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Versatile methods for the quantification of intracellular cholesterol are essential for understanding cellular physiology and for diagnosing disorders linked to cholesterol metabolism. Here we used Raman spectroscopy (RS) and surface-enhanced Raman spectroscopy (SERS) to measure changes in cholesterol after incubating human fibroblasts with increasing concentrations of cholesterol-methyl-β-cyclodextrin. RS and SERS were sensitive and accurate enough to detect high levels of cholesterol in fibroblasts from patients affected by type C Niemann-Pick disease (NPC), a lysosomal storage disorder characterized by the primary accumulation of cholesterol. Moreover, SERS was able to distinguish between fibroblasts from different NPC patients, demonstrating higher accuracy than RS and standard fluorescent labeling of cholesterol with filipin III. We show that the type of gold nanoparticles used as signal enhancer surfaces in our SERS measurements are internalized by the cells and are eventually found in lysosomes, the main site of accumulation of cholesterol in NPC fibroblasts. The higher sensitivity of SERS can thus be attributed to the specific trafficking of our gold nanoparticles into these organelles. Our results indicate that RS and SERS can be used as sensitive and accurate methods for the evaluation of intracellular cholesterol content, allowing for the potential development of an optical detection tool for the ex-vivo screening and monitoring of those diseases characterized by abnormal modification in cholesterol levels., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague-Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A (SCI + hDPSCs), and Lesion Treatment B (SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs (p < 0.01). IHC revealed that hDPSCs and MT-hDPSCs differentiated into neurons and astrocytes, with greater differentiation observed in the MT-hDPSCs compared to the hDPSCs (p < 0.01 and p < 0.05, respectively). Functional improvement was noted in both SCI + hDPSCs and SCI + MT-hDPSCs groups compared to SCI and Vehicle groups from Week 4 onward (p < 0.001). Significant differences were also observed between the SCI + hDPSCs and SCI + MT-hDPSCs groups starting from Week 7 (p < 0.01). Preconditioning hDPSCs with melatonin enhances engraftment, neuronal differentiation, and greater performance improvement compared to hDPSCs alone in the SCI animal model., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval and consent to participate The experimental protocols were approved by the Animal Experimentation Ethics Committee of Guilan University of Medical Sciences (IR.GUMS.REC.1400.175) and were conducted in accordance with the animal ethics guidelines of national health and research institutes. This study is reported following ARRIVE guidelines., (© 2024. The Author(s).)

Increased dietary salt intake is a well-established risk factor for hypertension and related cardiovascular diseases, involving complex vascular remodeling processes. However, the specific role of hypoxia-inducible factor-1α (HIF-1α) in vascular pathophysiology under high-salt conditions remains poorly understood. This study investigates the role of HIF-1α in high-salt-induced vascular remodeling using human aortic vascular smooth muscle cells (HA-VSMCs) cultured in vitro. HA-VSMCs were divided into three groups: high-salt with HIF-1α knockdown (shHIF-1α + HS), negative control (shcontrol), and high-salt (HS). Cell viability, migration, gene expression, and protein levels were evaluated. High-salt conditions significantly increased mRNA expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), angiotensin II type 1 receptor (AT1R), collagen I, and collagen III (p < 0.0001). HIF-1α knockdown partially attenuated these increases, particularly for α-SMA, SM22, and AT1R (p < 0.01). At the protein level, high-salt exposure markedly elevated expression of collagen III, HIF-1α, osteopontin (OPN), and angiotensin II (Ang II) (p < 0.0001). HIF-1α knockdown significantly reduced the high-salt-induced increases in collagen III and HIF-1α protein levels (p < 0.001) but had a limited effect on OPN and Ang II upregulation. Interestingly, SM22 protein expression was significantly decreased under high-salt conditions (p < 0.0001), an effect partially reversed by HIF-1α knockdown (p < 0.0001). These findings demonstrate that high-salt conditions induce complex changes in gene and protein expression in HA-VSMCs, with HIF-1α playing a crucial role in mediating many of these alterations. The study highlights the differential effects of HIF-1α on various markers of vascular remodeling and suggests that HIF-1α may be a potential therapeutic target for mitigating salt-induced vascular pathology. Further research is warranted to elucidate the mechanisms underlying the HIF-1α-dependent and -independent effects observed in this study., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The involvement of the mechanistic targets of rapamycin (mTOR) pathway in lipid metabolism has been recently elucidated. However, its specific role in the Meibomian gland, where lipid metabolism is significant, remains not fully understood. We investigated the role of mTOR signaling system in the lipogenesis and differentiation of human meibomian gland epithelial cells (HMGECs). Treatment of HMGECs with rapamycin resulted in a reduction in lipid synthesis and the expression of PPARγ and SREBP-1, the major regulators of lipid synthesis. The phosphorylation of p70S6kinase and AKT, which are downstream signals of mTOR complexes 1 and 2, respectively, decreased following rapamycin treatment. In addition, when both mTOR complex 1 and 2 were suppressed using siRNA, there was a significant reduction in the expression of PPARγ and SREBP-1, along with a decrease in lipid synthesis in HMGECs. Our findings suggest that inhibiting the mTOR pathway diminishes the differentiation and adipogenesis of meibomian gland epithelial cells, and both mTOR complexes 1 and 2 appear to play a role in this activity., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Perinatal stem cells have prominent applications in cell therapy and regenerative medicine. Among them, human Wharton's jelly mesenchymal stem/stromal cells (hWJMSCs) and human amniotic epithelial stem cells (hAESCs) have been widely used. However, the distinction in the therapeutic potential of hWJMSCs and hAESCs is poorly understood. In this study, we reported the phenotypic differences between these two distinct cell types and provided the first systematic comparison of their therapeutic potential in terms of immunomodulation, extracellular matrix (ECM) remodelling, angiogenesis and antioxidative stress using proteomics. The results revealed that the two cell types presented different protein expression profiles and were both promising candidates for cell therapy. Both types of cells demonstrated angiogenic and antifibrotic potential, whereas hAESCs presented superior immunological tolerance and antioxidant properties, which were supported by a series of relevant in vitro assays. Our study provides clues for the selection of appropriate cell types for diverse indications in cell therapy, which contributes to the advancement of their clinical translation and application., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Alveolar capillary barrier disruption induces local edema and inflammation that impairs pulmonary function and promotes alveolar destruction in COPD. This study aimed to determine how cigarette smoke modulated the serine-threonine phosphatase protein phosphatase 2 A (PP2A) to alter the barrier function of human lung microvascular endothelial cells (HLMVECs). Cigarette smoke exposure lowered overall PP2A activity and enhanced endothelial permeability in HLMVECs. However, directly decreasing PP2A activity with Fostriecin significantly reduced endothelial cell permeability. Protein fractionation studies determined that cigarette smoke diminished cytosolic PP2A activity but increased membrane and cytoskeletal activity. These changes coincided with the translocation of PP2A to the membrane, which reduced occludin phosphorylation in the membrane. Cigarette smoke decreased protein tyrosine phosphatase 1B (PTP1B) activity, a PP2A activator which also counters calcium intracellular influx. The decrease in PTP1B activity correlated with reduced calcium efflux in endothelial cells and these changes in calcium flux regulated PP2A activity. Indeed, culturing endothelial cells in low calcium medium prevented the decrease in cytosolic PP2A activity mediated by cigarette smoke. Together, these findings outline a mechanism whereby cigarette smoke acts via calcium to traffic PP2A from the cytosol to the membrane where it dephosphorylates occludin to increase endothelial cell permeability., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Exosomes derived from the stem cells of human exfoliated deciduous teeth (SHED) hold promise for tissue regeneration. Apoptotic cells release a variety of extracellular vesicles that affect intercellular communication. This study aimed to investigate the angiogenic effects of SHED-derived exosomes modified via apoptosis induction on human umbilical vein endothelial cells (HUVECs). Apoptosis was induced in SHED via serum starvation for 3 weeks and confirmed by the upregulation of the apoptotic genes, caspase 3 and 9, and via annexin V staining. The apoptotic SHED-derived exosomes were isolated, characterized, and subjected to proteomic analysis. In vitro experiments were performed to assess the effects of apoptotic SHED exosomes on the proliferation, migration, and tube formation of HUVECs. The apoptosis-induced SHED showed increased cell viability and decreased numbers of dead cells compared with those of conventional cultures while retaining their identity as mesenchymal stem cells positive for CD44, CD73, and CD90. The apoptotic SHED-derived exosomes exhibited characteristic features, such as standard size, cup-shaped morphology, and positive staining, for exosomal markers CD9, CD63, and CD81. Proteins associated with apoptosis, programmed cell death, and cellular senescence were downregulated in the apoptotic SHED exosomes, whereas those associated with extracellular matrix organization were upregulated, indicating positive angiogenesis. HUVECs treated with apoptotic SHED exosomes exhibited significantly enhanced proliferation and migration compared with those treated with normal SHED exosomes. The mesh-like structures in the apoptotic SHED exosomes exhibited significantly increased signs of angiogenesis. The findings of this study provide new insights into the potential use of apoptotic SHED-derived exosomes in regenerative medicine., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

