Diabetic SCID Mice Research Articles

Novel engineered B lymphocytes targeting islet-specific T cells inhibit the development of type 1 diabetes in non-obese diabetic Scid mice.

In this study, we report a novel therapeutic approach using B lymphocytes to attract islet-specific T cells in the non-obese diabetic (NOD) mouse model and prevent the development of autoimmune diabetes. Rather than using the antibody receptor of B cells, this approach utilizes their properties as antigen-presenting cells to T cells. Purified splenic B cells were treated with lipopolysaccharide, which increases regulatory B (Breg) cell function, then electroporated with mRNA encoding either chimeric MHC-I or MHC-II molecules covalently linked to antigenic peptides. Immunoregulatory functions of these engineered B cells (e-B cells) were tested by in vitro assays and in vivo co-transfer experiments with beta-cell-antigen-specific CD8+ or CD4+ T cells in NOD.Scid mice, respectively. The e-B cells expressing chimeric MHC-I-peptide inhibited antigen-specific CD8+ T-cell cytotoxicity in vitro. The e-B cells expressing chimeric MHC-II-peptide induced antigen-specific CD4+ T cells to express the regulatory markers, PD-1, ICOS, CTLA-4, Lag3, and Nrp1. Furthermore, e-B cells encoding the chimeric MHC-I and MHC-II peptide constructs protected NOD.Scid mice from autoimmune diabetes induced by transfer of antigen-specific CD8+ and CD4+ T cells. MHC-peptide chimeric e-B cells interacted with pathogenic T cells, and protected the host from autoimmune diabetes, in a mouse model. Thus, we have successfully expressed MHC-peptide constructs in B cells that selectively targeted antigen-specific cells, raising the possibility that this strategy could be used to endow different protective cell types to specifically regulate/remove pathogenic cells.

IN VIVO ANTI-CD45RB ANTIBODY-INDUCED REGULATORY T CELLS DEMONSTRATE SPECIES-SPECIFIC TOLERANCE TO DISCORDANT HUMAN XENOGENIC ISLETS

Organ transplantation-Sichuan Youth Science and Technology Innovation Research Team. Background: Although islet xenotransplantation has been proven to be a promising strategy for treatment of type 1 diabetes, it’s application in clinical setting would have to overcome acute cellular rejection and challenging species barrier. The aim of this study was to determine the role of Foxp3+ regulatory T cells (Treg) in anti-CD45RB induced dominant and specific tolerance to human islet cells in mice. Methods: One thousand and five hundred IEQ human islets were isolated from deceased donors and transplanted under the kidney capsule of B6 diabetic mice, B6µMT-/- mice and thymectomized µMT-/- mice. The recipients were then temporarily treated with low dose anti-CD45RB. CD4+Foxp3+Tregs were identified in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by FCM and immunohistochemistry. Additional anti-CD25 mAb was applied in the tolerant B6µMT-/-mice, which received another human islets simultaneously. The CD4+CD25+ Tregs sorted from the tolerant mice were adoptively transferred to diabetic Scid mice which were grafted with another human islets to assess the suppressive function. Results: In vitro, CD4+CD25-Foxp3-T cells which were activated with anti-CD3/anti-CD28 beads and cultured with anti-CD45RB demonstrated significant increase in Foxp3+ expression. In vivo, an increase of Foxp3+ T cells was observed in treated mice and more significant increase in tolerant mice, compared with a negligible increase in rejected mice. Anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice and 88.9% in µMT-/- mice, whereas none of xenografts were accepted in these mice treated simultaneously with anti-CD25. µMT-/-mice which were thymectomized and treated by anti-CD45 yielded 100% of grafts with long-term survival, which indicated that the transplant tolerance was thymus-derived Tregs independent. Moreover, the µMT-/-mice which tolerated to the first human islets accepted the re-transplanted second party of islets from another human, whereas the tolerance was hampered by anti-CD25 treatment. Adoptive cell transfer model showed the CD4+CD25+ Tregs from the mice tolerant to the first human islets prolonged survival of the second human islets, compared to natural Tregs (p < 0.05). Conclusions: These data suggest that anti-CD45RB-induced tolerance in islet xenotransplantation is mediated by Tregs expansion. These tolerant Tregs had the potent capacity to transfer dominant and species-specific tolerance. The National Natural Science Foundation of China (No. 81172832, 81771723). Sichuan Youth Science and Technology Foundation (2013JQ0020). Special Program for Sichuan Youth Science and Technology Innovation Research Team (2014TD0010).

