Normoglycemia In Diabetic Mice Research Articles

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels.

Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating β-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.

Co-transplantation of pancreatic islets and microvascular fragments effectively restores normoglycemia in diabetic mice

Insufficient revascularization of pancreatic islets is one of the major obstacles impairing the success of islet transplantation. To overcome this problem, we introduce in the present study a straightforward strategy to accelerate the engraftment of isolated islets. For this purpose, we co-transplanted 250 islets and 20,000 adipose tissue-derived microvascular fragments (MVF) from donor mice under the kidney capsule as well as 500 or 1000 islets with 40,000 MVF into the subcutaneous space of diabetic mice. We found that the co-transplantation of islets and MVF markedly accelerates the restoration of normoglycemia in diabetic recipients compared with the transplantation of islets alone. In fact, the transplantation of 250 islets with 20,000 MVF under the kidney capsule reversed diabetes in 88% of mice and the subcutaneous transplantation of 500 or 1000 islets with 40,000 MVF restored normoglycemia in 100% of mice. Moreover, diabetic mice receiving islets and MVF exhibited plasma insulin levels similar to nondiabetic control animals. Additional immunohistochemical analyses of the grafts revealed a significantly higher number of islet cells and microvessels in the co-transplantation groups. These findings demonstrate that the co-transplantation of islets and MVF is a promising strategy to improve the success rates of islet transplantation, which could be easily implemented into future clinical practice.

The loss of pancreatic islet NADPH oxidase (NOX)2 improves islet transplantation

Islet transplantation is a promising treatment strategy for type 1 diabetes mellitus (T1DM) patients. However, oxidative stress-induced graft failure due to an insufficient revascularization is a major problem of this therapeutic approach. NADPH oxidase (NOX)2 is an important producer of reactive oxygen species (ROS) and several studies have already reported that this enzyme plays a crucial role in the endocrine function and viability of β-cells. Therefore, we hypothesized that targeting islet NOX2 improves the outcome of islet transplantation. To test this, we analyzed the cellular composition and viability of isolated wild-type (WT) and Nox2−/− islets by immunohistochemistry as well as different viability assays. Ex vivo, the effect of Nox2 deficiency on superoxide production, endocrine function and anti-oxidant protein expression was studied under hypoxic conditions. In vivo, we transplanted WT and Nox2−/− islets into mouse dorsal skinfold chambers and under the kidney capsule of diabetic mice to assess their revascularization and endocrine function, respectively. We found that the loss of NOX2 does not affect the cellular composition and viability of isolated islets. However, decreased superoxide production, higher glucose-stimulated insulin secretion as well as expression of nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1 and superoxide dismutase 1 (SOD1) was detected in hypoxic Nox2−/− islets when compared to WT islets. Moreover, we detected an early revascularization, a higher take rate and restoration of normoglycemia in diabetic mice transplanted with Nox2−/− islets. These findings indicate that the suppression of NOX2 activity represents a promising therapeutic strategy to improve engraftment and function of isolated islets.

Cell Heterogeneity and Paracrine Interactions in Human Islet Function: A Perspective Focused in β-Cell Regeneration Strategies

The β-cell regeneration field has shown a strong knowledge boost in the last 10 years. Pluripotent stem cell differentiation and direct reprogramming from other adult cell types are becoming more tangible long-term diabetes therapies. Newly generated β-like-cells consistently show hallmarks of native β-cells and can restore normoglycemia in diabetic mice in virtually all recent studies. Nonetheless, these cells still show important compromises in insulin secretion, cell metabolism, electrical activity, and overall survival, perhaps due to a lack of signal integration from other islet cells. Mounting data suggest that diabetes is not only a β-cell disease, as the other islet cell types also contribute to its physiopathology. Here, we present an update on the most recent studies of islet cell heterogeneity and paracrine interactions in the context of restoring an integrated islet function to improve β-cell replacement therapies.

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels.

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin‐secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non‐prevascularized islet organoids. This is caused by paracrine signaling between the β‐cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood‐perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation.

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me2SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME2SO, 1M ME2SO + 1M EG, and 1M ME2SO + 0.5M EG). Next, fresh islets, 2M ME2SO islets, and 1M ME2SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME2SO (1M ME2SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME2SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME2SO + 0.5M EG), compared to islets preserved with 2M ME2SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.

