Regenerative Medicine For Diabetes Research Articles

Tissue engineering of decellularized pancreas scaffolds for regenerative medicine in diabetes.

Diabetes mellitus is a global disease requiring long-term treatment and monitoring. At present, pancreas or islet transplantation is the only reliable treatment for achieving stable euglycemia in Type I diabetes patients. However, the shortage of viable pancreata for transplantation limits the use of this therapy for the majority of patients. Organ decellularization and recellularization is emerging as a promising solution to overcome the shortage of viable organs for transplantation by providing a potential alternative source of donor organs. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have been performed, and show promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we provide an overview of the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with an outlook on the remaining work that needs to be done in order to realize the goal of using this technology to create bioengineered pancreata for transplantation in diabetes patients. STATEMENT OF SIGNIFICANCE: Pancreas or islet transplantation is a means of providing insulin-independence in diabetes patients. However, due to the shortage of viable pancreata, whole-organ decellularization and recellularization is emerging as a promising solution to overcome organ shortage for transplantation. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have shown promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we highlight the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with future work that needs to be done in order to realize clinical translation of bioengineered pancreata for transplantation in diabetes patients.

Pancreatic Organoids for Regenerative Medicine and Cancer Research.

In recent years, the development of ex vivo organoid cultures has gained substantial attention as a model to study regenerative medicine and diseases in several tissues. Diabetes and pancreatic ductal adenocarcinoma (PDAC) are the two major devastating diseases affecting the pancreas. Suitable models for regenerative medicine in diabetes and to accurately study PDAC biology and treatment response are essential in the pancreatic field. Pancreatic organoids can be generated from healthy pancreas or pancreatic tumors and constitute an important translational bridge between in vitro and in vivo models. Here, we review the rapidly emerging field of pancreatic organoids and summarize the current applications of the technology to tissue regeneration, disease modelling, and drug screening.

Generation of Functional Insulin-Producing Cells from Mouse Embryonic Stem Cells Through Protein Transduction of Transcription Factors.

In this chapter, we describe a simple and unique method for the differentiation of mouse embryonic stem cells into insulin-producing cells. In addition to cytokines and growth factors, key transcription factors for pancreatic development are applied in this method through protein transduction technology. Furthermore, a combination of nanofiber plates and laminin coatings improves the yield of differentiated cells. The insulin-producing cells derived through this method express marker genes of mature β-cells and have an ability to secrete insulin; therefore, these cells are useful for fundamental studies on pancreatic development, drug development, and regenerative medicine for diabetes.

Characterization of a Vimentinhigh /Nestinhigh proteome and tissue regenerative secretome generated by human pancreas-derived mesenchymal stromal cells.

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentinhigh /Nestinhigh cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease.

Generation of functional human pancreatic organoids by transplants of embryonic stem cell derivatives in a 3D-printed tissue trapper.

Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three-dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell derivatives including ES-derived pancreatic duodenal homeobox 1 (PDX1) + early PP, mesenchymal stem cells, and endothelial cells at an optimized cell ratio, on Matrigel. The PO was placed in a 3D-printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicated that, in contrast to corresponding early PP transplants, 3D PO developed more vascularization as indicated by greater area and number of vessels, a higher number of insulin-positive cells and improvement of human C-peptide secretions. Based on our findings, PO-derived β cells could be considered a novel strategy to study human β-cell development, novel therapeutics, and regenerative medicine for diabetes.

High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O2) conditions. Treatment with a high O2 condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O2 condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O2 condition activated Wnt signaling. Optimal stage-specific treatment with a high O2 condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O2 condition at a specific stage is useful for the efficient generation of insulin-producing cells.

