Cell-based Therapies For Diabetes Research Articles

Targeted mapping and utilization of the perihepatic surface for therapeutic beta cell replacement and retrieval in diabetic non-human primates

IntroductionSuccessful diabetes reversal using pancreatic islet transplantation by various groups illustrates the significant achievements made in cell-based diabetes therapy. While clinically, intraportal islet delivery is almost exclusively used, it is not without obstacles, including instant blood-mediated inflammatory reaction (IBMIR), relative hypoxia, and loss of function over time, therefore hindering long-term success. Here we demonstrate the perihepatic surface of non-human primates (NHPs) as a potential islet delivery site maximizing favorable characteristics, including proximity to a dense vascular network for adequate oxygenation while avoiding IBMIR exposure, maintenance of portal insulin delivery, and relative ease of accessibility through minimally invasive surgery or percutaneous means. In addition, we demonstrate a targeted mapping technique of the perihepatic surface, allowing for the testing of multiple experimental conditions, including a semi-synthetic hydrogel as a possible three-dimensional framework to improve islet viability.MethodsPerihepatic allo-islet cell transplants were performed in immunosuppressed cynomolgus macaques using a targeted mapping technique to test multiple conditions for biocompatibility. Transplant conditions included islets or carriers (including hydrogel, autologous plasma, and media) alone or in various combinations. Necropsy was performed at day 30, and histopathology was performed to assess biocompatibility, immune response, and islet viability. Subsequently, single-injection perihepatic allo-islet transplant was performed in immunosuppressed diabetic cynomolgus macaques. Metabolic assessments were measured frequently (i.e., blood glucose, insulin, C-peptide) until final graft retrieval for histopathology.ResultsTargeted mapping biocompatibility studies demonstrated mild inflammatory changes with islet-plasma constructs; however, significant inflammatory cell infiltration and fibrosis were seen surrounding sites with the hydrogel carrier affecting islet viability. In diabetic NHPs, perihepatic islet transplant using an autologous plasma carrier demonstrated prolonged function up to 6 months with improvements in blood glucose, exogenous insulin requirements, and HbA1c. Histopathology of these islets was associated with mild peri-islet mononuclear cell infiltration without evidence of rejection.DiscussionThe perihepatic surface serves as a viable site for islet cell transplantation demonstrating sustained islet function through 6 months. The targeted mapping approach allows for the testing of multiple conditions simultaneously to evaluate immune response to biomaterials at this site. Compared to traditional intraportal injection, the perihepatic site is a minimally invasive approach that allows the possibility for graft recovery and avoids IBMIR.

Genome editing for engineering stem cell-derived pancreatic β cells: recent trends and future perspectives

Rapid advancements in biotechnology have enabled the development of new therapeutic modalities for refractory diseases. Among these, stem cell-derived β (SC-β) cells are emerging as next-generation therapeutics for diabetes. However, several challenges must be overcome for these cell-based therapies to enter widespread use. Current SC-β cells exhibit functional immaturity, and transplanted cells face rejection by the host’s immune system. The cells also undergo apoptosis shortly after transplantation due to insufficient oxygen supply. Additionally, the use of stem cell-derived live cells may raise safety concerns, including the risk of teratoma formation. To address these challenges, genome-editing tools, particularly clustered regularly interspaced short palindromic repeats/Cas9 technologies, are being extensively explored owing to their ease of use, modularity, and robustness across diverse cell types. Herein, we provide a comprehensive overview of the various issues in the development of cell-based therapies for diabetes and discuss how genome-editing tools can enhance this emerging therapeutic modality. We also discuss the use of genome editing with SC-β cells for modeling genetic variants in diabetic β cells and correcting pathogenic variants to enable personalized diabetes therapy. Furthermore, we propose future research directions for leveraging genome-editing tools to improve the performance of cell-based therapeutics for diabetes.