The liver surface as a favorable site for islet cell sheet transplantation in type 1 diabetes model mice.

IntroductionIslet transplantation is one of the most promising therapeutic approaches for patients with severe type 1 diabetes mellitus (T1DM). Transplantation of engineered islet cell sheets holds great potential for treating T1DM as it enables the creation of stable neo-islet tissues. However, a large mass of islet cell sheets is required for the subcutaneous transplantation to reverse hyperglycemia in diabetic mice. Here, we investigated whether the liver surface could serve as an alternative site for islet cell sheet transplantation.MethodsDispersed rat islet cells (0.8 × 106 cells) were cultured on laminin-332-coated thermoresponsive culture dishes. After 2 days of cultivation, we harvested the islet cell sheets by lowering the culture temperature using a support membrane with a gelatin gel. We transplanted two recovered islet cell sheets into the subcutaneous space or onto the liver surface of severe combined immunodeficiency (SCID) mice with streptozocin-induced diabetes.ResultsIn the liver surface group, the non-fasting blood glucose level decreased rapidly within several days after transplantation. In marked contrast, the hyperglycemia state was maintained in the subcutaneous space transplantation group. The levels of rat C-peptide and insulin in the liver surface group were significantly higher than those in the subcutaneous space group. An immunohistological analysis confirmed that most of the islet cells engrafted on the liver surface were insulin-positive. The CD31-positive endothelial cells formed vascular networks within the neo-islets and in the surrounding tissues. In contrast, viable islet cells were not found in the subcutaneous space group.ConclusionsCompared with the subcutaneous space, a relatively small mass of islet cell sheets was enough to achieve normoglycemia in diabetic mice when the liver surface was selected as the transplantation site. Our results demonstrate that the optimization of the transplantation site for islet cell sheets leads to significant improvements in the therapeutic efficiency for T1DM.

Safety and Efficacy of Epigenetically Converted Human Fibroblasts Into Insulin-Secreting Cells: A Preclinical Study.

Type 1 Diabetes Mellitus (T1DM) is a chronic disease that leads to loss of insulin secreting β-cells, causing high levels of blood glucose. Exogenous insulin administration is not sufficient to mimic the normal function of β-cells and, consequently, diabetes mellitus often progresses and can lead to major chronic complications and morbidity. The physiological control of glucose levels can only be restored by replacing the β-cell mass.We recently developed a new strategy that allows for epigenetic conversion of dermal fibroblasts into insulin-secreting cells (EpiCC), using a brief exposure to the demethylating agent 5-aza-cytidine (5-aza-CR), followed by a pancreatic induction protocol. This method has notable advantages compared to the alternative available procedures and may represent a promising tool for clinical translation as a therapy for T1DM. However, a thought evaluation of its therapeutic safety and efficacy is mandatory to support preclinical studies based on EpiCC treatment.We here report the data obtained using human fibroblasts isolated from diabetic and healthy individuals, belonging the two genders. EpiCC were injected into 650 diabetic severe combined immunodeficiency (SCID) mice and demonstrated to be able to restore and maintain glycemic levels within the physiological range. Cells had the ability to self-regulate and not to cause hypoglycemia, when transplanted in healthy animals. Efficacy tests showed that EpiCC successfully re-established normoglycemia in diabetic mice, using a dose range that appeared clinically relevant to the concentration 0.6×106 EpiCC. Necropsy and histopathological investigations demonstrated the absence of malignant transformation and cell migration to organs and lymph nodes.The present preclinical study demonstrates safety and efficacy of human EpiCC in diabetic mice and supports the use of epigenetic converted cells for regenerative medicine of diabetes mellitus.