"Tissue engineering," is a concept that involves the use of scaffolds, cells, and growth factors/signaling molecules in the field of regenerative medicine. The present study was carried out to evaluate the attachment and depth of penetration of the Gingival Mesenchymal Stem Cells (GMSCs) onto the collagen scaffold preconditioned with Fetal Bovine Serum (FBS), Injectable Platelet Rich Fibrin (i-PRF) and a combination of FBS with i-PRF using histological analysis and environmental scanning electron microscopy (ESEM) respectively. In the present study, commercially available collagen membranes were used as scaffolds and divided into 3 groups where Group I was preconditioned with FBS, Group II was preconditioned with i-PRF, and Group III with a combination of FBS and i-PRF. Scaffolds were then seeded with GMSCs and incubated in a CO 2 incubator at 37 0 C for 7 days. Both histological and ESEM analysis showed proliferation, attachment, and depth of penetration along with the combination of cell morphological changes in all the three groups. In group I cells showed mild depth of penetration along with a round morphological appearance. In group II the cells showed moderate depth of penetration and rounded morphology. In group III maximum depth of penetration was seen along with the round and spheroidal morphology of cells. The combination of the growth media showed the best effect on cell attachment, depth of penetration, and cell morphology. This is the first report demonstrating the effect of the combination of collagen and i-PRF supports the proliferation of GMSCs. Since FBS alone and i-PRF alone exhibited similar cell proliferation it is recommended that i-PRF could be used in clinical scenarios making it xenofree., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Calcium oscillations in primary neuronal cultures and iPSCs have been employed to investigate arrhythmogenicity and epileptogenicity in drug development. Previous studies have demonstrated that Ca 2+ influx via NMDA and nicotinic acetylcholine receptors (nAChRs) modulates Ca 2+ oscillations. Nevertheless, there has been no comprehensive investigation into the impact of ischemia or nAChR-positive allosteric modulators (PAM) drugs on Ca 2+ oscillations at a level that would facilitate high-throughput screening. We investigated the effects of ischemia and nAChR subtypes or nAChR PAM agonists on Ca 2+ oscillations in high-density 2D and 3D-sphere primary neuronal cultures using 384-well plates with FDSS-7000. Ischemia for 1 and 2 h resulted in an increase in the frequency of Ca 2+ oscillations and a decrease in their amplitude in a time-dependent manner. The NMDA and AMPA receptor inhibition significantly suppressed Ca 2+ oscillation. Inhibition of NR2A or NR2B had the opposite effect on Ca oscillations. The potentiation of ischemia-induced Ca 2+ oscillations was significantly inhibited by the NMDA receptor antagonist, MK-801, and the frequency of these oscillations was suppressed by the NR2B inhibitor, Ro-256981. In the 3D-neurosphere, the application of an α7nAChR agonist increased the frequency of Ca 2+ oscillations, whereas the activation of α4β2 had no effect. The combination of nicotine and PNU-120596 (type II PAM) affected the frequency and amplitude of Ca 2+ oscillations in a manner distinct from that of type I PAM. These systems may be useful not only for detecting epileptogenicity but also in the search for neuroprotective agents against cerebral ischemia., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethics approval All experiments were performed following the Use of Laboratory Animals and ARRIVE guidelines. The Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine approved all animal protocols and experiments (Permission number: 30-091-001, 05-078-0000). Consent for publication All authors give their consent and approval for publication of this manuscript., (© 2024. The Author(s).)

