Nonclinical and quality assessment of cell therapy products: Report on the 4th Asia Partnership Conference of Regenerative Medicine, April 15, 2021.

The 4th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 15, 2021, to promote regulatory harmonization of regenerative medicine products throughout Asia. Recognizing domestic regulatory guidelines within each country and region, and their underpinning rationales, is an important initial step toward a convergence of regulations. The 4th APACRM consisted of an open dialog with regulatory agencies regarding nonclinical and quality settings for cell therapy products (CTPs) through industry presentations and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region, and specific regulatory schematics in Japan, were also introduced. The objective of this paper is to summarize the proceedings of the 4th APACRM for public dissemination and to foster further discussion in the future.

Non-clinical assessment of cell therapy products: the perspective from five Asian countries/regions based on regulatory guidelines and the underpinning rationales.

BACKGROUND AIMS: Cell-based regenerative medicine is an innovative field that can potentially alter the overall survival and quality of life of patients with devastating diseases. Several cell therapy products (CTPs) have been approved within the last two decades, and more are under development. The establishment of an effective developmental strategy in accordance with the regulatory bodies of each country/region is crucial for fast delivery of each respective CTP. In particular, facilitating investigational new drug (IND) approval is important for accelerating the transition from non-clinical to clinical research/trial phases. METHODS: Here the authors compared the non-clinical prerequisites for initiating clinical studies in five Asian countries/regions (India, China, Korea, Taiwan and Japan) from an industry viewpoint. The authors first identified the differences and tried to clarify the perspectives/considerations underpinning the different requirements. RESULTS: The authors' findings revealed that differences in regulations and development experiences, especially with CTPs, have led to clear differences in the non-clinical study package and its corresponding study design. CONCLUSIONS: By sharing experiences of the research and development of CTPs among Asian countries/regions and including not only industry but also regulatory authorities, we will be able to expedite cross-border IND approval and eventually contribute to the early delivery of innovative CTPs to many Asian patients.

A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation. METHODS AND RESULTS: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride. CONCLUSIONS: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Specific PERK inhibitors enhanced glucose-stimulated insulin secretion in a mouse model of type 2 diabetes.

BACKGROUND: We have reported that partial PERK attenuation using PERK inhibitors (PI) enhanced glucose-stimulated insulin secretion (GSIS) from pancreatic islets and mice through induction of ER chaperone BIP. Therefore, we investigated if PI would have the same effects in a diabetic condition as well. METHODS: GSK2606414 was treated to mouse islets under 20-mM glucose and 0.5-mM palmitate to examine GSIS. To generate a mouse model of type 2 diabetes mellitus (DM), male C57BL/6J mice were fed with high-fat diet and injected with streptozotocin. Several doses (6-16 mg/kg/day) of GSK2656157 and glimepiride were administrated to the mice for 8 weeks, and metabolic phenotypes were evaluated such as body weight, blood glucose levels, insulin secretion and sensitivity, and then changes in the pancreas were measured. RESULTS: High-glucose and palmitate treatment significantly increased PERK phosphorylation in the isolated islets. Suppression of GSIS and glucose-stimulated Ca2+ transit was also observed. PI at 40 nM which decreased PERK phosphorylation by 40% significantly recovered the GSIS and cytosolic calcium. In the mice where significant weight gain and prominent hyperglycemia were induced, PI at 10 mg/kg/day significantly enhanced GSIS and reduced blood glucose levels compared to the vehicle. The effects were similar to those by 10 mg/kg/day of glimepiride. Administration of PI did not induce changes in beta cell mass or pancreatic insulin contents, however, high dose PI decreased pancreatic weight. CONCLUSION: PI at low dose significantly enhanced GSIS in vitro and in vivo under metabolic stress and improved hyperglycemia in the mice mimicking type 2 DM, suggesting a potential as a new therapeutic approach for type 2 DM.

Recent progress of national banking project on homozygous HLA-typed induced pluripotent stem cells in South Korea.

