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Diabetes mellitus, both type 1 (T1D) and type 2 (T2D), has become the epidemic of the century and a major public health concern given its rising prevalence and the increasing adoption of a sedentary lifestyle globally. This multifaceted disease is characterized by impaired pancreatic beta cell function and insulin resistance (IR) in peripheral organs, namely the liver, skeletal muscle, and adipose tissue. Additional insulin target tissues, including cardiomyocytes and neuronal cells, are also affected. The advent of stem cell research has opened new avenues for tackling this disease, particularly through the regeneration of insulin target cells and the establishment of disease models for further investigation. Human-induced pluripotent stem cells (iPSCs) have emerged as a valuable resource for generating specialized cell types, such as hepatocytes, myocytes, adipocytes, cardiomyocytes, and neuronal cells, with diverse applications ranging from drug screening to disease modeling and, importantly, treating IR in T2D. This review aims to elucidate the significant applications of iPSC-derived insulin target cells in studying the pathogenesis of insulin resistance and T2D. Furthermore, recent differentiation strategies, protocols, signaling pathways, growth factors, and advancements in this field of therapeutic research for each specific iPSC-derived cell type are discussed. Full article

Organoids and microphysiological systems (MPSs) have emerged as physiologically relevant platforms that recapitulate key structural and functional features of human organs, tissues, and microenvironments. As one of the essential components that define the success of these systems, hydrogels play the central role of providing a three-dimensional, biomimetic scaffold that supports cell viability, spatial organization, and dynamic signaling. Natural polymer-based hydrogels, derived from materials such as collagen, gelatin, hyaluronic acid, and alginate, offer favorable properties including biocompatibility, degradability, and an extracellular matrix-like architecture. This review presents recent advances in the design and application of such hydrogels, focusing on crosslinking strategies (physical, chemical, and hybrid), the viscoelastic characteristics, and stimuli-responsive behaviors. The influence of these materials on cellular processes, such as stemness maintenance, differentiation, and morphogenesis, is critically examined. Furthermore, the applications of organoid culture and dynamic MPS platforms are discussed, highlighting their roles in morphogen delivery, barrier formation, and vascularization. Current challenges and future perspectives toward achieving standardized, scalable, and translational hydrogel systems are also addressed. Full article

According to the World Health Organization, musculoskeletal injuries affect more than 1.71 billion people around the world. These injuries are a major public health issue and the leading cause of disability. There has been a recent interest in hydrogels as a potential biomaterial for musculoskeletal tissue regeneration. This is due to their high water content (70–99%), ECM-like structure, injectability, and controllable degradation rates. Recent preclinical studies indicate that they can enhance regeneration by modulating the release of bioactive compounds, growth factors, and stem cells. Composite hydrogels that combine natural and synthetic polymers, like chitosan and collagen, have compressive moduli that are advantageous for tendon–bone healing. Some of these hydrogels can even hold up to 0.8 MPa of tensile strength. In osteoarthritis models, functionalized systems such as microspheres responsive to matrix metalloproteinase-13 have demonstrated disease modulation and targeted drug delivery, while intelligent in situ hydrogels have exhibited a 43% increase in neovascularization and a 50% enhancement in myotube production. Hydrogel-based therapies have been shown to restore contractile force by as much as 80%, increase myofiber density by 65%, and boost ALP activity in bone defects by 2.1 times in volumetric muscle loss (VML) models. Adding TGF-β3 or MSCs to hydrogel systems improved GAG content by about 60%, collagen II expression by 35–50%, and O’Driscoll scores by 35–50% in cartilage regeneration. Full article