Mesenchymal stem cells ameliorate β cell dysfunction of human type 2 diabetic islets by reversing β cell dedifferentiation.

BackgroundA physiological hallmark of patients with type 2 diabetes mellitus (T2DM) is β cell dysfunction. Despite adequate treatment, it is an irreversible process that follows disease progression. Therefore, the development of novel therapies that restore β cell function is of utmost importance.MethodsThis study aims to unveil the mechanistic action of mesenchymal stem cells (MSCs) by investigating its impact on isolated human T2DM islets ex vivo and in vivo.FindingsWe propose that MSCs can attenuate β cell dysfunction by reversing β cell dedifferentiation in an IL-1Ra-mediated manner. In response to the elevated expression of proinflammatory cytokines in human T2DM islet cells, we observed that MSCs was activated to secret IL-1R antagonist (IL-1Ra) which acted on the inflammed islets and reversed β cell dedifferentiation, suggesting a crosstalk between MSCs and human T2DM islets. The co-transplantation of MSCs with human T2DM islets in diabetic SCID mice and intravenous infusion of MSCs in db/db mice revealed the reversal of β cell dedifferentiation and improved glycaemic control in the latter.InterpretationThis evidence highlights the potential of MSCs in future cell-based therapies regarding the amelioration of β cell dysfunction.

48: Islets Loaded in Hydrogel Derived from Human Amniotic Membrane Reverse Diabetes in Mice

Background Development of macroencapsulation, neovascularized devices and biopolymer scaffolds that could be easily loaded with islets and implanted, with the aim of constructing a bioartificial pancreas may help to reduce the risks and improve the success rate of islet transplantation. Human amniotic membrane (HAM) is inexpensive and attractive as a biomaterial due to its structural similarities to islet extracellular matrix (ECM), and its immunomodulatory, anti-inflammatory and antifibrotic properties. The aim of our study was to develop hydrogel derived from HAM and assess whether it could support islet function in vitro and in vivo. Methods/ Materials The hydrogels were generated from HAM and accessed for porosity and ECM content. The protein content in HAM derived hydrogels and native HAM lysates were measured. To assess hydrogel impact on islet viability and function isolated rat islets were incorporated into the hydrogels and cultured for one week. The cell viability was evaluated by FDA/PI staining. To demonstrate islet function the glucose stimulated insulin secretion (GSIS) tests were performed using standard ELISA. Next, we assessed whether incorporation of islets into hydrogel could enhance engraftment and lead to better glycemic control in diabetic SCID mice. For this purpose 350 rat islets (IEQ) loaded into the hydrogels or islets alone (control) were transplanted into the epididymal fat of diabetic SCID mice. Blood glucose levels were monitored daily and intraperitoneal glucose tolerance tests (IPGTTs) were carried out. Grafts and serum were harvested at 1, 2, 6 and 12 weeks after transplantation to assess outcome. Results The ECM concentration in the hydrogel affected the pore size. Insulin and glucagon expression and viability of islets incorporated into hydrogel was significantly higher than that of islets in free-floating culture. In addition, significant enhancement of GSIS was observed from islets embedded in hydrogel as compared to controls. In vivo experiments showed that, transplantation of 350 IEQ embedded in hydrogel lead to enhanced engraftment, vascularization, viability and better glycaemic control compared to control mice transplanted with islets alone. Conclusions Incorporation of pancreatic islet into amnion-derived hydrogels enhances islet engraftment and is a valuable approach to improve islet transplantation outcomes. This work was supported by a grant from the Swiss National Science Foundation (Grant #310030_173138, to TB, EB, DB) and a grant from the European Foundation for Study of Diabetes (to EB and DB).