P41: 4D Manufacturing: Immunoprotective Materials for Islet Tissue Patches in Cell-Based Diabetes Treatments

Purpose: In order to cure diabetes on a large scale, researchers are working to develop insulin-producing cells differentiated from human pluripotent stem cells, encapsulating the tissues to protect the cells from immune attack, and implanting the cells for regulated insulin secretion. This project explored the synthesis and in vivo performance of a 4D biomaterial intended to protect implanted insulin-producing cells in the body. 3D printing of insulin-producing cells encapsulated within the biomaterial with additional timed-release anti-inflammatory agents enabled complex, personalized shapes and a 4D functional material. Methods: Anti-inflammatory agents were functionalized with varying permanent and degradable linkages to achieve a range of timed-release therapeutics to prevent immune system-mediated rejection. The molecules were crosslinked with a multi-arm polyethylene glycol (PEG) material in the subcutaneous space of immunocompetent C57BL/6 mice. After 30 days, tissue slices of the explant were examined using H&E staining revealing the thickness of biological encapsulation compared to alginate controls. Results: PEG-based implants with acetal linkages enabling the slow release (~30 days) of anti-inflammatory agents achieved the thinnest layer of biological encapsulation compared to the controls. Summary: Collectively, the described 4D biomaterial with other novel approaches and tools will enable the investigation of survival and function of a stem cell-based therapy for diabetes that could ultimately allow patients to live a diabetes-free lifestyle.

Insulin-producing cell clusters derived from human gingival mesenchymal stem cells as a model for diabetes research.

The human gingiva-derived mesenchymal stem cells (hGMSCs) possess a great potential to develop the cell-based therapy for diabetes due to its unscarred healing capacity and reparative potential. In this current study, we isolated, cultured and characterised the GMSCs and explored their potential to differentiate into Insulin Producing Cell Clusters (IPCCs). The cells derived from gingival tissues exhibited fibroblast-like morphology. The flow cytometric analysis revealed positive expression of CD73(97.43%), CD90(95.05%), and CD105(93.17%) and negative expression of CD34(0.05%), CD45(0.09%), and HLA-DR (0.025) surface markers. We then converted this adherent fibroblast-like GMSCs into floating IPCCs using a sequential three-step protocol containing a different combination of differentiating agents. Initially, the presence of insulin in IPCCs was confirmed by dithizone staining. Glucose-stimulated insulin secretion (GSIS) assay confirmed that IPCCs secrete insulin in response to glucose. Generated IPCCs express pancreatic markers such as insulin, pdx1, glucagon, GLUT4 and GLUT2 as evidenced by RT-PCR analysis. Our results unequivocally showed that IPCCs can be generated from gingiva which is a potential source of postnatal MSCs. Our results offer the IPCCs generated from hGMSCs a platform for screening anti-diabetic drugs and a new autologous source of tissue for islet transplantation for the treatment of diabetes. Our results unequivocally demonstrate for the first time that hGMSCs can be used as an attractive non-invasive tissue source for generating IPCCs, which can be employed in diabetes research for screening antidiabetic agents and also for transplantation in type 1 diabetic patients as autologous source without the need of immunosuppression.

The Ldb1 transcriptional co-regulator is required for establishment and maintenance of the pancreatic endocrine lineage.

Pancreatic islet cell development is regulated by transcription factors (TFs) that mediate embryonic progenitor differentiation toward mature endocrine cells. Prior studies from our lab and others showed that the islet-enriched TF, Islet-1 (Isl1), interacts with the broadly-expressed transcriptional co-regulator, Ldb1, to regulate islet cell maturation and postnhyperatal function (by embryonic day (E)18.5). However, Ldb1 is expressed in the developing pancreas prior to Isl1 expression, notably in multipotent progenitor cells (MPCs) marked by Pdx1 and endocrine progenitors (EPs) expressing Neurogenin-3 (Ngn3). MPCs give rise to the endocrine and exocrine pancreas, while Ngn3+ EPs specify pancreatic islet endocrine cells. We hypothesized that Ldb1 is required for progenitor identity in MPC and EP populations during development to impact islet appearance and function. To test this, we generated a whole-pancreas Ldb1 knockout, termed Ldb1ΔPanc , and observed severe developmental and postnatal pancreas defects including disorganized progenitor pools, a significant reduction of Ngn3-expressing EPs, Pdx1HI β-cells, and early hormone+ cells. Ldb1ΔPanc neonates presented with severe hyperglycemia, hypoinsulinemia, and drastically reduced hormone expression in islets, yet no change in total pancreas mass. This supports the endocrine-specific actions of Ldb1. Considering this, we also developed an endocrine-enriched model of Ldb1 loss, termed Ldb1ΔEndo . We observed similar dysglycemia in this model, as well as a loss of islet identity markers. Through in vitro and in vivo chromatin immunoprecipitation experiments, we found that Ldb1 occupies key Pdx1 and Ngn3 promoter domains. Our findings provide insight into novel regulation of endocrine cell differentiation that may be vital toward improving cell-based diabetes therapies.