As the global population continues to age, the incidence of neurodegenerative diseases and neural injuries is increasing, presenting major challenges for healthcare systems. Due to the brain’s limited regenerative capacity, there is an urgent need for strategies that promote neuronal repair and functional integration. Brain-derived neurotrophic factor (BDNF) is a key regulator of synaptic plasticity and neuronal development. In this study, we investigated whether constitutive BDNF expression in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) enhances their neurogenic and integrative potential in vitro. We found that NPCs engineered to overexpress BDNF produced neuronal cultures with increased numbers of mature and spontaneously active neurons, without altering the overall structure or organization of functional networks. Furthermore, BDNF-expressing neurons exhibited significantly greater axonal outgrowth, including directed axon extension in a compartmentalized microfluidic system, suggesting a chemoattractive effect of localized BDNF secretion. These effects were comparable to those observed with the early supplementation of recombinant BDNF. Our results demonstrate that sustained BDNF expression enhances neuronal maturation and axonal projection without disrupting network integrity. These findings support the use of BDNF not only as a therapeutic agent to improve cell therapy outcomes but also as a tool to accelerate the development of functional neural networks in vitro. Full article

Background: Often, neoadjuvant therapy, which relies on the induction of double-strand breaks (DSBs), is used prior to surgery to shrink tumors by inducing cancer cell apoptosis. However, recent studies have suggested that this treatment may also induce a fluctuating state between senescence and stemness in PA-1 embryonal carcinoma cells, potentially affecting therapeutic outcomes. Thus, the respective epigenetic pathways are up or downregulated over a time period of days. These fluctuations go hand in hand with changes in spatial DNA organization. Methods: By means of Single-Molecule Localization Microscopy in combination with mathematical evaluation tools for pointillist data sets, we investigated the organization of euchromatin and heterochromatin at the nanoscale on the third and fifth day after etoposide treatment. Results: Using fluorescently labeled antibodies against H3K9me3 (heterochromatin tri-methylation sites) and H3K4me3 (euchromatin tri-methylation sites), we found that the induction of DSBs led to the de-condensation of heterochromatin and compaction of euchromatin, with a peak effect on day 3 after the treatment. On day 3, we also observed the co-localization of euchromatin and heterochromatin, which have marks that usually occur in exclusive low-overlapping network-like compartments. The evaluation of the SMLM data using topological tools (persistent homology and persistent imaging) and principal component analysis, as well as the confocal microscopy analysis of H3K9me3- and H3K4me3-stained PA-1 cells, supported the findings that distinct shifts in euchromatin and heterochromatin organization took place in a subpopulation of these cells during the days after the treatment. Furthermore, by means of flow cytometry, it was shown that the rearrangements in chromatin organization coincided with the simultaneous upregulation of the stemness promotors OCT4A and SOX2 and senescence promotors p21Cip1 and p27. Conclusions: Our findings suggest potential applications to improve cancer therapy by inhibiting chromatin remodeling and preventing therapy-induced senescence. Full article

New approach methodologies (NAMs), including microphysiological systems (MPSs), can recapitulate structural and functional complexities of organs. Vanoxerine was reported to induce cardiac adverse events, including torsade de points (TdP), in a Phase III clinical trial. Despite earlier nonclinical animal models and Phase I–II clinical trials, events of QT prolongation or proarrhythmia were not observed. Here, we utilized cardiac NAMs to evaluate the functional consequences of vanoxerine treatment on human cardiac excitation–contraction coupling. The cardiac MPS used in this study was a microfabricated fluidic culture platform with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) capable of evaluating voltage, intracellular calcium handling, and contractility. Likewise, the hiPSC-CM comprehensive in vitro proarrhythmia assay (CiPA) was employed based on multielectrode array (MEA). Vanoxerine treatment delayed repolarization in a concentration-dependent manner and induced proarrhythmic events in both NAM platforms. The complex cardiac MPS displayed a frequency-dependent vanoxerine response such that EADs were eliminated at a faster pacing rate (1.5 Hz). Moreover, exposure analysis revealed a 99% vanoxerine loss in the cardiac MPS. TdP risk analysis demonstrated high to intermediate TdP risk at clinically relevant concentrations of vanoxerine and frequency-independent EAD events in the hiPSC-CM CiPA model. These findings demonstrate that nonclinical cardiac NAMs can recapitulate clinical outcomes, including detection of vanoxerine-induced delayed repolarization and proarrhythmic effects. Moreover, this work provides a foundation to evaluate the safety and efficacy of novel compounds to reduce the dependence on animal studies. Full article