Production, Characterization, and Function of Pseudoislets from Perinatal Canine Pancreas

We evaluated the cell composition and function of canine pancreatic pseudoislets (PIs) produced from 42- to 55-day-old fetuses, 1- to 21-day-old pups, and an adult dog pancreas. After mild collagenase treatment, partially digested tissues were cultured for 2–3 weeks. PI production started on culture day 3, was marked for 6 to 9 days, and then stopped. PI production was greatest with the neonatal specimens, reaching about 12 million aggregates per litter (55-day-old fetus) or per pancreas (1-day-old pup). Cell composition at all stages was similar to that in adult pancreatic islets, with predominant β cells, scant α cells and, most importantly, presence of δ cells. Among pancreatic markers assessed by quantitative real-time PCR (qRT-PCR) mRNA assay, insulin showed the highest expression levels in PIs from newborn and adult pancreas, although these were more than 1000 times lower than in adult islets. Pdx1 mRNA expression was high in PIs from 55-day-old pancreases and was lower at later stages. Consistent with the qRT-PCR results, the insulin content was far lower than reported in adult dog pancreatic islets. However, insulin release by PIs from 1-day-old pups was demonstrated and was stimulated by a high-glucose medium. PIs were transplanted into euglycemic and diabetic SCID mice. In euglycemic animals, the transplant cell composition underwent maturation and transplants were still viable after 6 months. In diabetic mice, the PI transplants produced insulin and partially controlled the hyperglycemia. These data indicate that PIs can be produced ex vivo from canine fetal or postnatal pancreases. Although functional PIs can be obtained, the production yield is most likely insufficient to meet the requirements for diabetic dog transplantation without further innovation in cell culture amplification.

Islet-Like Structures Generated In Vitro from Adult Human Liver Stem Cells Revert Hyperglycemia in Diabetic SCID Mice

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic β cells. HLSC-ILS showed endocrine granules similar to those seen in human β cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of β cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further β-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.

Shielding Human Islets with Human Amniotic Epithelial Cells Enhances Islet Engraftment and Revascularization

Background Hypoxia is a main cause of considerable islet loss during first days after intraportal transplantation. The aim of this study was to investigate whether shielding of human islets (HI) with human amniotic epithelial cells (hAECs), which are known to possess immunomodulatory, anti-inflammatory and regenerative properties could improve islet engraftment and survival. Methods Shielded islets were generated on microwells by mixing HI and hAECs at ratio of 1:800 (800 hAECs per HI). Next, 1200 shielded or neat islets, were transplanted under the kidney capsule of diabetic SCID mice. The ability of hAECs to adhere to human islets was analyzed by confocal microscopy. Islet function was assessed by dynamic insulin release in response to glucose in vitro. Blood glucose and weight were monitored regularly. Intravenous glucose tolerance test was performed 1 month after transplantation. Graft morphology and vascularisation were evaluated by immunohistochemistry. Results HI shielded with hAECs had greater cellular insulin content and increased glucose-stimulated insulin secretion. Transplantation of shielded islets resulted in considerably earlier normoglycemia and vascularization, improved glucose tolerance, and increased insulin content. Conclusion Co-transplantation of islets with hAECs had a profound impact on the remodelling process, maintaining islet organisation and improving islet revascularisation. Moreover, hAECs improved the capacity of islets to reverse hyperglycaemia.

Role of PI3K p110β in the differentiation of human embryonic stem cells into islet-like cells

To investigate the effects of the PI3K inhibitors on the differentiation of insulin-producing cells derived from human embryonic stem cells. Here, we report that human embryonic stem cells induced by phosphatidylinositol-3-kinase (PI3K) p110β inhibitors could produce more mature islet-like cells. Findings were validated by immunofluorescence analysis, quantitative real-time PCR, insulin secretion in vitro and cell transplantation for the diabetic SCID mice. Immunofluorescence analysis revealed that unihormonal insulin-positive cells were predominant in cultures with rare polyhormonal cells. Real-time PCR data showed that islet-like cells expressed key markers of pancreatic endocrine hormones and mature pancreatic β cells including MAFA. Furthermore, this study showed that the expression of most pancreatic endocrine hormones was similar between groups treated with the LY294002 (nonselective PI3K inhibitor) and TGX-221 (PI3K isoform selective inhibitors of class 1β) derivatives. However, the level of insulin mRNA in TGX-221-treated cells was significantly higher than that in LY294002-treated cells. In addition, islet-like cells displayed glucose-stimulated insulin secretion in vitro. After transplantation, islet-like cells improved glycaemic control and ameliorated the survival outcome in diabetic mice. This study demonstrated an important role for PI3K p110β in regulating the differentiation and maturation of islet-like cells derived from human embryonic stem cells.