Succinate receptor GPR91 is one of the G protein-coupled receptors (GPCRs) that interacts with various proteins to regulate diverse cellular functions such as cell morphology, apoptosis, and differentiation. In this study, we investigated whether the GPR91-mediated signaling pathway regulates mineralization in Porphyromonas gingivalis (P. gingivalis)-treated osteoblasts and to determine its potential role in osteoclast differentiation. Primary mouse osteoblasts from wild-type (WT) and GPR91 knockout (GPR91 -/- ) mice infected with P. gingivalis were used for in vitro experiments. The results showed that inhibition by 4C, a specific inhibitor, and GPR91 knockout promoted mineralization in P. gingivalis-infected osteoblasts. Surprisingly, GPR91 knockdown decreased the migration ability of osteoblasts. Moreover, compared with P. gingivalis-infected WT osteoblasts, GPR91 -/- osteoblasts exhibited decreased RANKL production, and conditioned media (CM) from bacteria-infected GPR91 -/- osteoblasts suppressed the formation of osteoclast precursors. Moreover, P. gingivalis mediated the role of GPR91 in osteoblast mineralization by activating the NF-κB pathway. These findings suggest that GPR91 activation reduces mineralization of P. gingivalis-infected osteoblasts and promotes osteoclastogenesis in macrophages. Therefore, targeting GPR91 may mitigate the loss of alveolar bone during bacterial infection., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval Each author has read and passed the final version and reached an agreement to ensure all features of the work are accurate., (© 2024. The Author(s).)

Adipose stem cell-derived exosomes (ADSC-EXO) have been demonstrated to promote osteogenic differentiation of bone marrow stem cells (BMSCs) and facilitate bone regeneration. The present study aims to investigate the effect of ADSC-EXO-loaded nano-hydroxyapatite/chitosan/poly-lactide-co-glycolide (nHA/CS/PLGA) scaffolds on maxillofacial bone regeneration using tissue engineering. ADSC-EXO was isolated and co-cultured with BMSCs, and the osteogenic differentiation of BMSCs was assessed through the detection of mineralized nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of COL1A1 and runt-related transcription factor 2 (RUNX2). The nHA/CS/PLGA scaffolds were fabricated and loaded with ADSC-EXO and BMSCs, and these tissue engineering complexes were applied to the maxillofacial bone defect region of rabbits to elucidate their bone regeneration effect. The osteogenic differentiation of BMSCs was markedly enhanced when they were co-cultured with ADSC-EXO. This was evidenced by an increase in the formation of mineralized nodule formation, ALP activity, and mRNA expression of COL1A1 and runt-related transcription factor 2 (RUNX2). In vivo experiments demonstrated that the application of ADSC-EXO and BMSCs loaded nHA/CS/PLGA scaffolds effectively repaired maxillofacial bone defects in rabbits. ADSC-EXO has been demonstrated to promote the osteogenic differentiation of BMSCs. The ADSC-EXO and BMSCs loaded nHA/CS/PLGA scaffolds have been shown to facilitate the regeneration of maxillofacial bone defects. This may serve as a potential therapeutic strategy for large-scale bone defects., Competing Interests: Declarations Ethics approval and consent to participate All animal experimental operations were approved by the China Medical University Committee for Laboratory Animal Welfare and Ethics (No. 2017036). All animal experiments were complied with the ARRIVE guidelines, and were carried out in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The detection of Leishmania tarentolae in sympatric areas where Leishmania infantum is endemic raised questions regarding the protective effect exerted in dogs by L. tarentolae when in coinfection. This study aimed monitoring the in vitro gene expression of pro- (IFN- γ; TNF-α; IL-12) and anti-inflammatory (IL-4; IL-6; IL-10) cytokines in primary canine macrophages infected by L. tarentolae and L. infantum in single and in co-infections. Macrophages differentiated from dog blood mononuclear cells were infected with the L. tarentolae field-isolated (RI-325) and laboratory (LEM-124) strains, with L. infantum laboratory strain (IPT1), or both. Infection and the number of amastigotes per infected cell were evaluated microscopically by counting a total of 200 cells between 4 and 96 h. Cytokine gene expression was analyzed by real-time PCR from infected macrophages mRNA. Single infections presented higher expression of the cytokines IL-4 and IL-6, and lower of IL-12. Co-infections induced a lower gene expression of IL-4 and IL-6, and a higher gene expression of IL-12, correlating with the low amastigote burden despite the slight increase of infected cells. Data highlight the potential protective effect of L. tarentolae against L. infantum in co-infection by the reduced anti-inflammatory and increased pro-inflammatory cytokines gene expression, opening new perspectives for a canine vaccine development exploiting the non-pathogenic L. tarentolae., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: As fat grafting is commonly used as a filler, Adipose-derived stem/stromal cells (ASC) have been reported to be key player in retention rate. Paracrine and differentiation potential of those cells confer them strong pro-angiogenic capacities. However, a full characterization of the influence of aging on ASC has not been reported yet. Here we've investigated the effect of age on paracrine function, stemness and angiogenic potential of ASC., Methods: ASC were extracted from young and old adult donors. We assessed stromal vascular fraction cell populations repartition, ASC stemness potential, capability to differentiate into mesenchymal lineages as well as their secretome. Angiogenic potential was assessed using a sprouting assay, an indirect co-culture of ASC and dermal microvascular endothelial cells (EC). Total vascular sprout length was measured, and co-culture soluble factors were quantified. Pro-angiogenic factors alone or in combination as well as ASC-conditioned medium (CM) were added to EC to assess sprouting induction., Results: Decrease of endothelial cells yield and percentage is observed in cells extracted from adipose tissue of older patients, whereas ASC percentage increased with age. Clonogenic potential of ASC is stable with age. ASC can differentiate into adipocytes, chondrocytes and osteoblasts, and aging does not alter this potential. Among the 25 analytes quantified, high levels of pro-angiogenic factors were found, but none is significantly modulated with age. ASC induce a significantly longer vascular sprouts compared to fibroblasts, and no difference was found between young and old ASC donors on that parameter. Higher concentrations of FGF-2, G-CSF, HGF and IL-8, and lower concentrations of VEGF-C were quantified in EC/ASC co-cultures compared to EC/fibroblasts co-cultures. EC/ASC from young donors secrete higher levels of VEGF-A compared to old ones. Neither soluble factor nor CM without cells are able to induce organized sprouts, highlighting the requirement of cell communication for sprouting. CM produced by ASC supporting development of long vascular sprouts promote sprouting in co-cultures that establish shorter sprouts., Conclusion: Our results show cells from young and old donors exhibit no difference in all assessed parameters, suggesting all patients could be included in clinical applications. We emphasized the leading role of ASC in angiogenesis, without impairment with age, where secretome is a key but not sufficient actor., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethics approval and consent to participate Written informed consent was obtained from the patient and surgical residue was collected according to French regulation and declared to research ministry., (© 2024. The Author(s).)

