Vav1 Sustains the Expression of Insulin, PDX1 and miR-375 During Differentiation of hiPSCs to β Cells: A Potential Target to Improve the In Vitro Generation of Insulin-Producing Cells Marina Pierantoni, Valentina Zamarian, Federica Brugnoli, Silvia Grassilli, Laura Monaco, et al. Tissue Engineering and Regenerative Medicine, 2026 Backround: Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising source of transplantable insulinproducing cells (IPCs) to restore insulin levels in Type 1 Diabetes (T1D) patients. Despite progress, obtaining fully functional β cells from hiPSCs remains challenging, underscoring the need to better understand the intracellular mechanisms involved. We investigated here the potential role of Vav1, a multidomain protein that we identified as crucial for the maturation of human biliary stem cells (hBTSCs) into β-like cells and in the trans-differentiation of pancreatic adenocarcinoma (PDAC) cells into IPCs; Methods: Levels and subcellular localization of Vav1 were investigated throughout a seven-step differentiation process of hiPSCs to β cells. Vav1expression was forcedly modulated in pancreatic progenitors, and the potential effects were evaluated on insulin production and on PDX1, miR-375, and Akt, key regulators of β cells generation; RESULTS. Vav1 showed dynamic modulation, with pancreatic precursor cells requiring adequate levels of the protein to generate IPCs. Results: Vav1 sustains the expression of PDX1, a primary regulator of insulin expression, and of its target miR-375, essential for determining β cell mass. Furthermore, Vav1 reduction correlated with increased activation of Akt, which regulates cell survival and insulin secretion in β cells and is down-regulated by miR- 375. Conclusion: Our findings suggest the existence of a Vav1/PDX1/miR-375/Akt axis as part of the complex network orchestrating the generation of functional β cells. These insights indicate that strategies aimed at specifically modulating Vav1 levels may positively impact the generation of IPCs in vitro and, ultimately, β cell replacement therapy for T1D.
Liraglutide Treatment Reverses Unconventional Cellular Defects in Induced Pluripotent Stem Cell–Derived β-Cells Harboring a Partially Functional WFS1 Variant Silvia Torchio, Gabriel Siracusano, Federica Cuozzo, Valentina Zamarian, Silvia Pellegrini, et al. Diabetes, 2025 Wolfram syndrome 1 (WS1) is a rare genetic disorder caused by WFS1 variants that disrupt wolframin, an endoplasmic reticulum-associated protein essential for cellular stress responses, Ca2+ homeostasis, and autophagy. Here, we investigated how the c.316-1G>A and c.757A>T WFS1 mutations, which yield partially functional wolframin, affect the molecular functions of β-cells and explored the therapeutic potential of the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide. Pancreatic β-cells obtained from patient-derived induced pluripotent stem cells (iPSCs) carrying this WFS1 variant exhibited reduced insulin processing and impaired secretory granule maturation, as evidenced by proinsulin accumulation and decreased prohormone convertase PC1/3. Moreover, they exhibited dysregulated Ca2+ fluxes due to altered transcription of Ca2+-related genes, including CACNA1D, and significantly reduced SNAP25 levels, leading to uncoordinated oscillations and poor glucose responsiveness. Affected cells also showed increased autophagic flux and heightened susceptibility to inflammatory cytokine-induced apoptosis. Notably, liraglutide treatment rescued these defects by normalizing Ca2+ handling, enhancing insulin processing and secretion, and reducing apoptosis, likely through modulation of the unfolded protein response. These findings underscore the importance of defining mutation-specific dysfunctions in WS1 and support targeting the GLP-1/GLP-1R axis as a therapeutic strategy. Article Highlights The molecular basis of WFS1-related mutations remains poorly investigated, and no definitive therapies exist for Wolfram syndrome 1. We dissected the molecular defects associated with c.316-1G>A and c.757A>T WFS1 mutations in patient-derived induced pluripotent stem cell islets and analyzed whether they are potential therapeutic targets of the glucagon-like peptide 1 receptor agonist liraglutide. We found impaired insulin granule maturation, altered Ca2+ fluxes, increased autophagic activity, and heightened susceptibility to inflammatory apoptosis in mutated cells. Liraglutide restored critical β-cell functions suggesting a route for personalized therapy based on WFS1 mutations.
Plasma small extracellular vesicles from dogs affected by cutaneous mast cell tumors deliver high levels of miR-21-5p Clarissa Zamboni, Valentina Zamarian, Damiano Stefanello, Roberta Ferrari, Luigi Auletta, et al. Frontiers in Veterinary Science, 2023 Small extracellular vesicles (sEV) are a class of extracellular vesicles (30–150 nm), delivering molecules including proteins, metabolites, and microRNAs (miRNAs), involved in physiological intercellular crosstalk and disease pathogenesis. The present pilot study aims are (I) to develop an easy and fast protocol for the isolation of sEV from plasma of mast cell tumor (MCT)-affected dogs; (II) to evaluate if miR-21-5p (sEV-miR-21-5p), a miRNA overexpressed by MCT, is associated with sEV. Seventeen dogs have been enrolled in the study: 4 healthy and 13 (6 with and 7 without nodal metastasis) MCT-affected dogs. sEV were isolated using size exclusion chromatography (SEC) (IZON column 35nm) and were characterized by Western blot, Nanoparticle tracking analysis, and transmission electron microscopy. sEV-miR-21-5p was quantified using digital PCR. sEV expressed the specific markers CD9 and TSG101, and a marker of mast cell tryptase. The sEV mean concentration and size were 2.68E + 10 particles/ml, and 99.6 nm, 2.89E + 10 particles/ml and 101.7 nm, and 3.21E + 10 particles/ml and 124 nm in non-metastatic, nodal metastatic, and healthy samples, respectively. The comparative analysis demonstrated that the level of sEV-miR-21-5p was significantly higher in dogs with nodal metastasis compared to healthy (P = 0.038) and without nodal metastasis samples (P = 0.007). In conclusion, the present work demonstrated that a pure population of sEV can be isolated from the plasma of MCT-affected dogs using the SEC approach and that the level of sEV-miR-21-5p is higher in nodal metastatic MCT-affected dogs compared with healthy and MCT-affected dogs without nodal involvement.