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.

Transplantation Can Reinitiate Proliferation of Terminally Differentiated Sertoli Cells.

Sertoli cells (SC) are immune privileged cells that survive allo- or xeno-transplantation long term, suggesting they could be used as a vehicle to deliver therapeutic proteins. To test this, “non-dividing” SC isolated from 27 day-old post-pubertal rats were transduced with a recombinant adenoviral vector containing modified human insulin cDNA and transplanted underneath the left kidney capsule of diabetic SCID mice. The adenovirus was chosen because it allows for robust expression of the protein of interest for an extended period of time (more than 2 months) in non-dividing cells including SC. Contrary to what was expected, blood glucose levels were lowered only transiently (less than10 days), and when the grafts were analyzed for insulin expression by immunostaining, a loss is insulin expression was observed. Surprisingly, when the grafts were immunostained with cell proliferation markers, PCNA (proliferating cell nuclear antigen) and KI-67, the SC within the grafts were positive, suggesting that the transient expression of insulin was due to loss of the epichromosomal adenoviral vector from the proliferating SC. This is surprising because, the dogma is that SC stop proliferating by puberty and become terminally differentiated. Some of the possible causes for resumption of proliferation by the SC could be viral transduction, cell isolation and culture, transplantation to kidney, and/or higher abdominal temperature at the transplant site. To test for the possible cause of proliferation, tissue samples were collected for histology (n=3) from cryptorchid testes (testes at abdominal temperature), various stages of SC isolation and culture, and transplants of non-diving, non-transduced SC in rats at various time-points between 1-55 days post-transplantation. The collected tissue sections were double immunostained for SC marker GATA-4, and cell proliferation marker KI-67. None of the GATA-4 positive SC stained positive for KI-67 in cryptorchid testes, tissue collected during SC isolation and culture. However, many of the GATA-4 positive SC stained positive for KI-67, between 1-14 days post-transplantation. Interestingly this resumption in proliferative ability of non-dividing SC was temporary, where SC stop proliferating within 19 days of transplantation and do not proliferate thereafter. These data indicate that transplantation of SC, but not the higher temperature, viral transduction, SC isolation or culture, is a potential cause for reinitiating proliferation of these non-dividing SC. Transplantation of SC could provide a useful model to study the regulation of SC proliferation in vivo.

Delivery of a Therapeutic Protein by Immune-Privileged Sertoli Cells

Immune-privileged Sertoli cells survive long term after allogeneic or xenogeneic transplantation without the use of immunosuppressive drugs, suggesting they could be used as a vehicle to deliver therapeutic proteins. As a model to test this, we engineered Sertoli cells to transiently produce basal levels of insulin and then examined their ability to lower blood glucose levels after transplantation into diabetic SCID mice. Mouse and porcine Sertoli cells transduced with a recombinant adenoviral vector containing furin-modified human proinsulin cDNA expressed insulin mRNA and secreted insulin protein. Transplantation of 5-20 million insulin-expressing porcine Sertoli cells into diabetic SCID mice significantly decreased blood glucose levels in a dose-dependent manner, with 20 million Sertoli cells decreasing blood glucose levels to 9.8 ± 2.7 mM. Similar results were obtained when 20 million insulin-positive, BALB/c mouse Sertoli cells were transplanted; blood glucose levels dropped to 6.3 ± 2.4 mM and remained significantly lower for 5 days. To our knowledge, this is the first study to demonstrate Sertoli cells can be engineered to produce and secrete a clinically relevant factor that has a therapeutic effect, thus supporting the concept of using immune-privileged Sertoli cells as a potential vehicle for gene therapy.