Vitamin D (vitD) deficiency is frequently observed in patients with pulmonary arterial hypertension (PAH) and, in these patients, low levels of vitD correlate with worse prognosis. The aim of this study was to examine the expression and the antiproliferative role of vitD receptor (VDR) and its signalling pathway in the human pulmonary vasculature. VDR presence and expression was analyzed in lungs, pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) from controls and PAH-patients. VDR expression and VDR-target genes were examined in PASMC treated with calcitriol. The antiproliferative effect of 48 h-calcitriol was studied in PASMC by MTT and BrdU assays. VDR is expressed in PASMC. It is downregulated in lungs and in PASMC, but not in PAEC, from PAH-patients compared to non-hypertensive controls. Calcitriol strongly upregulated VDR expression in PASMC and the VDR target genes KCNK3 (encoding TASK1), BIRC5 (encoding survivin) and BMP4. Calcitriol produced an antiproliferative effect which was diminished by silencing or by pharmacological inhibition of survivin or BMPR2, but not of TASK1. In conclusion, the expression of VDR is low in PAH-patients and can be rescued by calcitriol. VDR exerts an antiproliferative effect in PASMC by modulating survivin and the BMP signalling pathway., Competing Interests: Declarations Competing interests The authors declare no competing interests. Ethical approval Research using human samples were approved by the Ethics Committee of the Getafe Hospital (Madrid, Spain), Ciberes Biobank (Barcelona, Spain) and French institutional Ethics Committee (Protocol N8CO-08-003, ID RCB: 2008-A00485-50). Informed consent was obtained in all cases. Human explanted lung tissues from non-PAH subjects were obtained from non-tumor lung areas of the resection specimens from patients undergoing surgery for lung carcinoma or discarded for lung transplantation. The donor lungs included in this study were thoroughly reviewed by a pathologist. PAH samples were obtained from lung transplantation., (© 2024. The Author(s).)

The actin-binding protein filamin c (FLNc) is a key mediator in the response of skeletal muscle cells to mechanical stress. In addition to its function as a structural scaffold, FLNc acts as a signaling adaptor which is phosphorylated at S2234 in its mechanosensitive domain 20 (d20) through AKT. Here, we discovered a strong dephosphorylation of FLNc-pS2234 in cultured skeletal myotubes under acute mechanical stress, despite high AKT activity. We found that all three protein phosphatase 1 (PP1) isoforms are part of the FLNc d18-21 interactome. Enzymatic assays demonstrate that PP1 efficiently dephosphorylates FLNc-pS2234 and in vitro and in cells upon PP1 activation using specific modulators. FLNc-pS2234 dephosphorylation promotes the interaction with FILIP1, a mediator for filamin degradation. Altogether, we present a model in which dephosphorylation of FLNc d20 by the dominant action of PP1c prevails over AKT activity to promote the binding of the filamin degradation-inducing factor FILIP1 during acute mechanical stress., (© 2024. The Author(s).)

Meniscus injuries are common and while surgical strategies have improved, there is a need for alternative therapeutics to improve long-term outcomes and prevent post-traumatic osteoarthritis. Current research efforts in regenerative therapies and tissue engineering are hindered by a lack of understanding of meniscus cell biology and a poorly defined meniscus cell phenotype. This study utilized bulk RNA-sequencing to identify unique and overlapping transcriptomic profiles in cartilage, inner and outer zone meniscus tissue, and passaged inner and outer zone meniscus cells. The greatest transcriptomic differences were identified when comparing meniscus tissue to passaged monolayer cells (> 4,600 differentially expressed genes (DEGs)) and meniscus tissue to cartilage (> 3,100 DEGs). While zonal differences exist within the meniscus tissue (205 DEGs between inner and outer zone meniscus tissue), meniscus resident cells are more similar to each other than to either cartilage or passaged monolayer meniscus cells. Additionally, we identified and validated LUM, PRRX1, and SNTB1 as potential markers for meniscus tissue and ACTA2, TAGLN, SFRP2, and FSTL1 as novel markers for meniscus cell dedifferentiation. Our data contribute significantly to the current characterization of meniscus cells and provide an important foundation for future work in meniscus cell biology, regenerative medicine, and tissue engineering., (© 2024. The Author(s).)