Background: It is estimated that in Italy, there were 364,000 new diagnoses of neoplasms each year and that the overall incidence of blood cancers was 10% of these. Leukemia and lymphomas represented the ninth and eighth places, respectively, among the causes of death from neoplasia. Hematopoietic stem cell transplantation represented an effective treatment option for many of these malignancies, and not only that: benign and congenital diseases could also be treated. Objective: To assess knowledge among the Apulian population regarding stem cell donation and factors that could influence this choice, focusing especially on the knowledge of the residents of Puglia, Italy on how stem cells were harvested and their functions, their reasons for joining the National Registry, and the reasons that hold them back from making such a choice. Study Design: An observational and cross-sectional study was conducted, through snowball sampling methodology, until data saturation. An online survey was conducted, which included several Italian associations. The questionnaire administered contained five main sections, such as sociodemographic data, knowledge of the existence of National Registries and their adherence, the nationwide presence of various associations that promote donation, knowledge with respect to the structure, use and functions of stem cells, sources of procurement, such as bone marrow, peripheral blood and umbilical cord, and related procedures, beliefs, attitudes, values, and opinions of the Italian population regarding the topic, and degree of information and education regarding bone marrow donation. Results: A total of 567 Apulian citizens were enrolled. Of these, 75.3% were female and 96.8% were aged between 18 and 65 years. Most of participants were single (46.9%) and married (47.3%) and had a diploma (44.4%), and less had a degree (35.8%). Significant differences were recorded between gender, singles, and married participants, and participants with a diploma or a degree and the items proposed. Conclusions: A true culture of donation in our region was not clearly spread. Although something has been accomplished in recent years in terms of deceased donor donation, still a great deal needs to be achieved for living donation, which encountered a great deal of resistance. It has been deemed necessary to seek winning solutions to this issue in terms of communication and information campaigns, raising awareness and empowering citizens to express consciously their concerns about organs and tissues and to stand in solidarity with those who suffered. Full article

In modern society, obesity and its associated complications have emerged as serious public health concerns, primarily stemming from sedentary lifestyles and carbohydrate-rich diets. Due to the severe side effects often associated with pharmacological anti-obesity agents, emerging global efforts focus on preventive strategies, e.g., dietary modifications and weight gain-suppressing functional foods. In this context, plant-derived metabolites are extensively investigated for their beneficial anti-obesity effects. In this study, we evaluated how Rosa rugosa Thunb. flower bud extract affects fat metabolism in 3T3-L1 preadipocyte cells. The extract significantly inhibited adipocyte differentiation and intracellular triglyceride accumulation in 3T3-L1 cells, enhanced lipolysis, suppressed lipogenesis, and promoted energy metabolism in differentiated adipocytes. In vivo, it reduced body and organ weights and fat mass in high-fat diet-induced obese rats, along with marked adipocyte size and hepatic lipid accumulation reductions. In the epididymal adipose tissue, the extract similarly enhanced lipolytic activity, suppressed lipogenic enzyme expression, and stimulated energy expenditure. Taken together, our results demonstrate the potential of R. rugosa Thunb. flower bud extract in reducing fat accumulation through lipid metabolism modulation both in cellular and animal models. Further studies are warranted to identify the active constituents and evaluate the safety and efficacy of the extract in clinical applications. Full article

