expression of several miRNAs, including miR-375, in the pancreas of fetuses of mothers fed an LP diet. The expression of miR-375 remains augmented in neoformed islets derived from fetuses and in islets from adult (3-month-old) progeny of mothers fed an LP diet. miR-375 regulates the proliferation and insulin secretion of dissociated islet cells, contributing to the reduced β-cell mass and function of progeny of mothers fed an LP diet. Remarkably, miR-375 normalization in LP-derived islet cells restores β-cell proliferation and insulin secretion. Our findings suggest the existence of a developmental memory in islets that registers intrauterine protein restriction. Hence, pancreatic failure after in utero malnutrition could result from transgenerational transmission of miRNA misexpression in β-cells.

Apoptosis-Resistant and Highly Functional Islet Cell Sheets for Transplantation.

Background: Cell-based therapy using pancreatic islet cells has emerged as a promising new approach for the treatment of diabetes mellitus. Along with the conventional usage of the purified islets for transplantation, our group has recently established a novel dissociated islet cell-based approach, named islet cell sheet transplantation (Transplantation 30: 5943, 2011). In this approach, individually dissociated islet cells are cultured on a specially-defined culture surface to form a thin and contiguous monolayered islet cell sheet. Since it is known that islet cells are susceptible to apoptotic process during the dispersed cell status, the present study conduced a gene transduction of an anti-apoptotic gene FNK, during the culture period for achieving higher cell survival as well as higher functionality. Methods: Fiber knob-modified adenoviral vectors expressing FNK or GFP under the control of CAG promoter, Ad-CA-K7-GFP, Ad-CA-K7-FNK, were prepared. Purified rat islet cells were dissociated into single islet cells, followed by their culturing on laminin-5-coated and temperature-responsive polymer-immobilized plastic dishes. Cells were incubated with the Ad-vectors at day 1 for 1 hr at a previously defined vector dose, MOI=10. Apoptosis was then induced by inoculating hydrogen peroxide into culture medium at 400μM. Results: After infection with Ad-CA-K7-GFP, approximately 90% of the dispersed-cultured islet cells were found to be gene-transduced. There were no Ad-vector infection-mediated cellular damages. In an another set of experiment, cells were infected with Ad-CA-K7-FNK, and hydrogen peroxide were inoculated on day 2 for the induction of apoptosis. FNK-transduced cultured islet cells showed significantly less caspase expressions as well as remarkable resistance to the apoptosis. Finally, the FNK-transduced islet cells could be harvested as an intact cell sheet format that was expected to produce higher levels of insulin. Conclusions:The present study described a new proof-of-concept approach to engineer apoptosis-resistant and highly functional islet cell construct for transplantation.

Sustained Therapeutic Effects of Neo-Islet Tissue Engineering by Islet Sheet Transplantation

Background: Our group has recently established a procedure to fabricate contiguous layer of islet cells, namely islet sheets for generating new islet-based therapy for diabetes mellitus (DM). This islet sheet format can be transplanted to various sites without using any scaffold. Accordingly, those islet sheet transplantation procedures allow to create functional neo-islet tissues at subcutaneous space, in which islet could be protected from being exposed to several inflammatory responses after injecting into portal circulation. In this study, we report therapeutic values of the neo-islet tissue creation approach in subcutaneous space and tissue longevity were investigated. Methods and Results: Temperature-responsive culture dishes optimized for islet cell culturing were prepared by covalently immobilizing a temperature-responsive polymer poly(N-isopropylacrylamide) (PIPAAm) to plastic dishes followed by laminin-5 coating (Shimizu et al, Biomaterials, 30: 5943, 2009). Pancreatic islets were obtained from Lewis rats, and dissociated islet cells were obtained by incubating with trypsin-EDTA. Dissociated islet cells were then plated onto the PIPAAm dishes. After reaching confluency at day 2, islet cells were successfully harvested as a uniformly spread islet sheet by lowering the culture temperature from 37°C to 20°C for 20 min. The functional activities of the islet cell sheets were confirmed by histological examinations and insulin secretion assay in vitro. Transmission electron microscopic examinations showed that harvested islet sheet had a monolayered 2-D structure, which established and retained its structural cell-to-cell connections. We then conducted islet sheet transplantation procedure to create neo-islet tissues into subcutaneous space of Streptozotocin-induced diabetic SCID mice. Histological analyses revealed that strip-shaped neo-islet tissues in the subcutaneous space. This neo-islet engineering approach successfully converted the hyperglycemia to euglycemia in all the recipients and sustained the therapeutic effects for 300 days (length of the experiments). We then conducted intra-peritoneal glucose tolerant tests to the recipients, and found that the recipient SCID mice showed glucose changes that are identical to normal mice. Conclusions: The present study describes an approach to create functional neo-islet tissues in vivo by transplantation of islet sheets. Since sustained DM therapeutic values and prolonged longevity of the neo-islet tissues were confirmed, this approach could be a valuable alternative islet-based therapeutic mode for DM.