Inhibition of the mitochondrial oxidative phosphorylation (OXPHOS) system can lead to metabolic disorders and neurodegenerative diseases. In primary mitochondrial disorders, reactive astrocytes often accompany neuronal degeneration and may contribute to neurotoxic inflammatory cascades that elicit brain lesions. The influence of mitochondria to astrocyte reactivity as well as the underlying molecular mechanisms remain elusive. Here we report that mitochondrial Complex I dysfunction promotes neural progenitor cell differentiation into astrocytes that are more responsive to neuroinflammatory stimuli. We show that the SWItch/Sucrose Non-Fermentable (SWI/SNF/BAF) chromatin remodeling complex takes part in the epigenetic regulation of astrocyte responsiveness, since its pharmacological inhibition abrogates the expression of inflammatory genes. Furthermore, we demonstrate that Complex I deficient human iPSC-derived astrocytes negatively influence neuronal physiology upon cytokine stimulation. Together, our data describe the SWI/SNF/BAF complex as a sensor of altered mitochondrial OXPHOS and a downstream epigenetic regulator of astrocyte-mediated neuroinflammation., (© 2024. The Author(s).)

This study aimed to investigate the cytotoxicity and apoptotic activity of different endodontic sealers: Sealapex, Apexit Plus, AH Plus, MTA-Fillapex and TotalFill BC Sealer in the culture of human periodontal ligament fibroblast (HPDLF) cells. The sealers were mixed, set for 24 h, and then covered with culture medium to obtain extracts, which were diluted to 1:0, 1:1, 1:2, 1:4, and 1:8. Simultaneously, HPDLF cells (1 × 10 4 ) were seeded in 96-well plates and incubated for 24 h at 37 °C 5% CO 2 conditions. The cells were then exposed to 100 µL of diluted extract medium. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to check cell viability, while apoptosis was assessed by TUNEL assay. Statistical analyses were performed using Kruskal-Wallis and Dunn post hoc tests with Mann Whitney U. In MTT and TUNEL assay cells were treated with sealers both 24 and 72 h. All materials showed higher toxicity at 72 h compared to 24 h. AH Plus exhibited the highest cytotoxicity, followed by MTA-Fillapex, Apexit Plus, Sealapex, while TotalFill BC Sealer had the lowest cytotoxicity. Consuquently, it was considered that TotalFill BC Sealer had the lowest cytotoxic potency when compared to other sealers, so it can be considered biocompatible., (© 2024. The Author(s).)

Direct cell-type conversion of somatic cells into cell types of interest has garnered great attention because it circumvents rejuvenation and preserves the hallmarks of cellular aging (unlike induced pluripotent stem cells [iPSCs]) and is more suitable for modeling diseases with strong age-related and epigenetic contributions. Fibroblasts are commonly used for direct conversion; however, obtaining these cells requires highly invasive skin biopsies. Urine-derived cells (UDCs) are an alternative cell source and can be obtained via noninvasive procedures. Herein, induced motor neuron-like cells (iMNs) were generated from UDCs by transducing transcription factors involved in motor neuron (MN) differentiation. iMNs exhibited neuronal morphology, upregulation of pan-neuron and MN markers, and MN functionality, including spontaneous calcium oscillation and bungarotoxin-positive neuromuscular junction formation, when co-cultured with myotubes. Altogether, the findings of this study indicated that UDCs can be converted to functional MNs. This technology may allow us to understand disease pathogenesis and progression and discover biomarkers and drugs for MN-related diseases at the population level., (© 2024. Takeda Pharmaceutical Company, Ltd.)

Our study develops a generative adversarial network (GAN)-based method that generates faithful synthetic image data of human cardiomyocytes at varying stages in their maturation process, as a tool to significantly enhance the classification accuracy of cells and ultimately assist the throughput of computational analysis of cellular structure and functions. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were cultured on micropatterned collagen coated hydrogels of physiological stiffnesses to facilitate maturation and optical measurements were performed for their structural and functional analyses. Control groups were cultured on collagen coated glass well plates. These image recordings were used as the real data to train the GAN model. The results show the GAN approach is able to replicate true features from the real data, and inclusion of such synthetic data significantly improves the classification accuracy compared to usage of only real experimental data that is often limited in scale and diversity. The proposed model outperformed four conventional machine learning algorithms with respect to improved data generalization ability and data classification by incorporating synthetic data. This work demonstrates the importance of integrating synthetic data in situations where there are limited sample sizes and thus, effectively addresses the challenges imposed by data availability., (© 2024. The Author(s).)

Mast cells (MCs) are tissue-resident cells of hematopoietic origin that play an important role in host's defense mechanism against nematodes. However, excessive activation of these cells contributes to the development of certain allergic diseases. Immunoglobin E (IgE) is one of the well-known molecules that activate MCs. Even in the absence of specific antigens, the binding of highly cytokinergic IgE to FcεRI on MCs prolongs their survival and induces cytokine production without enhancing their degranulation. In the present study, we examined the effects of the members of the interleukin-10 (IL-10) family of cytokines on IgE-mediated MCs functions. The receptors including Il10r1, Il10r2, and Il20r2, but not Il20r1, Il22r1 or Il28r1, were constitutively expressed in mouse bone marrow cell-derived cultured MCs (BMCMCs), suggesting that IL-10 may influence MCs function. Indeed, we found that only IL-10 could influence upon BMCMCs function; IL-10 enhanced prolongation of survival, promoted IL-6 and/or IL-13 production dependently of STAT1 and STAT3, and suppressed tumor necrosis factor production independently of STAT1 and STAT3 on IgE-stimulated BMCMCs. Moreover, the IL-10-mediated enhancement of IL-6 production by IgE-stimulated BMCMCs promotes Th17 cell expansion. These results suggest that IL-10 has a dual role as an anti-inflammatory and pro-inflammatory cytokine in MCs functions., (© 2024. The Author(s).)