Microassay for Glucose-Induced Preproinsulin mRNA Expression to Assess Islet Functional Potency for Islet Transplantation

The capacity for insulin synthesis in islets is important for islet transplantation to succeed. We developed a microassay that evaluates the potency of human islets by measuring changes in glucose-induced human insulin gene (INS) expression using a single islet in octuplicate samples. Poly (A) messenger RNA (mRNA) was purified from a set of single handpicked human islets. Glucose-induced mature (postspliced) and premature (prespliced) insulin mRNA were quantified by reverse-transcriptase polymerase chain reaction using several insulin mRNA primers designed at different locations including, intron, exon, and an exon-intron junction. The synthesis of premature INS mRNA was significantly increased in islets exposed to high glucose for 16 vs. 4 hr (P<0.01), whereas mature INS mRNA showed no difference. Glucose-induced premature INS mRNA synthesis was attenuated in heat-damaged islets. Stimulation index (SI) calculated by normalizing premature by mature INS mRNA (SI_INS mRNA) positively correlated with SI of insulin release (SI_16h insulin) from the same set of islets during 16-hr incubation in high or low glucose media, and SI of glucose-mediated insulin release obtained from the same islet lot in a perifusion system (n=12). Furthermore, linear multiple regression analysis using SI_INS mRNA and SI_16h insulin predicted islet transplantation outcome in nonobese diabetic (NOD) scid mice (n=8). The measurement of glucose-induced premature INS mRNA normalized by mature INS mRNA can be used to assess the functional quality of human islets and may predict islet function after transplantation in type 1 diabetic patients.

Correlation of primary tumor engraftment in immune deficient mice and relapse rate in patients with early-stage non-small cell lung carcinoma (NSCLC)

11082 Background: Compared to cell lines, primary tumor xenografts potentially are more clinically relevant cancer models as they more closely reflect the phenotype and genotype of the original cancer. However, only a minority of tumors engraft successfully in severe combined immune deficient (scid) mice and can be passaged serially. Although xenograft models are used extensively, few studies have investigated whether tumors that engraft represent a distinct clinical subset. We hypothesized that NSCLC tumors with more aggressive clinical and histological features have greater engraftment capacity than those with a less aggressive phenotype. Methods: Fresh primary tumors were harvested from NSCLC patients who underwent curative resection. Tumor fragments were implanted into the subcutaneous tissue of non-obese diabetic-scid mice within 24 hrs of excision. Patient characteristics for tumors that engrafted (XG) and did not engraft (No-XG) were compared. Only tumors from patients with &gt;1-yr follow-up were evaluated for time to progression (TTP) and to correlate clinicopathological features with engraftment. Results: Between March 2005 and October 2008, 110 tumors were implanted. Of these, 45 (41%) engrafted and were passaged serially in vivo. The histological features of the primary were retained in 93% of XG tumors. Squamous cell carcinomas engrafted significantly more than adenocarcinomas (57% versus 26%, p=0.03). There were no significant differences in differentiation grade or clinical stage between the XG and No-XG groups. XG patients had significantly shorter TTP than the No-XG group (10.19 versus 18.64 months, p=0.003). In multivariate analysis the ability to form a xenograft was an independent predictor of relapse (HR 4.15 95% CI 1.152–14.94, p=0.03). Conclusions: Xenograft models can be established from the histological spectra of NSCLC encountered in the clinical setting and mimic closely the features of their primary tumors. The capacity of these tumors to engraft may be predictive of a more aggressive phenotype and poorer clinical outcome. No significant financial relationships to disclose.

The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells

Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6 h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell–scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.

Conversion of immortal liver progenitor cells into pancreatic endocrine progenitor cells by persistent expression of Pdx‐1

The conversion of expandable liver progenitor cells into pancreatic beta cells would provide a renewable cell source for diabetes cell therapy. Previously, we reported the establishment of liver epithelial progenitor cells (LEPCs). In this work, LEPCs were modified into EGFP/Pdx-1 LEPCs, cells with stable expression of both Pdx-1 and EGFP. Unlike previous work, with persistent expression of Pdx-1, EGFP/Pdx-1 LEPCs acquired the phenotype of pancreatic endocrine progenitor cells rather than giving rise to insulin-producing cells directly. EGFP/Pdx-1 LEPCs proliferated vigorously and expressed the crucial transcription factors involved in beta cell development, including Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1, MafA and endogenous Pdx-1, but did not secrete insulin. When cultured in high glucose/low serum medium supplemented with cytokines, EGFP/Pdx-1 LEPCs stopped proliferating and gave rise to functional beta cells without any evidence of exocrine or other islet cell lineage differentiation. When transplanted into diabetic SCID mice, EGFP/Pdx-1 LEPCs ameliorated hyperglycemia by secreting insulin in a glucose regulated manner. Considering the limited availability of beta cells, we propose that our experiments will provide a framework for utilizing the immortal liver progenitor cells as a renewable cell source for the generation of functional pancreatic beta cells.