Induced pluripotent stem cells (iPSCs) can be generated by introducing several factors into mature somatic cells. Banking of iPSCs can lead to wider application for treatment and research. In an economical view, it is important to store cells that can cover a high percentage of the population. Therefore, the use of homozygous human leukocyte antigen-iPSCs (HLA-iPSCs) is thought as a potential candidate for effective iPSC banking system for further clinical use. We screened the database stored in the Catholic Hematopoietic Stem Cell Bank of Korea and sorted the most frequent homozygous HLA types of the South Korean population. Blood cells with the selected homozygous HLA types were obtained and transferred to the GMP facility in the Catholic Institute of Cell Therapy. Cells were reprogrammed to iPSCs inside the facility and went through several quality controls. As a result, a total of 13 homozygous GMP-grade iPSC lines were obtained in the facility. The generated iPSCs showed high pluripotency and normal karyotype after reprogramming. Five HLA-homozygous iPSCs had the type that was included in the top five most frequent HLA types. Homozygous HLA-iPSCs can open a new opportunity for further application of iPSCs in clinical research and therapy.

Antifibrotic effect of rapamycin containing polyethylene glycol-coated alginate microcapsule in islet xenotransplantation.

Islet microencapsulation is an attractive strategy for the minimization or avoidance of life-long immunosuppression after transplantation. However, the clinical implementation of this technique is currently limited by incomplete biocompatibility. Thus, the aim of the present study was to demonstrate the improved biocompatibility of rapamycin-containing polyethylene glycol (Rapa-PEG)-coating on alginate microcapsules containing xenogeneic islets. The Rapa-PEG-coating on the alginate layer was observed using scanning electron microscopy (SEM) and the molecular cut-off weight of the microcapsules was approximately 70 kDa. The viabilities of the alginate-encapsulated and Rapa-PEG-coated alginate-encapsulated islets were lower than the viability of the naked islets just after encapsulation, but these the differences diminished over time in culture dishes. Rapa-PEG-coating on the alginate capsules effectively decreased the proliferation of macrophage cells compared to the non-coating and alginate coating of xenogeneic pancreas tissues. Glucose-stimulated insulin secretion did not significantly differ among the groups prior to transplantation. The random blood glucose levels of diabetic mice significantly improved following the transplantation of alginate-encapsulated and Rapa-PEG-coated alginate-encapsulated islets, but there were no significant differences between these two groups. However, there was a significant decrease in the number of microcapsules with fibrotic cell infiltration in the Rapa-PEG-coated alginate microcapsule group compared to the alginate microcapsule group. In conclusion, Rapa-PEG-coating might be an effective technique with which to improve the biocompatibility of microcapsules containing xenogeneic islets. Copyright © 2015 John Wiley & Sons, Ltd.

Suppression of ROS Production by Exendin-4 in PSC Attenuates the High Glucose-Induced Islet Fibrosis.

Pancreatic stellate cells (PSCs) play a major role to fibrotic islet destruction observed in diabetic patients and animal model of diabetes. Exendin-4 (Ex-4) is a potent insulinotropic agent and has been approved for the treatment of type 2 diabetes. However, there have been no reports demonstrating the effects of Ex-4 on pancreatic islet fibrosis. In this study, Ex-4 treatment clearly attenuated fibrotic islet destruction and improved glucose tolerance and islet survival. GLP-1 receptor expression was upregulated during activation and proliferation of PSCs by hyperglycemia. The activation of PKA pathway by Ex-4 plays a role in ROS production and angiotensin II (Ang II) production. Exposure to high glucose stimulated ERK activation and Ang II-TGF- ß1 production in PSCs. Interestingly, Ex-4 significantly reduced Ang II and TGF-ß1 production by inhibition of ROS production but not ERK phosphorylation. Ex-4 may be useful not only as an anti-diabetic agent but also as an anti-fibrotic agent in type 2 diabetes due to its ability to inhibit PSC activation and proliferation and improve islet fibrosis in OLETF rats.

Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells.

The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into ß-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and ß-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes.

The Paradoxical Effects of AMPK on Insulin Gene Expression and Glucose-Induced Insulin Secretion.

