Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..

Generation of Human Stem Cell-Derived Pancreatic Organoids (POs) for Regenerative Medicine.

Insulin-dependent diabetes mellitus or type 1 diabetes mellitus (T1DM) is an auto-immune condition characterized by the loss of pancreatic ß-cells. The curative approach for highly selected patients is the pancreas or the pancreatic islet transplantation. Nevertheless, these options are limited by a growing shortage of donor organs and by the requirement of immunosuppression.Xenotransplantation of porcine islets has been extensively investigated. Nevertheless, the strong xenoimmunity and the risk of transmission of porcine endogenous retroviruses, have limited their application in clinic. Generation of ß-like cells from stem cells is one of the most promising strategies in regenerative medicine. Embryonic, and more recently, adult stem cells are currently the most promising cell sources exploited to generate functional ß-cells in vitro. A number of studies demonstrated that stem cells could generate functional pancreatic organoids (POs), able to restore normoglycemia when implanted in different preclinical diabetic models. Nevertheless, a gradual loss of function and cell dead are commonly detected when POs are transplanted in immunocompetent animals. So far, the main issue to be solved is the post-transplanted islet loss, due to the host immune attack. To avoid this hurdle, nanotechnology has provided a number of polymers currently under investigation for islet micro and macro-encapsulation. These new approaches, besides conferring PO immune protection, are able to supply oxygen and nutrients and to preserve PO morphology and long-term viability.Herein, we summarize the current knowledge on bioengineered POs and the stem cell differentiation platforms. We also discuss the in vitro strategies used to generate functional POs, and the protocols currently used to confer immune-protection against the host immune attack (micro- and macro-encapsulation). In addition, the most relevant ongoing clinical trials, and the most relevant hurdles met to move towards clinical application are revised.

Extracellular Vesicles: New Players in Lymphomas.

Lymphomas are heterogeneous diseases, and the term includes a number of histological subtypes that are characterized by different clinical behavior and molecular phenotypes. Valuable information on the presence of lymphoma cell-derived extracellular vesicles (LCEVs) in the bloodstream of patients suffering from this hematological cancer has recently been provided. In particular, it has been reported that the number and phenotype of LCEVs can both change as the disease progresses, as well as after treatment. Moreover, the role that LCEVs play in driving tumor immune escape has been reported. This makes LCEVs potential novel clinical tools for diagnosis, disease progression, and chemoresistance. LCEVs express surface markers and convey specific molecules in accordance with their cell of origin, which can be used as targets and thus lead to the development of specific therapeutics. This may be particularly relevant since circulating LCEVs are known to save lymphoma cells from anti-cluster of differentiation (CD)20-induced complement-dependent cytotoxicity. Therefore, effort should be directed toward investigating the feasibility of using LCEVs as predictive biomarkers of disease progression and/or response to treatment that can be translated to clinical use. The use of liquid biopsies in combination with serum EV quantification and cargo analysis have been also considered as potential approaches that can be pursued in the future. Upcoming research will also focus on the identification of specific molecular targets in order to generate vaccines and/or antibodies against LCEVs. Finally, the removal of circulating LCEVs has been proposed as a simple and non-invasive treatment approach. We herein provide an overview of the role of LCEVs in lymphoma diagnosis, immune tolerance, and drug resistance. In addition, alternative protocols that utilize LCEVs as therapeutic targets are discussed.

The effect of G-CSF and AMD3100 on mice treated with streptozotocin: Expansion of alpha-cells and partial islet protection.

Administration of streptozotocin (STZ) is one of the most used experimental models of diabetes (STZ-DT). STZ induces beta-cell damage in pancreatic islets. It is known that hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow to damaged tissues. In this work, we evaluated the effects of the hematopoietic mobilizers G-CSF (250µg/kg; for five consecutive days) and AMD3100 (5mg/kg; single s.c injection) in mice treated with STZ (175mg/kg). Mice injected with STZ showed a significant reduction in the number and area of islets and in the number of beta- and alpha-cells. Concurrently, they had hyperglycemia (blood glucose over 300mg/dl) associated with very low levels of insulin in plasma. The number and area of islets from STZ-DT mice treated with G-CSF and/or AMD3100 were similar to the controls. However, these mice had neither a reduction of hyperglycemia nor an improvement in the insulin levels. Analysis of islet cellularity showed a large reduction in beta-cells with a significant expansion of alpha-cells. These results indicate that G-CSF and AMD3100 induce partial protection of islet tissues and expansion of alpha-cells in mice treated with STZ but do not protect beta-cells from the damage induced by this compound.

A Versatile Model of Microfluidic Perifusion System for the Evaluation of C-Peptide Secretion Profiles: Comparison Between Human Pancreatic Islets and HLSC-Derived Islet-Like Structures.

A robust and easy-to-use tool for the ex vivo dynamic evaluation of pancreatic islet (PI) function is essential for further development of novel cell-based therapeutic approaches to treating diabetes. Here, we developed four different glucose perifusion protocols (GPPs) in a microfluidic perifusion system (MPS), based entirely on commercially available components. After validation, the GPPs were used to evaluate C-peptide secretion profiles of PIs derived from different donors (healthy, obese, and type 2 diabetic) and from human liver stem-cell-derived islet-like structures (HLSC-ILS). Using this device, we demonstrated that PIs derived from healthy donors displayed a physiological C-peptide secretion profile as characterized by the response to (a) different glucose concentrations, (b) consecutive pulses of high-glucose concentrations, (c) a glucose threshold ranging from 5-8 mM, and (d) a constant high-glucose perifusion in a biphasic manner. Moreover, we were able to detect a dysregulated secretion profile in PIs derived from both obese and type 2 diabetes mellitus (T2DM) donors. Finally, we also evaluated the kinetic secretion profiles of HLSC-ILS, demonstrating that, nonetheless, with a lower amplitude of secretion compared to PI derived from healthy donors, they were already glucose-responsive on day seven post-differentiation. In conclusion, we have provided evidence that our MPS is a versatile device and may represent a valuable tool to study insulin-producing cells in vitro.

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

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

Stem Cell-Derived Extracellular Vesicles and Immune-Modulation.

Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system.