Background Insulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation. Results The transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process. Conclusions Transdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.

Diabetes mellitus (DM) is a complex metabolic disease with many specifically related complications. Early diagnosis of this disease could prevent the progression to overt disease and its related complications. There are several limitations to using existing biomarkers, and between 24% and 62% of people with diabetes remain undiagnosed and untreated, suggesting a large gap in current diagnostic practices. Early detection of the percentage of insulin-producing cells preceding loss of function would allow for effective therapeutic interventions that could delay or slow down the onset of diabetes. MicroRNAs (miRNAs) could be used for early diagnosis, as well as for following the progression and the severity of the disease, due to the fact of their pancreatic specific expression and stability in various body fluids. Thus, many studies have focused on the identification and validation of such groups or "signatures of miRNAs" that may prove useful in diagnosing or treating patients. Here, we summarize the findings on miRNAs as biomarkers in diabetes and those associated with direct cellular reprogramming strategies, as well as the relevance of miRNAs that act as a bidirectional switch for cell therapy of damaged pancreatic tissue and the studies that have measured and tracked miRNAs as biomarkers in insulin resistance are addressed.

Autologous cells replacement therapy by liver to pancreas transdifferentiation (TD) allows diabetic patients to be also the donors of their own therapeutic tissue. Aim: To analyze whether the efficiency of the process is affected by liver donors' heterogeneity with regard to age, gender and the metabolic state. Materials & methods: TD of liver cells derived from nondiabetic and diabetic donors at different ages was characterized at molecular and cellular levels, in vitro. Results: Neither liver cells proliferation nor the propagated cells TD efficiency directly correlate with the age (3-60 years), gender or the metabolic state of the donors. Conclusion: Human liver cells derived from a wide array of ages and metabolic states can be used for autologous cells therapies for diabetics.

Background: Liver cells represent an attractive source of cells for autologous regenerative medicine. The present study assesses the liver cells' stability during in vitro expansion, as a prerequisite for therapeutic use. Results: The human liver cell cultures in this study were propagated efficiently in vitro for at least 12 passages. No significant changes in morphology, intracellular ultrastructures and characteristic markers expression were found during in vitro expansion of cells from all analyzed donors. However, expanded cells derived from male donors of >60 years old, lost the Y chromosome. Conclusion: Liver-derived cell cultures adopt a proliferative, stable mesenchymal phenotype, through an epithelial to mesenchymal transition process. The molecular and phenotypic changes of the cells during propagation are uniform, despite the heterogeneity of the different donors. Loss of Y chromosome occurs after cells' propagation in elder male donors.

Background: Although pancreatic islet transplantation therapy is ideal for diabetes patients, several hurdles have prevented it from becoming a standard treatment, including donor shortage and low engraftment efficacy. In this study, we prepared insulin-producing cells trans-differentiated from adult human liver cells as a new islet source.Also, cell sheets formation couldimprove differentiation efficiencyand graft survival. Methods: Liver cells were expanded in vitro and trans-differentiated to IPCs using adenovirus vectors carrying human genes for PDX1 , NEUROD1 and MAFA . IPCs were seeded on temperature-responsive culture dishes to form cell sheets. Differentiation efficiency were confirmed by ß cell-specific gene expression, insulin production, and immunohistochemistry. IPCs suspension was injected by portal vein (PV), and IPCs sheet was transplanted on the liver surface of the diabetic nude mouse.The therapeutic effect of IPC sheet was evaluated by comparing blood glucose control, weight gain, histological evaluation and hepatotoxicity with IPCs injection group. Also, cell biodistribution was assessed by in vivo / ex vivo fluorescence image tagging. Results: Insulin gene expression and protein production were significantly increased on IPC sheets compared with those in IPCs cultured on conventional culture dishes. Transplanted IPC sheets displayed significantly higher engraftment efficiency and fewer transplanted cells in other organs than injected IPCs, and also lower liver toxicity, improved blood glucose levels, and weight gain. Immunohistochemical analyses of liver tissue revealed positive staining for PDX1 and insulin at 1, 2 and 4 weeks after IPCs transplantation. Conclusions: In conclusion, cell sheet formation enhanced the differentiation function and maturation of IPCs in vitro . Additionally, parameters for clinical application such as distribution, therapeutic efficacy, and toxicity were favorable. The cell sheet technique may be used with IPCs derived from various cell sources in clinical applications.