Delayed functional maturation of neonatal porcine islets in recipients under strict glycemic control

The aim of this study was to compare the functional maturation of neonatal porcine islet (NPI) grafts exposed to long-term hyperglycemia with those implanted under euglycemic conditions. mice Neonatal porcine islets were transplanted under the left renal capsule of diabetic SCID mice (group H), or in diabetic SCID mice who were also implanted with 500 BALB/c islets under the right renal capsule (group N). On day 42, the right kidneys were removed in both groups. No animals in group H achieved euglycemia within 3 weeks after transplantation. Thus, these mice were exposed to long-term hyperglycemia. Mice in group N became euglycemic immediately after transplantation, however after removal of BALB/c grafts on day 42 they exhibited significantly higher blood glucose levels than in group H and showed glucose intolerance after glucose administration. Cellular insulin content of NPI grafts harvested on day 58 or 72 was significantly lower in group N mice compared to group H. These results suggest that tight control of glycemia reduces the functional maturation of NPI grafts.

Normal Relationship of β- and Non–β-Cells Not Needed for Successful Islet Transplantation

Islets are composed mostly of beta-cells, and therefore stem cell research has concentrated on generating purified beta-cells, neglecting the other endocrine cell types in the islet. We investigated the presence of endocrine non-beta-cells after islet transplantation. In addition, we studied whether the transplantation of pure beta-cells, in volumes similar to that used in islet transplantation, would suffice to reverse hyperglycemia in diabetic mice. Rat islets were dispersed and beta-cells were purified by fluorescence-activated cell sorting according to their endogenous fluorescence. After reaggregation, 600 islet equivalents of the purified beta-cell aggregates were implanted into diabetic SCID mice. In mice implanted with beta-cell-enriched aggregates, the hyperglycemia was reversed and good graft function over a 12-week period was observed with regard to glucose and insulin levels, glucose tolerance tests, and graft insulin content. The endocrine cell composition of the beta-cell-enriched aggregates remained constant; before and 12 weeks after transplantation, the beta-cell-enriched aggregates comprised 95% beta-cells and 5% endocrine non-beta-cells. However, islet grafts, despite originally having comprised 75% beta-cells and 25% endocrine non-beta-cells, comprised just 5% endocrine non-beta-cells after transplantation, indicating a loss of these cells. beta-Cell-enriched aggregates can effectively reverse hyperglycemia in mice, and transplanted intact islets are depleted in non-beta-cells. It is therefore likely that islet non-beta-cells are not essential for successful islet transplantation.

Improving islet transplantation by gene delivery of hepatocyte growth factor (HGF) and its downstream target, protein kinase B (PKB)/Akt

Clinical studies have demonstrated that islet transplantation may be a useful procedure to replace beta cell function in patients with Type 1 diabetes. Islet transplantation faces many challenges, including complications associated with the procedure itself, the toxicity of immunosuppression regimens, and to the loss of islet function and insulin-independence with time. Despite the current successes, and residual challenges, these studies have pointed out an enormous scarcity of islet tissue that precludes the use of islet transplantation in a clinical setting on a wider scale. To address this problem, many research groups are trying to identify different islet growth factors and intracellular molecules capable of improving islet graft survival and function, therefore reducing the number of islets needed for successful transplantation. Among these growth factors, hepatocyte growth factor (HGF), a factor known to improve transplantation of a variety of organs/cells, has shown promising results in increasing islet graft survival and reducing the number of islets needed for successful transplantation in four different rodent models of islet transplantation. Protein kinase B (PKB)/Akt, a pro-survival intracellular signaling molecule is known to be activated in the beta cell by several different growth factors, including HGF. PKB/Akt has also shown promising results for improving human islet graft survival and function in a minimal islet mass model of islet transplantation in diabetic SCID mice. Increasing our knowledge on how HGF, PKB/Akt and other emerging molecules work for improving islet transplantation may provide substrate for future therapeutic approaches aimed at increasing the number of patients in which beta cell function can be successfully replaced.