The activation of AMP-activated protein kinase (AMPK) is known to repress the expression of the insulin gene and glucose-stimulated insulin secretion (GSIS). However, the mechanisms by which this occurs, as well as the effects of AMPK activation on glucolipotoxicity-induced ß-cell dysfunction, have not been elucidated. To investigate the effects of 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) and peroxisome proliferator-activated receptorγ-coactivator-1α (PGC-1α) on ß-cell-specific genes under glucolipotoxic conditions, we performed real-time PCR and measured insulin secretion by primary islets. To study these effects in vivo, we administered AICAR for 10 days (1 mg/g body weight) to 90% pancreatectomized hyperglycemic mice. The exposure of isolated rat and human islets to glucolipotoxic conditions and the overexpression of PGC-1α suppressed insulin and NEUROD1 mRNA expression. However, the expression of these genes was preserved by AICAR treatment and by PGC-1α inhibition. Exposure of isolated islets to glucolipotoxic conditions for 3 days decreased GSIS, which was also well maintained by AICAR treatment and by PGC-1α inhibition. The administration of AICAR to 90% pancreatectomized hyperglycemic mice improved glucose tolerance and insulin secretion. These results indicate that treatment of islets with an AMPK agonist under glucolipotoxic conditions protects against glucolipotoxicity-induced ß-cell dysfunction. A better understanding of the functions of molecules such as PGC-1α and AMPK, which play key roles in intracellular fuel regulation, could herald a new era for the treatment of patients with type 2 diabetes mellitus by providing protection against glucolipotoxicity.

Long-term Efficacy and Biocompatibility of Encapsulated Islet Transplantation With Chitosan-Coated Alginate Capsules in Mice and Canine Models of Diabetes.

BACKGROUND: Clinical application of encapsulated islet transplantation is hindered by low biocompatibility of capsules leading to pericapsular fibrosis and decreased islet viability. To improve biocompatibility, we designed a novel chitosan-coated alginate capsules and compared them to uncoated alginate capsules. METHODS: Alginate capsules were formed by crosslinking with BaCl2, then they were suspended in chitosan solution for 10 minutes at pH 4.5. Xenogeneic islet transplantation, using encapsulated porcine islets in 1,3-galactosyltransferase knockout mice, and allogeneic islet transplantation, using encapsulated canine islets in beagles, were performed without immunosuppressants. RESULTS: The chitosan-alginate capsules showed similar pore size, islet viability, and insulin secretory function compared to alginate capsules, in vitro. Xenogeneic transplantation of chitosan-alginate capsules demonstrated a trend toward superior graft survival (P = 0.07) with significantly less pericapsular fibrosis (cell adhesion score: 3.77 ± 0.41 vs 8.08 ± 0.05; P < 0.001) compared to that of alginate capsules up to 1 year after transplantation. Allogeneic transplantation of chitosan-alginate capsules normalized the blood glucose level up to 1 year with little evidence of pericapsular fibrotic overgrowth on graft explantation. CONCLUSIONS: The efficacy and biocompatibility of chitosan-alginate capsules were demonstrated in xenogeneic and allogeneic islet transplantations using small and large animal models of diabetes. This capsule might be a potential candidate applicable in the treatment of type 1 diabetes mellitus patients, and further studies in nonhuman primates are required.

Successful xenotransplantation with re-aggregated and encapsulated neonatal pig liver cells for treatment of mice with acute liver failure.