Standardization & analytical tool innovation in the cell & gene therapy field

David McCall, Editor, Cell and Gene Therapy Insights, speaks to Fouad Atouf, Vice President, Global Biologics, United States PharmacopeiaFouad Atouf is Vice President, Global Biologics, for USP. He leads all scientific activities related to the development and maintenance of documentary and reference standards for biologics and advanced therapies as well as the oversees biologics laboratories in USP–US and USP–India. Dr Atouf has been at USP for over 15 years and served in a variety of scientific leadership roles including being the regional champion for the Middle East and North Africa Region, where he helped facilitate, programs designed to enhance the understanding of the role of regulations and standards in the registration of medicinal products. Dr Atouf has a strong background and experience in the development and standardization of cellular and tissue-based products. Prior to joining USP in 2006, his research at the US National Institutes of Health focused on developing methods for the in vitro generation of cell-based therapies for diabetes. Dr Atouf is the author of numerous publications in peer-reviewed journals and a frequent speaker at national and international scientific conferences. Dr Atouf earned his Master’s degree in Biochemistry and his PhD in Cell Biology from the Pierre & Marie Curie University, Paris, France.

Inducible Pluripotent Stem Cells as a Potential Cure for Diabetes.

Over the last century, diabetes has been treated with subcutaneous insulin, a discovery that enabled patients to forego death from hyperglycemia. Despite novel insulin formulations, patients with diabetes continue to suffer morbidity and mortality with unsustainable costs to the health care system. Continuous glucose monitoring, wearable insulin pumps, and closed-loop artificial pancreas systems represent an advance, but still fail to recreate physiologic euglycemia and are not universally available. Islet cell transplantation has evolved into a successful modality for treating a subset of patients with ‘brittle’ diabetes but is limited by organ donor supply and immunosuppression requirements. A novel approach involves generating autologous or immune-protected islet cells for transplant from inducible pluripotent stem cells to eliminate detrimental immune responses and organ supply limitations. In this review, we briefly discuss novel mechanisms for subcutaneous insulin delivery and define their shortfalls. We describe embryological development and physiology of islets to better understand their role in glycemic control and, finally, discuss cell-based therapies for diabetes and barriers to widespread use. In response to these barriers, we present the promise of stem cell therapy, and review the current gaps requiring solutions to enable widespread use of stem cells as a potential cure for diabetes.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles in the Treatment of Diabetic Foot Ulcers: Application and Challenges.

Diabetic foot ischemia and ulcer (DFU) persists as a serious diabetes mellitus complication in spite of increased understanding of the pathophysiology and the cellular and molecular responses. Contributing to this pessimistic situation is the lack of effective treatments that are slow to heal the deep chronic wounds and microvascular obstruction. Mesenchymal stromal cells (MSCs) have been tested as a promising cell-based therapy for diabetes in vitro and in vivo, which is able to accelerate wound closure with increased epithelialization, granulation tissue formation and angiogenesis by differentiation into skin cells and paracrine pathways to repair injured cells. The secretomes of MSCs, including cytokines, growth factors, chemokines, and extracellular vesicles containing mRNA, proteins and microRNAs, have immunomodulatory and regenerative effects. This review will shed new light on the therapeutic potential of MSC-derived extracellular vesicles (MSC-EVs) for the treatment of diabetes-induced lower limb ischemia and ulcers. The identification of underlying mechanisms for MSC-EVs regulation on impaired diabetic wound healing might provide a new direction for MSC-centered treatment for diabetic lower limb ischemia and ulcers. Immunomodulatory and angiogenic effects of MSC-derived extracellular vesicles on diabetic foot ulcer.

High-Dimensional Design-Of-Experiments Extracts Small-Molecule-Only Induction Conditions for Dorsal Pancreatic Endoderm from Pluripotency.