Insulin-Producing Cells Derived from Human Embryonic Stem Cells: Comparison of Definitive Endoderm- and Nestin-Positive Progenitor-Based Differentiation Strategies

Human embryonic stem cells (hESCs) are pluripotent and capable of undergoing multilineage differentiation into highly specialized cells including pancreatic islet cells. Thus, they represent a novel alternative source for targeted therapies and regenerative medicine for diabetes. Significant progress has been made in differentiating hESCs toward pancreatic lineages. One approach is based on the similarities of pancreatic β cell and neuroepithelial development. Nestin-positive cells are selected as pancreatic β cell precursors and further differentiated to secrete insulin. The other approach is based on our knowledge of developmental biology in which the differentiation protocol sequentially reproduces the individual steps that are known in normal β cell ontogenesis during fetal pancreatic development. In the present study, the hESC cell line PKU1.1 was induced to differentiate into insulin-producing cells (IPCs) using both protocols. The differentiation process was dynamically investigated and the similarities and differences between both strategies were explored. Our results show that IPCs can be successfully induced with both differentiation strategies. The resulting IPCs from both protocols shared many similar features with pancreatic islet cells, but not mature, functional β cells. However, these differently-derived IPC cell types displayed specific morphologies and different expression levels of pancreatic islet development-related markers. These data not only broaden our outlook on hESC differentiation into IPCs, but also extend the full potential of these processes for regenerative medicine in diabetes.

Current status of regeneration of pancreatic β-cells.

Newly generated insulin-secreting cells for use in cell therapy for insulin-deficient diabetes mellitus require properties similar to those of native pancreatic β-cells. Pancreatic β-cells are highly specialized cells that produce a large amount of insulin, and secrete insulin in a regulated manner in response to glucose and other stimuli. It is not yet explained how the β-cells acquire this complex function during normal differentiation. So far, in vitro generation of insulin-secreting cells from embryonic stem cells, induced-pluripotent stem cells and adult stem/progenitor-like cells has been reported. However, most of these cells are functionally immature and show poor glucose-responsive insulin secretion compared to that of native pancreatic β-cells (or islets). Strategies to generate functional β-cells or a whole organ in vivo have also recently been proposed. Establishing a protocol to generate fully functional insulin-secreting cells that closely resemble native β-cells is a critical matter in regenerative medicine for diabetes. Understanding the physiological processes of differentiation, proliferation and regeneration of pancreatic β-cells might open the path to cell therapy to cure patients with absolute insulin deficiency.

GATA6 inactivating mutations are associated with heart defects and, inconsistently, with pancreatic agenesis and diabetes

To the Editor: Pancreatic agenesis is an extremely rare cause of permanent neonatal diabetes mellitus (NDM) in humans. It can be associated with severe intrauterine growth retardation as well as a plethora of abnormalities or malformations in the heart, biliary tract, gut, thyroid or brain [1–3]. To date, mutations in three genes have been shown to cause pancreatic agenesis, namely, PDX1 [1], PTF1A [2], and most recently GATA6 [3], all three of which encode transcription factors. The GATA6 study reported heterozygous coding mutations in a dozen individuals with permanent NDM presumed to be due to pancreatic agenesis [3]. The genetic evidence supporting the pathogenicity of these mutations seemed quite strong as the reported mutations had arisen de novo and none of these mutations was present in the 1,000 Genome Project database [3]. The authors concluded that GATA6 may have a key role in human pancreatic development, with strong implications for beta cell regenerative medicine in diabetes [3]. In the

Pluripotent Stem Cell-Derived Pancreatic β-Cells: Potential for Regenerative Medicine in Diabetes

Diabetes mellitus, which affects 346 million people, is one of the leading causes of death worldwide. Pancreatic β-cells, existing in the islets of Langerhans, play central roles in the progression of diabetes. An efficient strategy to produce functional pancreatic β-cells is important for both transplantation therapy and disease modeling of diabetes. Human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, provide unlimited starting materials to generate differentiated cells for regenerative studies. Significant progress has been made in human embryonic/induced pluripotent stem cell differentiation in the last several years. However, efficient generation of mature pancreatic β-cells with complete functional capabilities has not yet been accomplished. Here, we review recent successes as well as the technical and theoretical challenges in the use of pluripotent stem cell-derived pancreatic β-cells for disease modeling and replacement therapy of diabetes.

Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

BackgroundRegarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.Methodology/Principal FindingsIn the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin.Conclusions/SignificanceProteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.