Pancreatic islet transplantation is the fundamental treatment for insulin-dependent diabetes; however, donor shortage is a major hurdle in its use as a standard treatment. Accordingly, differentiated insulin-producing cells (DIPCs) are being developed as a new islet source. Differentiation efficiency could be enhanced if the spheroid structure of the natural islets could be recapitulated. Here, we fabricated DIPC spheroids using concave microwells, which enabled large-scale production of spheroids of the desired size. We prepared DIPCs from human liver cells by trans-differentiation using transcription factor gene transduction. Islet-related gene expression and insulin secretion levels were higher in spheroids compared to those in single-cell DIPCs, whereas actin–myosin interactions significantly decreased. We verified actin–myosin-dependent insulin expression in single-cell DIPCs by using actin–myosin interaction inhibitors. Upon transplanting cells into the kidney capsule of diabetic mouse, blood glucose levels decreased to 200 mg/dL in spheroid-transplanted mice but not in single cell-transplanted mice. Spheroid-transplanted mice showed high engraftment efficiency in in vivo fluorescence imaging. These results demonstrated that spheroids fabricated using concave microwells enhanced the engraftment and functions of DIPCs via actin–myosin-mediated cytoskeletal changes. Our strategy potentially extends the clinical application of DIPCs for improved differentiation, glycemic control, and transplantation efficiency of islets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the caused disease - coronavirus disease 2019 (COVID-19), has affected so far >6,000,000 people worldwide, with variable grades of severity, and has already inflicted >350,000 deaths. SARS-CoV-2 infection seems severely affected by background diseases such as diabetes mellitus and its related complications, that seem to be favoring the most severe manifestations of SARS-CoV-2 and, therefore, require special attention in clinical care units. The present literature review focus on addressing several hypotheses explaining why diabetic patients could develop multi-organ failure in severe acute respiratory syndrome coronavirus (SARS-CoV) infections. Undoubtedly, as diabetes related complications are present it is expected to emphasize the severity of the COVID-19. Dermatological complications can occur and worsen in diabetic patients, and diseases such as acanthosis nigricans and psoriasis are prone to more severe manifestations of COVID-19. Approaches to treat SARS-CoV-2 infected patients, based on different solutions i.e. plasma therapy, use of antiviral compounds, development of vaccines or new therapeutic agents are ongoing.

Transdifferentiation is the direct reprogramming of adult cells into alternate cell types with different function. Liver to pancreas transdifferentiation (TD) induced by ectopic expression of pancreatic transcription factors (pTFs) was first described by our group both in vivo (1) and in human liver cells in vitro (2). Aim: Disclose the mechanism that mediate the developmental reprogramming process of adult human liver cells into endocrine pancreatic cells. Determine the developmental barriers that restrict this process efficiency and suggest modalities to increase the process efficiency. Finally, the clinical and industrial translation of adult cells reprogramming will be discussed.

Background Insulin-dependent diabetes is a multifactorial disorder that could be theoretically cured by functional pancreatic islets and insulin-producing cell (IPC) implantation. Regenerative medicine approaches include the potential for growing tissues and organs in the laboratory and transplanting them when the body cannot heal itself. However, several obstacles remain to be overcome in order to bring regenerative medicine approach for diabetes closer to its clinical implementation; the cells generated in vitro are typically of heterogenic and immature nature and the site of implantation should be readily vascularized for the implanted cells to survive in vivo. The present study addresses these two limitations by analyzing the effect of co-implanting IPCs with vasculature promoting cells in an accessible site such as subcutaneous. Secondly, it analyzes the effects of reconstituting the in vivo environment in vitro on the maturation and function of insulin-producing cells. Methods IPCs that are generated by the transdifferentiation of human liver cells are exposed to the paracrine effects of endothelial colony-forming cells (ECFCs) and human bone marrow mesenchymal stem cells (MSCs), which are the “building blocks” of the blood vessels. The role of the vasculature on IPC function is analyzed upon subcutaneous implantation in vivo in immune-deficient rodents. The paracrine effects of vasculature on IPC maturation are analyzed in culture. Results Co-implantation of MSCs and ECFCs with IPCs led to doubling the survival rates and a threefold increase in insulin production, in vivo. ECFC and MSC co-culture as well as conditioned media of co-cultures resulted in a significant increased expression of pancreatic-specific genes and an increase in glucose-regulated insulin secretion, compared with IPCs alone. Mechanistically, we demonstrate that ECFC and MSC co-culture increases the expression of CTGF and ACTIVINβα, which play a key role in pancreatic differentiation. Conclusions Vasculature is an important player in generating regenerative medicine approaches for diabetes. Vasculature displays a paracrine effect on the maturation of insulin-producing cells and their survival upon implantation. The reconstitution of the in vivo niche is expected to promote the liver-to-pancreas transdifferentiation and bringing this cell therapy approach closer to its clinical implementation.

Here, we review recent findings in the field of generating insulin-producing cells by pancreatic transcription factor (pTF)-induced liver transdifferentiation (TD). TD is the direct conversion of functional cell types from one lineage to another without passing through an intermediate stage of pluripotency. We address potential reasons for the restricted efficiency of TD and suggest modalities to overcome these challenges, to bring TD closer to its clinical implementation in autologous cell replacement therapy for insulin-dependent diabetes. Liver to pancreas TD is restricted to cells that are a priori predisposed to undergo the developmental process. In vivo, the predisposition of liver cells is affected by liver zonation and hepatic regeneration. The TD propensity of liver cells is related to permissive epigenome which could be extended to TD-resistant cells by specific soluble factors. An obligatory role for active Wnt signaling in continuously maintaining a “permissive” epigenome is suggested. Moreover, the restoration of the pancreatic niche and vasculature promotes the maturation of TD cells along the β cell function. Future studies on liver to pancreas TD should include the maturation of TD cells by 3D culture, the restoration of vasculature and the pancreatic niche, and the extension of TD propensity to TD-resistant cells by epigenetic modifications. Liver to pancreas TD is expected to result in the generation of custom-made “self” surrogate β cells for curing diabetes.