Salivary miR-21 is a potential biomarker for canine mast cell tumors Valentina Zamarian, Damiano Stefanello, Roberta Ferrari, Lavinia E. Chiti, Valeria Grieco, et al. Veterinary Pathology, 2023 MicroRNAs (miRNAs) are a class of noncoding RNA molecules playing a crucial role in tumor modulation targeting mRNA. This study aimed to validate the diagnostic potential of a panel of 3 miRNAs previously identified in canine mast cell tumors (MCTs), miR-21, miR-379, and miR-885, as markers of lymph node involvement in terms of histological absence (nonmetastatic: HN0; premetastatic: HN1) and presence (early-metastatic: HN2; overt-metastatic: HN3) of metastasis, in the saliva of mast cell tumor (MCT)-affected dogs by quantitative polymerase chain reaction (PCR). Forty-seven saliva samples were analyzed: 36 from MCT-affected dogs (12 subcutaneous [3 HN0-1 and 9 HN2-3] and 24 cutaneous [9 HN0-1 and 15 HN2-3—MCT]) and 11 from healthy dogs. MCT-group effects were investigated using analysis of variance (ANOVA). The origin of the tumor affected the expression of salivary miR-21 ( P = .011) with an increase in cases with subcutaneous MCTs compared with the healthy group ( P = .0005) and those with cutaneous MCTs ( P = .004). Salivary miR-21 was higher in the HN2-3 class compared with the healthy group ( P = .004). Salivary miR-885 was not affected by the presence of MCT, while miR-379 was not detected in saliva. The diagnostic potential of salivary miR-21 in discriminating MCT-affected dogs from the healthy group (AUC = 0.8917), cutaneous from subcutaneous (AUC = 0.8111), and subcutaneous HN0-1 (AUC = 0.7250) and HN2-3 (AUC = 0.9750) classes from healthy samples was demonstrated by receiver operating characteristic curve analysis. Overall, salivary miR-21 was identified as a promising tool, representing a novel approach to detecting MCT-associated epigenetic alterations in a minimally invasive manner.
Treating iPSC-Derived β Cells with an Anti-CD30 Antibody–Drug Conjugate Eliminates the Risk of Teratoma Development upon Transplantation Silvia Pellegrini, Valentina Zamarian, Elisa Landi, Alessandro Cospito, Marta Tiffany Lombardo, et al. International Journal of Molecular Sciences, 2022 Insulin-producing cells derived from induced pluripotent stem cells (iPSCs) are promising candidates for β cell replacement in type 1 diabetes. However, the risk of teratoma formation due to residual undifferentiated iPSCs contaminating the differentiated cells is still a critical concern for clinical application. Here, we hypothesized that pretreatment of iPSC-derived insulin-producing cells with an anti-CD30 antibody–drug conjugate could prevent in vivo teratoma formation by selectively killing residual undifferentiated cells. CD30 is expressed in all human iPSCs clones tested by flow cytometry (n = 7) but not in iPSC-derived β cells (iβs). Concordantly, anti-CD30 treatment in vitro for 24 h induced a dose-dependent cell death (up to 90%) in human iPSCs while it did not kill iβs nor had an impact on iβ identity and function, including capacity to secrete insulin in response to stimuli. In a model of teratoma assay associated with iβ transplantation, the pretreatment of cells with anti-CD30 for 24 h before the implantation into NOD-SCID mice completely eliminated teratoma development (0/10 vs. 8/8, p < 0.01). These findings suggest that short-term in vitro treatment with clinical-grade anti-CD30, targeting residual undifferentiated cells, eliminates the tumorigenicity of iPSC-derived β cells, potentially providing enhanced safety for iPSC-based β cell replacement therapy in clinical scenarios.
Strategies to Improve the Safety of iPSC-Derived β Cells for β Cell Replacement in Diabetes Silvia Pellegrini, Valentina Zamarian, Valeria Sordi Transplant International, 2022 Allogeneic islet transplantation allows for the re-establishment of glycemic control with the possibility of insulin independence, but is severely limited by the scarcity of organ donors. However, a new source of insulin-producing cells could enable the widespread use of cell therapy for diabetes treatment. Recent breakthroughs in stem cell biology, particularly pluripotent stem cell (PSC) techniques, have highlighted the therapeutic potential of stem cells in regenerative medicine. An understanding of the stages that regulate β cell development has led to the establishment of protocols for PSC differentiation into β cells, and PSC-derived β cells are appearing in the first pioneering clinical trials. However, the safety of the final product prior to implantation remains crucial. Although PSC differentiate into functional β cells in vitro, not all cells complete differentiation, and a fraction remain undifferentiated and at risk of teratoma formation upon transplantation. A single case of stem cell-derived tumors may set the field back years. Thus, this review discusses four approaches to increase the safety of PSC-derived β cells: reprogramming of somatic cells into induced PSC, selection of pure differentiated pancreatic cells, depletion of contaminant PSC in the final cell product, and control or destruction of tumorigenic cells with engineered suicide genes.