SummaryThe derivation of endoderm and descendant organs, such as pancreas, liver, and intestine, impacts disease modeling and regenerative medicine. Use of TGF-β signaling agonism is a common method for induction of definitive endoderm from pluripotency. By using a data-driven, High-Dimensional Design of Experiments (HD-DoE)-based methodology to address multifactorial problems in directed differentiation, we found instead that optimal conditions demanded BMP antagonism and retinoid input leading to induction of dorsal foregut endoderm (DFE). We demonstrate that pancreatic identity can be rapidly, and robustly, induced from DFE and that such cells are of dorsal pancreatic identity. The DFE population was highly competent to differentiate into both stomach organoids and pancreatic tissue types and able to generate fetal-type β cells through two subsequent differentiation steps using only small molecules. This alternative, rapid, and low-cost basis for generating pancreatic insulin-producing cells may have impact for the development of cell-based therapies for diabetes.

Effect of FIGF overexpression on liver cells transforming to insulin-producing cells

Limitation in the number of insulin-producing pancreatic β-cells is a typical feature of diabetes. It has been indicated that activating pancreatic transcription factors can promote the transformation of hepatocytes into insulin-secreting β-like cells, indicating that direct hepatocyte differentiation seems promising as a treatment for diabetes. Nevertheless, the reprogramming efficiency still remains low. Our previous study found that the expression of c-fos-induced growth factor (FIGF) was increased in the pancreatic tissues in partial pancreatectomy mice compared to that in normal mice. Here, we observed that treatment with Ad-FIGF was found to enhance MafA and Ngn3-induced reprogramming of BNL CL.2 cells to β-like cells with the ability of secreting insulin. And FIGF overexpression increased the levels of histone H3/H4 acetylation at MafA and Ngn3 promoter regions in BNL CL.2 cells. Importantly, in vivo study further confirmed that forced expression of FIGF facilitated the insulin expression and decreased the blood glucose levels in STZ mice. These results strengthen the possibility of developing cell-based therapies for diabetes through utilizing β-like cells derived from non-insulin-secreting cells.

Live Cell Monitoring and Enrichment of Stem Cell-Derived β Cells Using Intracellular Zinc Content as a Population Marker.

Our laboratory and others have developed protocols to generate glucose‐responsive stem cell–derived β cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell‐based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell–derived β cells and gene‐editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell–derived β cells. This purification of SC‐β cells is based on intracellular zinc content and is a simple method to complement other approaches for generating and assaying these cells. © 2019 The Authors. Basic Protocol: Fluorescent labeling and isolation of stem cell‐derived β cells

Tea for type 1 and type 2 diabetes

Cell Engineering Cell therapy is a promising approach for treating diabetes. Yin et al. developed an elegant control system by engineering cells to respond to protocatechuic acid, a metabolite in green tea. Orally ingested protocatechuic acid regulated blood glucose by triggering secretion of insulin or a short variant of human glucagon–like peptide 1 from engineered cells implanted in mouse and nonhuman primate models of type 1 and type 2 diabetes. This study demonstrates the versatility of synthetic biology for developing remotely controlled cell-based therapies for diabetes. Sci. Transl. Med. 11 , eaav8826 (2019).

Sufu- and Spop-mediated downregulation of Hedgehog signaling promotes beta cell differentiation through organ-specific niche signals

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However, efficient stem cell to beta cell differentiation has proven difficult, possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals, we isolated pancreatic and gastrointestinal stromal cells, and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas, whereas stroma-specific activation of signaling via loss of Hedgehog regulators, Sufu and Spop, impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly, inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation, altogether revealing the requirement for organ-specific regulation of stromal niche signals.

Small molecules and extrinsic factors promoting differentiation of stem cells into insulin-producing cells

In the modern world, type-2 diabetes mellitus has become a leading public healthcare problem, due to major risks of morbidity and mortality. Prevalence has increased significantly in recent decades. Treatment involves oral hypoglycemic agents or insulin replacement therapy. Development is ongoing for cell-based diabetes therapies using stem cells with the potential to differentiate into insulin-producing cells (IPCs): embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and stem cells from adult pancreas, liver, central nervous system, bone marrow and adipose tissue. Successful induction of iPSCs, however, depends on the quantity and quality of available stem cells and the development of adapted protocols determining the environment of extrinsic factors and involvement of small molecules. Validating such new cell therapies must be founded on this experimental rationale.