Activation of Liver-Resident DX5 Negative NK Cells after Intraportal Islet Transplantation Prevent the Engraftment of Secondary Transplanted Islets

It has been recently reported that liver-resident DX5− natural killer (NK) cells have potent memory functions, which include differentiation into a long-lived memory subset and robust antigen-specific expansion to a secondary challenge. We have previously demonstrated that liver NK cells play a pivotal role in islet graft loss during the early phase after intraportal syngeneic islet transplantation (IT). In this study, we investigated the mechanism of liver-resident DX5− NK cells activation after IT and the influence of primary IT on the engraftment of secondarily transplanted islets. To evaluate the mechanism of NK cells activation early after IT, liver NK cells from C57BL/6 mice were cultured for 24 h in the presence of TNFα, IFNγ, and IL-1β, mimicking instant blood-mediated inflammatory reaction. Furthermore, we established a multiple IT model for evaluating the memory functions of NK cells in vivo; 400 islets were secondarily transplanted following primary IT with 200 islets. Surface molecules on liver NK cells were analyzed to evaluate the phenotypical and functional alteration of liver NK cells from the early to the late phase after IT. The population of DX5− NK cells significantly expanded 2.4 times compared to naive condition (p < 0.01) after the cytokine stimulation dominantly associated with TNFα, and the significant increase of both CD69 and CXCR3 expression on liver NK cells was observed (24 h/fresh ratio; 2.0, p < 0.01; 1.5, p < 0.01, respectively). In in vivo assay, the population of DX5− NK cells also promptly expanded and the expression of CD69, TRAIL, and CXCR3 on liver NK cells were significantly increased at 24 h after IT, meanwhile this phenomenon was restored by the administration of anti-TNFα antibody. At the late phase after IT, a significant and persistent increase of DX5− NK cells, expression of CD69, TRAIL, and CXCR3 on liver NK cells was observed compared to naive condition (35 d/0 d ratio; 2.2, p < 0.01, 2.3, p < 0.01, 3.7, p < 0.01, 2.0, p < 0.05, respectively). The syngeneic 400 islets of primary IT mostly achieved normoglycemia in diabetic mice, whereas the same mass of secondary IT failed to induce normoglycemia in the recipients received syngeneic 200 islets of primary IT. Our findings indicate that the liver-resident DX5− NK cells significantly and persistently expanded even after syngeneic IT, and those memory-like NK cells may target both of originally engrafted islets and even secondary transplanted islets.

Mold-casted non-degradable, islet macro-encapsulating hydrogel devices for restoration of normoglycemia in diabetic mice.

Islet transplantation is a potential cure for diabetic patients, however this procedure is not widely adopted due to the high rate of graft failure. Islet encapsulation within hydrogels is employed to provide a three-dimensional microenvironment conducive to survival of transplanted islets to extend graft function. Herein, we present a novel macroencapsulation device, composed of PEG hydrogel, that combines encapsulation with lithography techniques to generate polydimethylsiloxane (PDMS) molds. PEG solutions are mixed with islets, which are then cast into PDMS molds for subsequent crosslinking. The molds can also be employed to provide complex architectures, such as microchannels that may allow vascular ingrowth through pre-defined regions of the hydrogel. PDMS molds allowed for the formation of stable gels with encapsulation of islets, and in complex architectures. Hydrogel devices with a thickness of 600 μm containing 500 islets promoted normoglycemia within 12 days following transplantation into the epididymal fat pad, which was sustained over the two-month period of study until removal of the device. The inclusion of microchannels, which had a similar minimum distance between islets and the hydrogel surface, similarly promoted normoglycemia. A glucose challenge test indicated hydrogel devices achieved normoglycemia 90 min post-dextrose injections, similar to control mice with native pancreata. Histochemical staining revealed that transplanted islets, identified as insulin positive, were viable and isolated from host tissue at 8 weeks post-transplantation, yet devices with microchannels had tissue and vascular ingrowth within the channels. Taken together, these results demonstrate a system for creating non-degradable hydrogels with complex geometries for encapsulating islets capable of restoring normoglycemia, which may expand islet transplantation as a treatment option for diabetic patients. Biotechnol. Bioeng. 2016;113: 2485-2495. © 2016 Wiley Periodicals, Inc.

Long-term persistence and development of induced pancreatic beta cells generated by lineage conversion of acinar cells.

Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, the survival and function of lineage-reprogrammed cells in vivo over the long term has not been examined. Here, using an improved method for in vivo conversion of adult mouse pancreatic acinar cells toward beta cells, we show that induced beta cells persist for up to 13 months (the length of the experiment), form pancreatic islet-like structures and support normoglycemia in diabetic mice. Detailed molecular analyses of induced beta cells over 7 months reveal that global DNA methylation changes occur within 10 d, whereas the transcriptional network evolves over 2 months to resemble that of endogenous beta cells and remains stable thereafter. Progressive gain of beta-cell function occurs over 7 months, as measured by glucose-regulated insulin release and suppression of hyperglycemia. These studies demonstrate that lineage-reprogrammed cells persist for >1 year and undergo epigenetic, transcriptional, anatomical and functional development toward a beta-cell phenotype.