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach

We examined a clinical model of ex vivo transdifferentiation of primary adult hepatocytes to insulin-secreting cells for the treatment of type 1 diabetes. Isolated rat hepatocytes were transduced in primary culture with a human lentivirus containing pancreatic duodenal homeobox 1 (PDX1, now known as insulin promoter factor 1, homeodomain transcription factor [IPF1]). Insulin expression and secretion of the newly engineered cells were assessed in vitro by RT-PCR, in situ hybridisation, immunostaining and radioimmunoassay. PDX1-transduced hepatocytes were further studied in vivo by injecting them under the renal capsule of diabetic SCID mice. Isolated rat hepatocytes were efficiently transduced with the lentiviral vector, as assessed by green fluorescent reporter gene expression. The transduced cells exhibited insulin at both mRNA (RT-PCR, in situ hybridisation) and protein levels (immunostaining and radioimmunoassay). Moreover, insulin secretion by the engineered cells was dependent on glucose and sulfonylurea. Other beta cell genes, including those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), glucokinase (Gck), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), the potassium inwardly-rectifying channel, subfamily J, member 11 (Kcnj11) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) were also expressed. The PDX1-transduced hepatocytes expressed several pancreatic transcription factors related to early pancreatic endocrine development (endogenous Pdx1, neurogenic differentiation factor 1 [Neurod1], and NK6 transcription factor related, locus 1 [Nkx6-1]) as well as the late-stage pancreatic transcription factors (paired box gene 4 [Pax4], paired box gene 6 [Pax6], and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A [Mafa]). Transplantation of 3 x 10(6) transdifferentiated liver cells under the renal capsule of seven streptozotocin-induced diabetic SCID mice resulted in significant reduction of non-fasting blood glucose levels from 30.7 +/- 1.3 to 8.7 +/- 3.7 mmol/l (mean +/- SEM, p = 0.01), in 6 to 8 weeks. Removal of the graft resulted in severe hyperglycaemia. Ex vivo lentiviral-mediated PDX1 expression in isolated adult liver cells represents a potential model for type 1 diabetes mellitus therapy.

Hemin Activation Ameliorates HIV-1 Infection via Heme Oxygenase-1 Induction

Hemin, a critical component of hemoglobin, is an active ingredient of a biologic therapeutic approved by the Food and Drug Administration for the treatment of acute porphyries. This report describes a biological function of this molecule in inducing host defense against HIV-1 infection via heme oxygenase-1 (HO-1) induction. Treatment of monocytes with hemin substantially inhibited HIV replication, as evident by nearly undetectable viral RNA and cell-free HIV-1 p24 protein in a dose-dependent manner. Hemin exposure of these cells before infection, at the time of infection, or after infection caused >90% reduction of HIV DNA with substantially low levels of HIV-1 p24 and HIV-associated cytopathic effects. In addition, hemin treatment significantly suppressed infection of both monocytes and T cells inoculated with R5, X4, R5X4 tropic strains, and reverse transcriptase-resistant, azidothymidine-resistant, ddC/ddI-resistant, nivirapine-resistant, and other clinical HIV isolates. Intraperitoneal administration of hemin 4 days after HIV infection reduced viral load in the serum of human PBMC-reconstituted nonobese diabetic SCID mice by >6-fold. Suppression of HIV replication in hemin-activated cells correlated with the induction of HO-1 and was attenuated by tin protoporphyrin (SnPP) IX, an inhibitor of HO-1 activity, suggesting a pivotal role of this endogenous enzyme in the regulation of HIV infection. Hemin-induced HO-1 induction in the CCR-5, CXCR-4, and CD4 coexpressing GHOST(3) cells was consistent with the inhibition of Tat-dependent activation of long terminal repeat promoter leading to reduced GFP expression. These findings suggest an important role of hemin-induced HO-1 activity as a host defense mechanism against HIV-1 infection.