Co-Transplant of Parathyroid Gland and Stem Cell-Derived Insulin-Producing Cells Enhances Graft Survival through Release of Pro-Angiogenic and Pro-Survival Factors

Introduction Ischemia dramatically impairs survival and function of islets after transplantation. Human Stem Cell-derived Beta Cells are currently being tested in clinical trials for replacement therapy. Although stem cell technology represents an unlimited source of beta cells, it is paramount to reduce the effects of ischemia to optimize outcome. Parathyroid glands (PTG) are known to engraft in the subcutaneous and intra-muscular sites with high success rate. The objective of this study is to investigate if PTG can protect beta-cell grafts. We hypothesize that PTG protect beta cells by both accelerating neo-vasculogenesis and by producing tropic factors that sustain beta cell viability before revascularization. Methods Stem Cell-derived Beta Cells were produced from a human embryonic stem cell line with a GFP transgene knocked into the insulin locus and a constitutively expressed luciferase transgene. Insulin-producing GFP+ cells were enriched by FACS and reaggregated to form enhanced beta clusters (eBCs). eBC were transplanted under the subcutaneous space (SQ) of NSG mice and graft mass was monitored using bioluminescence imaging. Human PTG were finely cut and co-transplanted with eBC. PTG-derived CD34+ and CD34- cells were FACS purified, cultured for 24 hours and supernatants were assessed for cytokines using Luminex. Effects of PTG-derived factors on eBCs viability after challenges of hypoxia and nutrient deprivation were assessed in vitro. Results and Discussion eBC graft mass decreased by 60% in two weeks after transplant under the SQ. However, when eBC grafts were co-transplanted with PTG, bioluminescent signal increased up to 150% (Figure 1 A and B). We further investigated the mechanisms of PTG protection. We found PTG-derived CD34+ cells produced CCL2, CXCL12, VEGF-A, and PDGF-AA (Figure 2), which have all been previously implicated in neo-vasculogenesis. Consistently, PTG co-transplant led to a dramatic increase in the ingrowth of CD31+ cells. Additionally, PTG secreted parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and γ-aminobutyric acid (GABA). These factors improved eBC survival during in vitro-simulated ischemia challenge using nutrient-deprivation and/or hypoxia (Figure 3). Conclusions Our results show that PTG co-transplantation preserves beta cell graft survival. PTG-derived CD34+ cells promote neovascularization of eBC grafts by producing angiogenic factors. PTG may also have direct cytoprotective effects on eBCs by producing PTH, PTHrP, and GABA. These properties of PTG may be harnessed to minimize ischemic injury to eBC after transplant to realize the therapeutic potential of stem cell-based therapy for diabetes.

Monogenic diabetes: Implementation of translational genomic research towards precision medicine.

Various forms of early onset non-autoimmune diabetes are recognized as monogenic diseases, each subtype being caused by a single highly penetrant gene defect at the individual level. Monogenic diabetes (MD) is clinically and genetically heterogeneous, including maturity onset diabetes of the young and infancy-onset and neonatal diabetes mellitus, which are characterized by functional defects of insulin-producing pancreatic β-cells and hyperglycemia early in life. Depending on the genetic cause, MD differs in the age at diabetes onset, the severity of hyperglycemia, long-term diabetic complications, and extrapancreatic manifestations. In this review we discuss the many challenges of molecular genetic diagnosis of MD in the face of a substantial genetic heterogeneity, as well as the clinical benefit and cost-effectiveness of an early genetic diagnosis, as demonstrated by simulation models based on lifetime complications and treatment costs. We also discuss striking examples of proof-of-concept of genomic medicine, which have enabled marked improvement in patient care and long-term clinical management. Recent advances in genome editing and pluripotent stem cell reprogramming technologies provide new opportunities for in vitro diabetes modeling and the discovery of novel drug targets and cell-based diabetes therapies. A review of these future directions makes the case for exciting translational research to further our understanding of the pathophysiology of early onset diabetes.

Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation—Pdx1, Neurogenin3, and MafA—efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2′-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2′-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Minireview: Directed Differentiation and Encapsulation of Islet β-Cells-Recent Advances and Future Considerations.