Generation of insulin‐producing cells from human stem cells (207.3)

Recent advances in directing human embryonic stem cells (hESC) towards endodermal organs have identified conditions under which hESC differentiate into pancreatic progenitor cells in cell culture. Transplantation of these progenitor cells into immune compromised mice allows for the generation of insulin‐producing, ß‐like cells capable of restoring normoglycemia in diabetic mice. However, the generating of functional ß‐cells under cell culture conditions has proven difficult. Using a combination of 2D and 3D culture methods and optimized culture conditions we have generated single hormone‐positive, ß‐like cells that show signs of glucose responsive insulin secretion. Furthermore, I will discuss efforts related to identifying signals provided by the pancreatic mesenchyme that guide ß‐cell formation and can be used to optimize cell culture conditions.

Generation of Functional Insulin-Producing Cells From Mouse Embryonic Stem Cells Through 804G Cell-Derived Extracellular Matrix and Protein Transduction of Transcription Factors

Embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications to regenerative medicine for diabetes; however, a useful and safe way to generate pancreatic β cells has not been developed. In this study, we tried to establish an effective method of differentiation through the protein transduction of three transcription factors (Pdx1, NeuroD, and MafA) important to pancreatic β cell development. The method poses no risk of unexpected genetic modifications in target cells. Transduction of the three proteins induced the differentiation of mouse ES and mouse iPS cells into insulin-producing cells. Furthermore, a laminin-5-rich extracellular matrix efficiently induced differentiation under feeder-free conditions. Cell differentiation was confirmed with the expression of the insulin 1 gene in addition to marker genes in pancreatic β cells, the differentiated cells secreted glucose-responsive C-peptide, and their transplantation restored normoglycemia in diabetic mice. Moreover, Pdx1 protein transduction had facilitative effects on differentiation into pancreatic endocrine progenitors from human iPS cells. These results suggest the direct delivery of recombinant proteins and treatment with laminin-5-rich extracellular matrix to be useful for the generation of insulin-producing cells.

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice

Background aimsThe success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol. MethodsWe compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets. ResultsSHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3–4 weeks, which persisted for >60 days. ConclusionsOur results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.

Immuno-Isolation of Pancreatic Islet Allografts Using Pegylated Nanotherapy Leads to Long-Term Normoglycemia in Full MHC Mismatch Recipient Mice

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of “stealth” islets encapsulated within a thin cage of pegylated material of 100–200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice. We first demonstrated that pegylation of the islet surface, plus or minus nanoparticles, improved long-term islet viability in vitro compared to non-pegylated (naked) control islets. Moreover, pegylation of the islets with nanoparticles was compatible with glucose-stimulated insulin secretion and insulin biogenesis. We next looked for functionality of the created “stealth” DBA/2 (H-2d) islets in vivo by comparing glycemic profiles across 4 groups of streptozotozin-induced diabetic C57BL/6 (H-2b) recipients of (i) naked islets; (ii) pegylated islets; (iii) pegylated islets with nanoparticles (empty); and (iv) pegylated islets with nanoparticles loaded with a cargo of leukemia inhibitory factor (LIF), a factor both promotes adaptive immune tolerance and regulates pancreatic β cell mass. Without any other treatment, normoglycemia was lost after 17 d (+/−7.5 d) in control group. In striking contrast, recipients in groups (ii), (iii), and (iv) showed long-term (>100 d) normoglycemia involving 30%; 43%, and 57% of the recipients in each respective group. In conclusion, construction of “stealth” islets by pegylation-based nanotherapeutics not only supports islet structure and function, but also effectively isolates the islets from immune-mediated destruction. The added value of nanoparticles to deliver immune modulators plus growth factors such as LIF expands the potential of this novel therapeutic approach to cell therapy for diabetes.

Therapeutic siRNA silencing in inflammatory monocytes in mice

Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.