BACKGROUND: Hepatocyte transplantation is a promising therapy for acute liver failure. Cell therapy using xenogeneic sources has emerged as an alternative treatment for patients with organ failure due to the shortage of transplantable human organs. The purpose of this study was to improve the survival of mice with acute liver failure by transplanting encapsulated neonatal pig re-aggregated liver cells (NPRLC). METHODS: Liver injury was induced in C57/BL6 male mice by the injection of 600 mg/kg of acetaminophen. Xenogeneic liver cells were isolated from a neonatal pig and processed via re-aggregation and encapsulation to improve the efficiency of the xenogeneic liver cell transplantation. The neonatal pig liver showed abnormal lobule structure. Isolated cells were re-aggregated and intraperitoneally transplanted into acute liver failure mice models. RESULTS: Re-aggregated cells showed significantly enhanced viability and significantly greater synthesis of albumin and urea than cells cultured in monolayers. Further, we observed improved serum levels of ALT/AST, and the survival rate of mice with acute liver failure was improved by the intraperitoneal transplantation of encapsulated hepatocytes (48,000 equivalent (Eq) per mouse). CONCLUSIONS: This study shows that using encapsulated NPRLCs improves the efficacy of xenogeneic liver cell transplantation for the treatment of mice with acute liver failure. Therefore, this may be a good strategy for bridge therapy for the treatment of acute liver failure in humans.

Reversal of Hypoglycemia Unawareness with a Single-donor, Marginal Dose Allogeneic Islet Transplantation in Korea: A Case Report.

Pancreatic islet transplantation is a physiologically advantageous and minimally invasive procedure for the treatment of type 1 diabetes mellitus. Here, we describe the first reported case of successful allogeneic islet transplantation alone, using single-donor, marginal-dose islets in a Korean patient. A 59-yr-old patient with type 1 diabetes mellitus, who suffered from recurrent severe hypoglycemia, received 4,163 islet equivalents/kg from a single brain-death donor. Isolated islets were infused intraportally without any complications. The immunosuppressive regimen was based on the Edmonton protocol, but the maintenance dosage was reduced because of mucositis and leukopenia. Although insulin independence was not achieved, the patient showed stabilized blood glucose concentration, reduced insulin dosage and reversal of hypoglycemic unawareness, even with marginal dose of islets and reduced immunosuppressant. Islet transplantation may successfully improve endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus.

Autophagy deficiency in ß cells blunts incretin-induced suppression of glucagon release from α cells.

Incretin-based therapy such as GLP-1 receptor agonists and DPP-4 inhibitors for type 2 diabetes mellitus is characterized by glucose-dependent insulin secretion and glucose-inhibited glucagon secretion. Recently, autophagy deficiency in islet ß cells has been shown to contribute to the pathogenesis of type 2 diabetes mellitus however, with the role of incretin has not been established. To evaluate the role of autophagy in incretin effects, 8-week-old male ß cell-specific Atg7 knockout (Atg7(Δß cell)) mice and wild-type mice were administered vildagliptin for 12 weeks. Vildagliptin treatment improved glucose intolerance and hypoinsulinemia; however, it failed to suppress serum glucagon levels after glucose loading in the Atg7(Δß cell) mice. Ex vivo glucose-induced glucagon suppression was also blunted in the islets from vildagliptin-treated Atg7(Δß cell) mice. The α cell mass was not affected by ß cell autophagy deficiency or vildagliptin. However, glucagon mRNA expression was significantly increased by vildagliptin in the autophagy-deficient islets, and was significantly reduced by vildagliptin in wild-type islets. Pancreatic glucagon contents were not in agreement with the changes in mRNA expression, suggesting a dysregulation in glucagon translation and secretion. In vitro studies revealed that glucose-stimulated cAMP production was impaired in the autophagy-deficient islets exposed to exendin-4. Taken together, the results suggest that the constitutive autophagy in ß cells could regulate incretin-induced glucagon expression and release in α cells, and that cAMP may play a role in this process.

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway.

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.

Targeting PGC-1α to overcome the harmful effects of glucocorticoids in porcine neonatal pancreas cell clusters.