Changes in MicroRNA Expression Contribute to Pancreatic β-Cell Dysfunction in Prediabetic NOD Mice

During the initial phases of type 1 diabetes, pancreatic islets are invaded by immune cells, exposing β-cells to proinflammatory cytokines. This unfavorable environment results in gene expression modifications leading to loss of β-cell functions. To study the contribution of microRNAs (miRNAs) in this process, we used microarray analysis to search for changes in miRNA expression in prediabetic NOD mice islets. We found that the levels of miR-29a/b/c increased in islets of NOD mice during the phases preceding diabetes manifestation and in isolated mouse and human islets exposed to proinflammatory cytokines. Overexpression of miR-29a/b/c in MIN6 and dissociated islet cells led to impairment in glucose-induced insulin secretion. Defective insulin release was associated with diminished expression of the transcription factor Onecut2, and a consequent rise of granuphilin, an inhibitor of β-cell exocytosis. Overexpression of miR-29a/b/c also promoted apoptosis by decreasing the level of the antiapoptotic protein Mcl1. Indeed, a decoy molecule selectively masking the miR-29 binding site on Mcl1 mRNA protected insulin-secreting cells from apoptosis triggered by miR-29 or cytokines. Taken together, our findings suggest that changes in the level of miR-29 family members contribute to cytokine-mediated β-cell dysfunction occurring during the initial phases of type 1 diabetes.

Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells

Efficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known. We developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells. Mouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells. Culture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

Cell–cell communication mimicry with poly(ethylene glycol) hydrogels for enhancing β-cell function

A biomimetic hydrogel platform was designed to signal encapsulated cells using immobilized cell-cell communication cues, with a focus on enhancing the survival and function of encapsulated pancreatic β-cells to treat type 1 diabetes. When MIN6 cells, a pancreatic β-cell line, were encapsulated in poly(ethylene glycol) (PEG) hydrogels, their survival and glucose responsiveness to insulin were highly dependent on the cell-packing density. A minimum packing density of 10(7) cells/mL was necessary to maintain the survival of encapsulated β-cells without the addition of material functionalities (e.g., cell adhesion ligands). While single cell suspensions can improve diffusion-limited mass transfer, direct cell-cell interactions are limited. Thus, thiolated EphA5-Fc receptor and ephrinA5-Fc ligand were conjugated into PEG hydrogels via a thiol-acrylate photopolymerization to render an otherwise inert PEG hydrogel bioactive. The biomimetic hydrogels presented here can provide crucial cell-cell communication signals for dispersed β-cells and improve their survival and proliferation. Together with the cell-adhesive peptide RGDS, the immobilized fusion proteins (EphA5-Fc and ephrinA5-Fc) synergistically increased the survival of both MIN6 β-cells and dissociated islet cells, both at a very low cell-packing density (< 2 × 10(6) cells/mL). This unique gel platform demonstrates new strategies for tailoring biomimetic environments to enhance the encapsulation of cells that require cell-cell contact to survive and function.