Neurodegenerative diseases are often accompanied by neuroinflammation and impairment of the blood-brain barrier (BBB) mediated by activated glial cells through their release of proinflammatory molecules. To study the effects of glial cells on mouse brain endothelial cells (mBECs), we developed an in vitro BBB model with inflammation by preactivating mixed glial cells (MGCs) with lipopolysaccharide (LPS) before co-culturing with mBECs to study the influence of molecules released by activated MGCs. The response of the mBECs to activated MGCs was compared to direct stimulation with LPS. The cytokine profile of activated MGCs was analyzed together with their effects on the mBEC's integrity, expression of tight junction proteins, adhesion molecules, and BBB-specific transport proteins. Stimulation of MGCs significantly upregulated mRNA expression and secretion of several pro-inflammatory cytokines. Co-culturing mBECs with pre-stimulated MGCs significantly affected the barrier integrity of mBECs similar to direct stimulation with LPS. The gene expression levels of tight junction proteins were unaltered, but tight junction proteins revealed rearrangements with respect to subcellular distribution. Compared to direct stimulation with LPS, the expression of cell-adhesion molecules was significantly increased when mBECs were co-cultured with prestimulated MGCs and thus pre-activating MGCs transforms mBECs into a proinflammatory phenotype., (© 2024. The Author(s).)

Hypertrophic scar (HTS) formation is a pathological fibrotic skin disease, with no satisfactory treatments available currently. Inducing apoptosis of HTS-derived fibroblasts (HSFs) are becoming promising approaches. In this research, we aim to improve the technology with co-delivery COX-2 and TGF-β1 siRNAs and further investigate the underlying mechanism. Firstly, the HSFs were transfected with 1 µg/ml COX-2 and/or TGF-β1 siRNAs, and proved that the apoptosis of HSFs was greater induced by COX-2/TGF-β1 siRNAs than either COX-2 or TGF-β1 siRNA alone by flow cytometry. To investigate the impact of co-silencing TGF-β1 and COX-2 mRNA expression in vivo, we established HTSs model in rat tails. Our results confirmed that co-silencing of TGF-β1 and COX-2 mRNA expression could significantly alleviate the HTS formation in vivo. Furthermore, we explored the potential molecular mechanism and revealed that the protein levels of TP53, Bcl-2 and Caspase-3 were downregulated while Bax and Cleaved Caspase-3 were upregulated in the COX-2/TGF-β1 siRNA groups compared with HKP group. Taken together, our results demonstrated that simultaneous silencing of COX-2 and TGF-β1 expression by siRNAs induced HSF apoptosis through a TP53 mediated caspase pathway. Therefore, COX-2/TGF-β1 siRNAs might serve as a novel and effective therapeutic alternative for HTSs treatments., (© 2024. The Author(s).)

The present study was conducted to observe the protective effects of c9, t11- conjugated linoleic acid (CLA) on oxidative stress and inflammation in bovine mammary epithelial cells (BMECs) exposed to H 2 O 2 . The BMECs were treated with different concentrations of H 2 O 2 for 8 h, 600 µmol/L was determined to be the damage concentration. Using different concentrations of c9, t11-CLA to process BMECs for 24 h, 50 and 100 µmol/L were determined to be the effective concentrations for subsequent analyses. Thus, four BMEC groups were established: Control group; H 2 O 2 group; 50 µmol/L c9, t11-CLA + H 2 O 2 group; 100 µmol/L c9, t11-CLA + H 2 O 2 group. We observed that the H 2 O 2 group exhibited significantly lower total antioxidant activity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and significantly higher secretions of malondialdehyde (MDA), interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and expressions of IL-1β, IL-6, and IL-8 than the control group (p < 0.05). Pretreatment with c9, t11-CLA enhanced SOD, CAT, and GPx activities and SOD mRNA expression and repressed IL-6 and IL-8 secretion and expression in H 2 O 2 -treated BMECs (p < 0.05). In conclusion, c9, t11-CLA treatment efficiently enhanced antioxidant capacity and decreased inflammation induced by H 2 O 2 in BMECs., (© 2024. The Author(s).)

To evaluate the levels of YKL40, IL-6(interleukin-6), IL-8(interleukin-8), IL-10(interleukin-10), TNF-α (tumor necrosis factor-α) in OSAS (obstructive sleep apnea syndrome)children and explore the mechanism of YKL40 promoting inflammatory factors overexpression in tonsils. qPCR and ELISA were used to identify the expression of YKL40, IL-6, IL-8, IL-10, and TNF-α in the tonsils of OSAS children. Primary tonsil lymphocytes (PTLCs) were cultured and recombinant human YKL40(rhYKL40)was used to stimulate PTLCs in different concentrations and at different time points. The activation of NF-κB in PTLCs was screened by western blotting. Relative mRNA of YKL40, IL-6, IL-8, TNF-α was over expressed in OSAS-derived tonsil tissue and the levels of YKL40, IL-6, IL-8, and TNF-α was increased in OSAS-derived protein supernatant of tonsil tissue.The relative mRNA expression of IL-6, IL-8 and TNF-α increased under the treatment of YKL40 (100 ng/mmol for 24 h). The phosphorylation of p65 in NF-κB pathway was stimulated in the process. The levels of YKL40, IL-6, IL-8, and TNF-α increases in OSAS children, and YKL40 promotes the overexpression of IL-6, IL-8 and TNF-α in PTLCs via NF-κB pathway. The result implements that inflammation may play an important role in the pathogenesis of OSAS in children., (© 2024. The Author(s).)