BACKGROUND: Peroxisome proliferator-activated receptor gamma-coactivator-1α (PGC-1α) has recently been implicated as a crucial factor in the glucocorticoid-suppressed expansion and transdifferentiation of porcine neonatal pancreatic cell clusters (NPCCs). However, the molecular mechanism has not been clarified. METHODS: We investigated whether the suppression of PGC-1α expression protects against ß-cell dysfunction induced by dexamethasone (Dx) treatment in vitro and in vivo and determined the mechanism of action of PGC-1α in porcine NPCCs. RESULTS: The reduction in Pdx-1 gene expression caused by either Dx treatment or PGC-1α overexpression was normalized by siPGC-1α. Nuclear FOXO1 and cytoplasmic Pdx-1 were detected after Dx treatment. However, FOXO1 was observed in the cytoplasm, and Pdx-1 was observed in the nucleus after siPGC-1α. Suppression of PGC-1α by siPGC-1α improved the Dx-induced repression of insulin secretion and insulin content. In vivo studies showed that the glucose level in the Ad-siPGC-1α-infected group was significantly lower than that in the Dx-treated group. Insulin expression in the graft tissue disappeared in the Dx-injected group. However, the siPGC-1α- and Dx-treated group showed increased insulin expression and an increase in graft mass, ß-cell mass, and ß-cell % in the graft. Conversely, the Dx-induced ductal cystic area was markedly reduced in the siPGC-1α- and Dx-treated group. CONCLUSIONS: Our results suggest that the transdifferentiation of porcine NPCCs into ß cells is influenced by the duration of the Dx treatment, which might result from the suppression of key pancreatic transcription factors. PGC-1α is an attractive target for modulating the deleterious effects of glucocorticoids on pancreatic stem cells.

Generation of functional insulin-producing cells from neonatal porcine liver-derived cells by PDX1/VP16, BETA2/NeuroD and MafA.

Surrogate ß-cells derived from stem cells are needed to cure type 1 diabetes, and neonatal liver cells may be an attractive alternative to stem cells for the generation of ß-cells. In this study, we attempted to generate insulin-producing cells from neonatal porcine liver-derived cells using adenoviruses carrying three genes: pancreatic and duodenal homeobox factor1 (PDX1)/VP16, BETA2/NeuroD and v-maf musculo aponeurotic fibrosarcoma oncogene homolog A (MafA), which are all known to play critical roles in pancreatic development. Isolated neonatal porcine liver-derived cells were sequentially transduced with triple adenoviruses and grown in induction medium containing a high concentration of glucose, epidermal growth factors, nicotinamide and a low concentration of serum following the induction of aggregation for further maturation. We noted that the cells displayed a number of molecular characteristics of pancreatic ß-cells, including expressing several transcription factors necessary for ß-cell development and function. In addition, these cells synthesized and physiologically secreted insulin. Transplanting these differentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the reversal of hyperglycemia, and more than 18% of the cells in the grafts expressed insulin at 6 weeks after transplantation. These data suggested that neonatal porcine liver-derived cells can be differentiated into functional insulin-producing cells under the culture conditions presented in this report and indicated that neonatal porcine liver-derived cells (NPLCs) might be useful as a potential source of cells for ß-cell replacement therapy in efforts to cure type I diabetes.

A case of cystoid macular edema associated with Paclitaxel chemotherapy.

We encountered a patient with cystoid macular edema (CME) secondary to paclitaxel use. A 57-year-old man presented with gradual decreased bilateral vision. His chemotherapeutic regimen consisted of bevacizumab, paclitaxel (175 mg/m(2) for 5 months), and carboplatin. Optical coherence tomography imaging revealed bilateral CME greater than 500 µm. However, one year later, visual acuity was improved, best-corrected Snellen visual acuity was 40 / 80 in each eye, and CME was spontaneously improved. Our study confirmed that macular edema associated with paclitaxel use shows spontaneous resolution and improvement of visual acuity after a change of chemotherapeutic regimen.

Expression of Stat3 and indoleamine 2, 3-dioxygenase in cornea keratocytes as factor of ocular immune privilege.