Trimodal Gadolinium-Gold Microcapsules Containing Pancreatic Islet Cells Restore Normoglycemia in Diabetic Mice and Can Be Tracked by Using US, CT, and Positive-Contrast MR Imaging

To develop microcapsules that immunoprotect pancreatic islet cells for treatment of type I diabetes and enable multimodal cellular imaging of transplanted islet cells. All animal experiments were approved by the institutional animal care and use committee. Gold nanoparticles functionalized with DTDTPA (dithiolated diethylenetriaminepentaacetic acid):gadolinium chelates (GG) were coencapsulated with pancreatic islet cells by using protamine sulfate as a clinical-grade alginate cross linker. Conventional poly-l-lysine-cross-linked microcapsules and unencapsulated islets were included as controls. The viability and glucose responsiveness of islet cells were assessed in vitro, and in vivo insulin (C-peptide) secretion was monitored for 6 weeks in (streptozotocin-induced) diabetic mice with (n = 7) or without (n = 8) intraabdominally engrafted islet cells. Five nondiabetic mice were included as controls. Differences between samples were calculated by using a nonparametric Wilcoxon Mann-Whitney method. To adjust for multiple comparisons, a significance level of P < .01 was chosen. Generalized estimating equations were used to model cell function over time. Three mice with engrafted capsules were imaged in vivo with high-field-strength (9.4-T) magnetic resonance (MR) imaging, micro-computed tomography (CT), and 40-MHz ultrasonography (US). Encapsulated human pancreatic islets were functional in vitro for at least 2 weeks after encapsulation. Blood glucose levels in the diabetic mice transplanted with GG-labeled encapsulated mouse βTC6 insulinoma cells returned to normal within 1 week after transplantation, and normoglycemia was sustained for at least 6 weeks without the use of immunosuppressive drugs. GG microcapsules could be readily visualized with positive-contrast high-field-strength MR imaging, micro-CT, and US both in vitro and in vivo. Cell encapsulation with GG provides a means of trimodal noninvasive tracking of engrafted cells.

Cryopreserved Agarose-Encapsulated Islets As Bioartificial Pancreas: A Feasibility Study

A bioartificial pancreas in which islets of Langerhans (islets) are encapsulated within a semipermeable membrane, such as agarose, has been proposed for treating type I diabetic mellitus. However, the long-term storage for providing a convenient and easily accessible supply still remains an issue. We investigated cryopreservation as a potential method of long-term storage for agarose-encapsulated islets (Mic-islets). The morphology, insulin secretion, and histochemical staining of cryopreserved Mic-islets were analyzed. Streptozotocin-induced diabetic mice were transplanted intraperitoneally with 1000, 2000, and 3000 Mic-islets after cryopreservation in KYO-1 vitrification solution. Blood glucose levels were measured and immunohistochemical analyses were performed at 41 days posttransplantation. Transplanted cryopreserved Mic-islets restored normoglycemia in diabetic mice. The mean (+/-SD) normoglycemic periods were 32.0+/-13.2 days and 46.3+/-13.3 days for recipients of 1000 (n=5) and 2000 (n=4) cryopreserved Mic-islets, respectively, whereas the mean normoglycemic period was 53.2+/-16.7 days for recipients of 1000 noncryopreserved Mic-islets (n=7). These data indicate that cryopreserved Mic-islets transplanted as a bioartificial pancreas successfully controlled blood glucose levels for extended periods. Cryopreserved agarose-encapsulated islets could successfully control the blood glucose level for a long period as a bioartificial pancreas.

Growing organs for transplantation from embryonic precursor tissues

Our recent data pinpoint a window of time in human and pig kidney organogenesis that may be optimal for transplantation into mature recipients. 'Window' transplants are defined by their remarkable ability to grow, differentiate and undergo vascularization, achieving successful organogenesis of urine-producing miniature kidneys. The transplanted tissue shows no evidence of trans-differentiation into non-renal cell types or tumorogenicity, and displays reduced immunogenicity compared to its adult counterparts. Very recently, we demonstrated that this approach can be extended to transplantation of embryonic pig liver, pancreas, and lung tissue. Furthermore, it was demonstrated that E42 pancreatic tissue is optimally suited for induction of normoglycemia in diabetic mice. These results emphasize the importance of selecting precursors of the correct gestational age for optimal growth and function, and with reduced immunogenicity, and provide a proof of principle for the curative potential of E42 embryonic pig pancreatic tissue for transplantation in diabetic patients.