Diabetes mellitus has rapidly become a 21st century epidemic with the promise to create vast economic and health burdens, if left unchecked. The 2 major forms of diabetes arise from unique causes, with outcomes being an absolute (type 1) or relative (type 2) loss of functional pancreatic islet β-cell mass. Currently, patients rely on exogenous insulin and/or other pharmacologies that restore glucose homeostasis. Although these therapies have prolonged countless lives over the decades, the striking increases in both type 1 and type 2 diabetic diagnoses worldwide suggest a need for improved treatments. To this end, islet biologists are developing cell-based therapies by which a patient's lost insulin-producing β-cell mass is replenished. Pancreatic or islet transplantation from cadaveric donors into diabetic patients has been successful, yet the functional islet demand far surpasses supply. Thus, the field has been striving toward transplantation of renewable in vitro-derived β-cells that can restore euglycemia. Challenges have been numerous, but progress over the past decade has generated much excitement. In this review we will summarize recent findings that have placed us closer than ever to β-cell replacement therapies. With the promise of cell-based diabetes therapies on the horizon, we will also provide an overview of cellular encapsulation technologies that will deliver critical protection of newly implanted cells.

Swertisin an Anti-Diabetic Compound Facilitate Islet Neogenesis from Pancreatic Stem/Progenitor Cells via p-38 MAP Kinase-SMAD Pathway: An In-Vitro and In-Vivo Study.

Transplanting islets serves best option for restoring lost beta cell mass and function. Small bio-chemical agents do have the potential to generate new islets mass, however lack of understanding about mechanistic action of these small molecules eventually restricts their use in cell-based therapies for diabetes. We recently reported “Swertisin” as a novel islet differentiation inducer, generating new beta cells mass more effectively. Henceforth, in the present study we attempted to investigate the molecular signals that Swertisin generate for promoting differentiation of pancreatic progenitors into islet cells. To begin with, both human pancreatic progenitors (PANC-1 cells) and primary cultured mouse intra-islet progenitor cells (mIPC) were used and tested for Swertisin induced islet neogenesis mechanism, by monitoring immunoblot profile of key transcription factors in time dependent manner. We observed Swertisin follow Activin-A mediated MEPK-TKK pathway involving role of p38 MAPK via activating Neurogenin-3 (Ngn-3) and Smad Proteins cascade. This MAP Kinase intervention in differentiation of cells was confirmed using strong pharmacological inhibitor of p38 MAPK (SB203580), which effectively abrogated this process. We further confirmed this mechanism in-vivo in partial pancreatectomised (PPx) mice model, where we could show Swertisin exerted potential increase in insulin transcript levels with persistent down-regulation of progenitor markers like Nestin, Ngn-3 and Pancreatic Duodenal Homeobox Gene-1 (PDX-1) expression, within three days post PPx. With detailed molecular investigations here in, we first time report the molecular mode of action of Swertisin for islet neogenesis mediated through MAP Kinase (MEPK-TKK) pathway involving Ngn-3 and Smad transcriptional regulation. These findings held importance for developing Swertisin as potent pharmacological drug candidate for effective and endogenous differentiation of islets in cell based therapy for diabetes.

Development of large-scale size-controlled adult pancreatic progenitor cell clusters by an inkjet-printing technique.

The generation of transplantable β-cells from pancreatic progenitor cells (PPCs) could serve as an ideal cell-based therapy for diabetes. Because the transplant efficiency depends on the size of islet-like clusters, it becomes one of the key research topics to produce PPCs with controlled cluster sizes in a scalable manner. In this study, we used inkjet printing to pattern biogenic nanoparticles, i.e., mutant tobacco mosaic virus (TMV), with different spot sizes to support the formation of multicellular clusters by PPCs. We successfully achieved TMV particle patterns with variable features and sizes by adjusting the surface wettability and printing speed. The spot sizes of cell-adhesive TMV mutant arrays were in the range of 50-150 μm diameter. Mouse PPCs were seeded on the TMV-RGD (arginine-glycine-aspartate)-patterned polystyrene (PS) substrate, which consists of areas that either favor (TMV-RGD) or prohibit (bare PS) cell adhesion. The PPCs stably attached, proliferated on top of the TMV-RGD support, thus resulting in the formation of uniform and confluent PPC clusters. Furthermore, the aggregated PPCs also maintained their multipotency and were positive for E-cadherin, indicating that the formation of cell-cell junctions is critical for enhanced cell-cell contact.