Airway inflammation, a protective response in the human body, can disrupt normal organ function when chronic, as seen in chronic obstructive pulmonary disease (COPD) and asthma. Chronic bronchitis induces goblet cell hyperplasia and metaplasia, obstructing airflow. Traditional animal testing is often replaced by in vitro three-dimensional cultures of human epithelial cells to assess chronic cell responses. However, these cells are cultured horizontally, differing from the tubular structure of the human airway and failing to accurately reproduce airway stenosis. To address this, we developed the Bronchus-on-a-Chip (BoC) system. The BoC uses a novel microfluidic design in a standard laboratory plate, embedding 62 chips in one plate. Human bronchial epithelial cells were cultured against a collagen extracellular matrix for up to 35 days. Characterization included barrier integrity assays, microscopy, and histological examination. Cells successfully cultured in a tubular structure, with the apical side air-lifted. Epithelial cells differentiated into basal, ciliated, and secretory cells, mimicking human bronchial epithelium. Upon exposure to inducers of goblet cell hyperplasia and metaplasia, the BoC system showed mucus hyperproduction, replicating chronic epithelial responses. This BoC system enhances in vitro testing for bronchial inflammation, providing a more human-relevant and high-throughput method., (© 2024. The Author(s).)

Abnormal accumulation of hyperphosphorylated tau protein plays a pivotal role in a collection of neurodegenerative diseases named tauopathies, including Alzheimer's disease (AD). We have recently conceptualized the design of hetero-bifunctional chimeras for selectively promoting the proximity between tau and phosphatase, thus specifically facilitating tau dephosphorylation and removal. Here, we sought to optimize the construction of tau dephosphorylating-targeting chimera (DEPTAC) and obtained a new chimera D14, which had high efficiency in reducing tau phosphorylation both in cell and tauopathy mouse models, while showing limited cytotoxicity. Moreover, D14 ameliorated neurodegeneration in primary cultured hippocampal neurons treated with toxic tau-K18 fragments, and improved cognitive functions of tauopathy mice. These results suggested D14 as a cost-effective drug candidate for the treatment of tauopathies., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Osteoarthritis (OA) is a degenerative disease of the joints, prevalent worldwide. Polydeoxyribonucleotide (PDRN) is used for treating knee OA. However, the role of PDRN in IL-1β-induced inflammatory responses in human bone marrow-derived mesenchymal stem cells (hBMSCs) remains unknown. Here, we investigated the role of PDRN in IL-1β-induced impairment of chondrogenic differentiation in hBMSCs. hBMSCs treated with PDRN showed a large micromass, enhanced safranin O and alcian blue staining intensity, and increased expression of chondrogenic genes in IL-1β-induced inflammatory responses, in addition to regulation of catabolic and anabolic genes. In addition, PDRN treatment suppressed the expression of inflammatory cytokines and mitigated IL-1β-induced apoptosis in hBMSCs. Mechanistically, PDRN treatment increased the formation of cyclic adenosine monophosphate (cAMP) and upregulated the phosphorylation of cAMP-dependent protein kinase A (PKA)/cAMP response element binding protein (CREB) through the adenosine A2A receptor in hBMSCs and thus blocked the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway. Thus, IL-1β-induced expression of inflammatory cytokines in hBMSCs was directly reduced by adenosine A2A receptor activation. Based on our results, we suggest that PDRN may be a promising MSC-based therapeutic agent for OA., (© 2024. The Author(s).)

The interactions between myogenic cells and adipocytes play an important role in improving carcass traits and the efficiency of energy utilization. However, there are few reports about the interaction between them mediated by small extracellular vesicles (sEV). In this study, sEV derived from porcine primary skeletal muscle stem cells (MuSCs) was found to be involved in the inhibition of porcine primary adipocyte viability, triglyceride content, Oil Red O enrichment and the expression of adipogenic genes. When the MuSCs were treated with insulin (INS) and oleic acid (OA), the effects of their secreted sEVs on adipose precursor cells were reversed, suggesting that the signaling effects of sEV are related to their own heterogeneity. Further by component heterogeneity analysis, miR-146a-5p was found to be enriched in sEVs of MuSCs and to regulate and suppress adipogenesis through its heterogeneity. This study provides an important mechanism and molecular target for small extracellular vesicles to regulate the interaction between muscle and adipose tissue and improve carcass traits at the intercellular level., (© 2024. The Author(s).)

Induced pluripotent stem cell (iPSC) technology, in combination with electrophysiological characterization via multielectrode array (MEA), has facilitated the utilization of iPSC-derived motor neurons (iPSC-MNs) as highly valuable models for underpinning pathogenic mechanisms and developing novel therapeutic interventions for motor neuron diseases (MNDs). However, the challenge of MN adherence to the MEA plate and the heterogeneity presented in iPSC-derived cultures raise concerns about the reproducibility of the findings obtained from these cellular models. We discovered that one novel factor modulating the electrophysiological activity of iPSC-MNs is the extracellular matrix (ECM) used in the coating to support in vitro growth, differentiation and maturation of iPSC-MNs. The current study showed that two coating conditions, namely, Poly-L-ornithine/Matrigel (POM) and Polyethyleneimine (PEI) strongly promoted attachment of iPSC-MNs on MEA culture dishes compared to three other coating conditions, and both facilitated the maturation of iPSC-MNs as characterized by the detection of extensive electrophysiological activities from the MEA plates. POM coating accelerated the maturation of the iPSC-MNs for up to 5 weeks, which suits modeling of neurodevelopmental disorders. However, the application of PEI resulted in more even distribution of the MNs on the culture dish and reduced variability of electrophysiological signals from the iPSC-MNs in 7-week cultures, which permitted the detection of enhanced excitability in iPSC-MNs from patients with amyotrophic lateral sclerosis (ALS). This study provides a comprehensive comparison of five coating conditions and offers POM and PEI as favorable coatings for in vitro modeling of neurodevelopmental and neurodegenerative disorders, respectively., (© 2024. The Author(s).)