With the rise in immunocompromised individuals and patients with immune-related comorbidities such as COVID-19, the rate of fungal infections is growing. This increase, along with the current plateau in antifungal drug development, has made understanding the pathogenesis and dissemination of these organisms more pertinent than ever. The mouse model of fungal infection, while informative on a basic scientific level, has severe limitations in terms of translation to the human disease. Here we present data supporting the implementation of the human cerebral organoid model, which is generated from human embryonic stem cells and accurately recapitulates relevant brain cell types and structures, to study fungal infection and dissemination to the central nervous system (CNS). This approach provides direct insight into the relevant pathogenesis of specific fungal organisms in human tissues where in vivo models are impossible. With this model system we assessed the specific brain tropisms and cellular effects of fungal pathogens known to cross the blood–brain barrier (BBB), such as Cryptococcus neoformans. We determined the effects of this fungal pathogen on the overall gross morphology, cellular architecture, and cytokine release from these model organoids. Furthermore, we demonstrated that C. neoformans penetrates and invades the organoid tissue and remains present throughout the course of infection. These results demonstrate the utility of this new model to the field and highlight the potential for this system to elucidate fungal pathogenesis to develop new therapeutic strategies to prevent and treat the disseminated stages of fungal diseases such as cryptococcal meningitis. Full article

Mutations in PKD1 and PKD2 cause autosomal dominant polycystic kidney disease (ADPKD), the most common renal genetic disease, leading to the dysregulation of renal tubules and the development of cystic growth that compromises kidney function. Despite significant advances in recent decades, there remains a considerable unmet clinical need, as current therapeutics are not effective at slowing or halting disease progression. Although preclinical animal models have been used extensively, the translatability of such findings is uncertain and human-relevant disease models are urgently needed. The advent of pluripotent stem cells (PSCs) and their ability to more accurately recapitulate organ architecture and function has allowed for the study of renal disease in a more physiological and human-relevant setting. To date, many research groups have studied ADPKD using PSC-derived kidney organoids, identifying many dysregulated pathways and screening drug candidates that may yield effective therapies in the clinic. In this review article, we discuss in detail the development of PSC-derived kidney organoids as ADPKD models and how they have advanced our understanding of the disease’s pathogenesis, as well as their limitations and potential strategies to address them. Full article

An investigation into the biological mechanisms initiated in wounded skin following the application of mesenchymal stem cells (MSCs) and nanoparticles (NPs) (Ag, ZnO), either alone or combined, was performed in mice, with the aim of determining the optimal approach to accelerate the healing process. This combined treatment was hypothesized to be beneficial, as it is associated with the production of molecules supporting the healing process and antimicrobial activity. The samples were collected seven days after injury. When compared with untreated wounded animals (controls), the combined (MSCs+NPs) treatment induced the expression of Sprr2b, encoding small proline-rich protein 2B, which is involved in keratinocyte differentiation, the response to tissue injury, and inflammation. Pathways associated with keratinocyte differentiation were also affected. Ag NP treatment (alone or combined) modulated DNA methylation changes in genes involved in desmosome organization. The percentage of activated regulatory macrophages at the wound site was increased by MSC-alone and Ag-alone treatments, while the production of nitric oxide, an inflammatory marker, by stimulated macrophages was decreased by both MSC/Ag-alone and MSCs+Ag treatments. Ag induced the expression of Col1, encoding collagen-1, at the injury site. The results of the MSC and NP treatment of skin wounds (alone or combined) suggest an induction of processes accelerating the proliferative phase of healing. Thus, MSC-NP interactions are a key factor affecting global mRNA expression changes in the wound. Full article