Cell and gene therapies have the potential to revolutionize medicine. Bioprocessing for these therapies still faces many challenges during scale-up. The utilization of scale-down unit operations from bioreactors to downstream purification is very useful in defining the design space and preventing costly scale-up errors. Two case studies focusing on viral vectors will be presented to demonstrate the challenges and solutions associated with process development of such therapeutics.

Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta-like, insulin-producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver-to-pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD-predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral-specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD-predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD-resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. Conclusion: Liver-to-pancreas TD occurs in defined predisposed cells. These cells' predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).

Cell-based therapies for the treatment of diabetes, generally aim to provide long-term glucose regulated-insulin delivery using insulin producing cells. The delivery platform is crucial for the therapeutic outcome as well as for immunoisolation of the entrapped cells. We have developed a novel artificial pancreas encapsulation platform for the treatment of diabetes that is based on solubilized whole porcine pancreatic extracellular matrix (ECM). These unique capsules were used to entrap human liver cells and mesenchymal stem cells that were induced to differentiate into glucose-regulated insulin-producing cells. We demonstrate that the ECM-microcapsule platform provides a natural fibrous 3D niche, supporting cell viability and differentiation, while significantly improving insulin delivery. In vivo, ECM-encapsulated cells were shown to be non-immunogenic, and most importantly, to significantly improve the glycemic control in diabetic mouse preclinical model, thus establishing a proof-of-concept for this new cell-based insulin delivery platform.

Diabetes is a major international health problem with over 370 million diabetics globally and an estimated 552 million by 2030. Orgenesis has developed an autologous cell therapy approach that allows the diabetic patient to be a donor of their own therapeutic tissue. Starting with a small biopsy from a patient's liver, the biopsied cells are expanded in flatware, trans-differentiated into autologous insulin producing (AIP) cells using adenovirus vectors and then infused back into the patient for long term amelioration of insulin dependency. To commercialize this cell therapy approach, Orgenesis and Pall have combined their respective expertise to develop a manufacturing process for both autologous cell expansion and viral vector production using two single-use large-scale bioreactor systems. Using the Xpansion® 200 single-used bioreactor, Orgenesis now has a robust, industrialized process to amplify the human adult liver-derived cells from 10 million per biopsy to 1.8 billion cells required to treat a diabetic patient . For large scale adenovirus production, the fixed-bed iCellis® 500 single-used bioreactor is used. . Using iCELLis®, an optimized manufacturing processes for three adenovirus vectors has been developed using the predictive small-scale iCellis® Nano bioreactor which was then scaled to the large-scale iCellis® 500 bioreactor. A high yield of 1.0 × 1016 total infectious virus particles was produced in an iCellis® 500 bioreactor using a bioreactor with 66 m2 of cell growth surface area by optimizing various key process parameters. The crude harvest material was purified by a Pall developed industrial purification downstream process. The purified adenovirus were fully functional and successfully transduced the target liver cells. As a result, Orgenesis is moving forward with this process for pre-clinical and clinical testing of AIP cells for the treatment of diabetes.

Diabetes is a major global health problem with the WHO reporting over 422 million affected persons and 3.7 million diabetes related deaths annually worldwide. Orgenesis is developing a cell therapy approach that genetically modifies a patient's own liver cells to secrete insulin. To bring this approach into pharmaceutical development, Orgenesis and Pall have combined their respective expertise to develop a manufacturing strategy for large scale adenoviral vector production in the packed-bed iCellis® 500 single-use bioreactor together with the development of a downstream purification industrialized process. In this study, we describe scale-up of the optimized adenovirus serotype 5 production process that was developed using a predictive small scale iCellis® Nano bioreactor. By optimizing culture and infection parameters such as HEK293 cell seeding density, multiplicity of infection, time of infection, day of harvest and media circulation parameters, yield was increased from 1.6E9 to 1.4E10 infectious virus particles per cm2 fixed bed. This yield makes the iCellis® bioreactor a promising scalable technology for the production of adenovirus products. The current density gradient based purification method for adenovirus is a time consuming, inefficient and non-manufacturing process. Here, an optimized rapid AEX membrane chromatography step was used for purification. Following depth and sterile filtration, clarified harvest is processed over a Mustang Q membrane in bind/elute mode. Eluted material is immediately concentrated and buffer exchanged into the final formulation buffer. Final product is then sterile filtered and vialed for potency studies. Purified adenovirus hPDX-1 was fully functional and successfully transduced the target liver derived cells. The next step of the study will incorporate the viral trans-differentiation step into the cGMP autologous patient cell expansion process in Pall's Xpansion® 200 bioreactor.