PURPOSE: Ocular immune privilege is a multifactorial phenomenon evolutionally selected to prevent immunogenic inflammation from disrupting the visual axis and causing blindness. Here, we investigated the role of signal transducers and activators of transcription (Stat3) and indoleamine 2,3-dioxygenase (IDO) in ocular immune privilege in corneal stromal cells. METHODS: Human keratocytes were isolated and cultured in vitro, and Stat3 and IDO expression on keratocytes was investigated by reverse transcription polymerase chain reaction (RT-PCR). The active form of Stat3 was detected by flow-cytometry, and IDO enzyme activity following IFN-γ stimulation of keratocytes was measured by tryptophan to kynurenine conversion with photometric determination of kynurenine concentration in the supernatant. RESULTS: Stat3 was constitutively expressed in cultured keratocytes and up-regulated following IFN-γ stimulation. The active form of Stat3 was also up-regulated following IFN-γ stimulation. IDO expression and enzyme activity was markedly induced following IFN-γ stimulation, but this induction was prevented by the IDO specific inhibitor, 1-methyl tryptophan (1-MT). CONCLUSIONS: On the basis of this study, Stat3 and IDO may act as a factor of ocular immune privilege in corneal keratocytes. Thus, focus on these inhibitory molecules should be considered in studies aimed at developing therapeutic agents for controlling ocular inflammatory or immune diseases.

Both sitagliptin analogue & pioglitazone preserve the beta-cell proportion in the islets with different mechanism in non-obese and obese diabetic mice.

In this study, the effects of sitagliptin analogue (SITA) or pioglitazone (PIO) treatment on glucose homeostasis and Β-cell dynamics in animal models of type 2 diabetes--Akita and db/db mice were evaluated. After 4-6 weeks of treatment, both SITA and PIO were shown to lower non-fasting glucose levels and reduced glycemic excursion in the intraperitoneal glucose tolerance test. In addition, both drugs preserved normal islet structure and the proportion of Β-cells in the islets. Compared to the controls, SITA treatment induced a higher Β-cell proliferation rate in Akita mice and a lower rate of apoptosis in db/db mice, whereas PIO treatment induced a lower rate of apoptosis in db/db mice and reduced proliferation rates in Akita mice. In conclusion, both SITA and PIO appear to exert some beneficial effects on the islet structure in addition to glycemic control via different mechanisms that involve Β-cell dynamics in Akita and db/db mice. [BMB reports 2011; 44(11): 713-718].

Adenoviruses Expressing PDX-1, BETA2/NeuroD and MafA Induces the Transdifferentiation of Porcine Neonatal Pancreas Cell Clusters and Adult Pig Pancreatic Cells into Beta-Cells.

BACKGROUND: A limitation in the number of insulin-producing pancreatic beta-cells is a special feature of diabetes. The identification of alternative sources for the induction of insulin-producing surrogate beta-cells is a matter of profound importance. PDX-1/VP16, BETA2/NeuroD, and MafA overexpression have been shown to influence the differentiation and proliferation of pancreatic stem cells. However, few studies have been conducted using adult animal pancreatic stem cells. METHODS: Adult pig pancreatic cells were prepared from the non-endocrine fraction of adult pig pancreata. Porcine neonatal pancreas cell clusters (NPCCs) were prepared from neonatal pigs aged 1-2 days. The dispersed pancreatic cells were infected with PDX-1/VP16, BETA2/NeuroD, and MafA adenoviruses. After infection, these cells were transplanted under the kidney capsules of normoglycemic nude mice. RESULTS: The adenovirus-mediated overexpression of PDX-1, BETA2/NeuroD and MafA induced insulin gene expression in NPCCs, but not in adult pig pancreatic cells. Immunocytochemistry revealed that the number of insulin-positive cells in NPCCs and adult pig pancreatic cells was approximately 2.6- and 1.1-fold greater than those in the green fluorescent protein control group, respectively. At four weeks after transplantation, the relative volume of insulin-positive cells in the grafts increased in the NPCCs, but not in the adult porcine pancreatic cells. CONCLUSION: These data indicate that PDX-1, BETA2/NeuroD, and MafA facilitate the beta-cell differentiation of NPCCs, but not adult pig pancreatic cells. Therefore PDX-1, BETA2/NeuroD, and MafA-induced NPCCs can be considered good sources for the induction of pancreatic beta-cells, and may also have some utility in the treatment of diabetes.