Cutaneous wound healing is a complex process involving multiple cellular and molecular events, and current treatments often face limitations in efficacy and safety. Stem-cell therapy, particularly using mesenchymal stem cells (MSCs), has emerged as a promising approach to enhance wound repair through both direct cell replacement and paracrine signaling. This study investigates the therapeutic potential of human chorionic villus mesenchymal stem cells (hCV-MSCs) and their secretory factors in enhancing cutaneous wound healing. Utilizing a rat model, we combined the local administration of hCV-MSC-laden PEGDA/SA/Col-I hydrogel with the systemic delivery of their secretome, aiming to leverage the complementary mechanisms of cellular and cell-free therapies. Our findings demonstrate that hCV-MSCs delivered via PEGDA/SA/Col-I hydrogel significantly accelerated wound closure compared to controls, with near-complete closure observed by day 20. Histological analysis revealed enhanced keratinocyte maturation (increased KRT10/KRT14 ratio) and a higher density of CD31⁺ blood vessels, indicating improved re-epithelialization and angiogenesis. A mass spectrometry analysis of the hCV-MSC secretome identified 849 proteins, with enrichment in pathways related to ECM organization, cell adhesion, and immune regulation. Key proteins such as ANXA1, SERPINE1, and WNT5A were implicated in wound-healing processes. Combination therapy with systemic secretome administration further accelerated wound closure and enhanced collagen deposition, keratinocyte maturation, and vascularization compared to hCV-MSCs alone. Our results highlight the promising application of hCV-MSCs and their secretome in cutaneous wound healing, paving the way for innovative therapeutic strategies that integrate both local and systemic regenerative approaches. Full article

Two-dimensional culture systems have been used for a long time in the research field but their disadvantages make it difficult to reproduce the in vivo environment. Three-dimensional culture systems overcome these limitations, simulating the physiological context of an organism, from the molecular level to the cellular, tissue, and organ complexity levels. This review focuses on 3D cellular models, such as spheroids and tumoroids, which reproduce tumor heterogeneity and microenvironments. It also includes 3D cultures of mesenchymal stem cells (MSCs), particularly those derived from teeth. In conclusion, 3D models are profoundly impacting the biomedical field by offering more accurate in vitro platforms for drug development and disease modeling, thereby significantly reducing the reliance on animal testing and leading to the advancement of personalized and regenerative medicine. Full article

This paper conducts a literature review on the role of natural killer cells in haploidentical hematopoietic stem cell transplantation. Theoretical concepts related to KIR genes are introduced regarding their structure, nomenclature, genetic organization, polymorphism, and inheritance pattern, types of KIR proteins and receptors, HLA ligands for KIR receptors, and the definition of different NK alloreactivity prediction models for the donor of haploidentical hematopoietic stem cell transplantation and the recipient. These models include the following and consider incompatibility: ligand–ligand, receptor–ligand, gene–gene, and KIR haplotype models or the KIR-B donor group. These models consider the presence or absence of specific ligands or receptors and/or KIR genes in the donor and recipient to predict alloreactivity. Determining the best model for predicting KIR alloreactivity and its significance in donor selection algorithms for haploidentical transplantation is still under investigation. Full article

Umbilical cord mesenchymal stem cells (UCMSCs)-derived small extracellular vehicles (sEVs) are reported to offer therapeutic effects in regenerative medicine, but they lack safety and biodistribution profiles to support smooth translation at the clinical stage and regulatory requirements. Our study aimed to determine the safety and biodistribution profile in a healthy animal model before application in the metabolic syndrome model. Method: Healthy male Sprague Dawley (SD) rats were given an intravenous (IV) injection of normal saline (control group) or pooled fetal UCMSCs-derived sEVs (treated group) every three weeks for 90 days. Morbidity and mortality observation (daily), physical measurements (weekly), selected serum biochemistry (every three weeks), and hematology (every three weeks) were performed for 90 days. Acute toxicity (on day 14) and sub-chronic toxicity (on day 90) were assessed for gross necropsy, relative organ weight, and histopathological assessment of lungs, liver, spleen, kidney, and lymph nodes. Separately, a biodistribution study was conducted with the sEVs preparations labeled with PKH26 fluorescent dye, given intravenously to the rats. The organs were harvested 24 h post-injection. There were no drastic changes in either group’s morbidity or mortality, physical, hematological, and biochemistry evaluation. The histopathological assessment concluded moderate (focal) inflammation in the treated group’s kidneys and signs of recovery from the inflammation and vascular congestion in the liver. A biodistribution study revealed a higher accumulation of sEVs in the spleen. Multiple IV injections of the pooled fetal UCMSCs-derived sEVs in healthy male SD rats were deemed safe. The sEVs were abundantly distributed in the spleen 24 h post-injection. Full article