Decidualization denotes the process of inflammatory reprogramming of endometrial stromal cells (EnSC) into specialized decidual cells (DC). During this process, EnSC are subjected to endoplasmic reticulum (ER) stress as well as acute cellular senescence. Both processes contribute to the proinflammatory mid-luteal implantation window and their dysregulation has been implicated in reproductive failure. Here, we evaluated the link between ER stress, decidual differentiation and senescence. In-silico analysis identified HSPA5 gene, codifying the ER chaperone BiP, as a potentially critical regulator of cell fate divergence of decidualizing EnSC into anti-inflammatory DC and pro-inflammatory senescent decidual cells (snDC). Knockdown of HSPA5 in primary EnSC resulted both in decreased expression of DC marker genes and attenuated induction of senescence associated β-galactosidase activity, a marker of snDC. Stalling of the decidual reaction upon HSPA5 knockdown was apparent at 8 days of differentiation and was preceded by the upregulation of ER stress associated proteins IRE1α and PERK. Further, HSPA5 knockdown impaired colony-forming unit activity of primary EnSC, indicative of loss of cellular plasticity. Together, our results point to a key role for HSPA5/BiP in decidual transformation of EnSCs and highlight the importance of constraining ER stress levels during this process., (© 2024. The Author(s).)

Microfluidics cell encapsulation offers a way to mimic a 3D microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Several studies have demonstrated the advantages of graphene oxide (GO) as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young's modulus of the alginate and alginate-slGO was obtained as 1414 kPa and 985.9 kPa, respectively. Live/dead assay and fluorescence microscopy images illustrate that slGO can maintain the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the encapsulated hBMSCs, so that microgels containing hBMSCs gradually became opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration. This finding offers a new method for creating injectable tissue transplants that are minimally invasive., (© 2024. The Author(s).)

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM., (© 2024. The Author(s).)

Haemophilia is associated with reduced bone mass and mineral density. Due to the rarity of the disease and the heterogeneity among the studies, the pathogenesis of bone loss is still under investigation. We studied the effects of coagulation factors on bone cells and characterized in a pilot study the osteoclastogenic potential of patients' osteoclast precursors. To evaluate the effect of coagulation factors on osteoclasts, we treated Healthy Donor-Peripheral Blood Mononuclear Cells (HD-PBMC) with Factor VIII (FVIII), von Willebrand Factor (VWF), FVIII/VWF complex, activated Factor IX (FIXa), activated Factor X (FXa) and Thrombin (THB). FVIII, VWF, FVIII/VWF, FXa and THB treatments reduced osteoclast differentiation of HD-PBMC and VWF affected also bone resorption. Interestingly, PBMC isolated from patients with moderate/severe haemophilia showed an increased osteoclastogenic potential due to the alteration of osteoclast precursors. Moreover, increased expression of genes involved in osteoclast differentiation/activity was revealed in osteoclasts of an adult patient with moderate haemophilia. Control osteoblasts treated with the coagulation factors showed that FVIII and VWF reduced ALP positivity; the opposite effect was observed following THB treatment. Moreover, FVIII, VWF and FVIII/VWF reduced mineralization ability. These results could be important to understand how coagulation factors deficiency influences bone remodeling activity in haemophilia., (© 2024. The Author(s).)

Preclinical methods are needed for screening potential Alzheimer's disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. Long-term potentiation (LTP), which is a correlate of learning and memory, was induced in mature human iPSC-derived cortical neurons cultured on microelectrode arrays utilizing circuit patterns connecting two adjacent electrodes. We demonstrated an LTP system that modeled the MCI and pre-MCI stages of Alzheimer's and validated this functional system utilizing four AD therapeutics, which was also verified utilizing patch-clamp electrophysiology. LTP was induced by tetanic electrical stimulation, and LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aβ 42 ) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aβ 42 -induced LTP impairment. The results illustrate the utility of the system as a validated platform to model MCI AD pathology, and potentially for the pre-MCI phase before significant neuronal death. This system also has the potential to become an ideal platform for high-content therapeutic screening for other neurodegenerative diseases., (© 2024. The Author(s).)

Human bone marrow mesenchymal stem cells (hBMSCs) are adult stem cells residing in the bone marrow, characterized by their capacity for multi-directional differentiation, self-renewal, migration, and engraftment. Serving as seed cells, BMSCs play a pivotal role in the regeneration of bone defects. Hence, investigating the transcription factors and signaling pathways involved in the regulation of osteogenic differentiation in BMSCs holds significant importance. Recent research has unveiled that certain circular RNAs (circRNAs) can function as molecular sponges, influencing the osteogenic differentiation process of mesenchymal stem cells. However, many circRNAs remain undiscovered, and their precise mechanisms remain elusive. Therefore, the objective of this study is to construct an osteogenic differentiation-related circRNA-miRNA-mRNA network in hBMSCs. Subsequently, through bioinformatics analysis, we constructed a ceRNA network related to the osteogenic differentiation ability of hBMSCs, comprising 22 circRNAs, 17 miRNAs, and 15 mRNAs. The potential circRNA-miRNA-mRNA axes, including the role of hsa&#95;circ&#95;0001600 in promoting the osteogenic differentiation of hBMSCs through the targeted regulation of hsa-miR-542-3p, were validated through in vitro experiments., (© 2024. The Author(s).)

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, immune dysregulation, and systemic fibrosis. Research on SSc has been hindered largely by lack of relevant models to study the progressive nature of the disease and to recapitulate the cell plasticity that is observed in this disease context. Generation of models for fibrotic disease using pluripotent stem cells is important for recapitulating the heterogeneity of the fibrotic tissue and are a potential platform for screening anti-fibrotic drugs. We previously reported a novel in-vitro model for fibrosis using induced pluripotent stem cell-derived mesenchymal cells (iSCAR). Here we report the generation of a "scar-like phenotype" when iPSC derived mesenchymal cells are cultured on hydrogel that mimicks a wound healing/scarring response (iSCAR). First, we performed RNA sequencing (RNA-seq) based transcriptome profiling of iSCAR culture at 48 h and 13 days to characterize early and latestage scarring phenotypes. The next generation RNA-seq of these iSCAR culture at different timepoints detected expression 92% of early "scar associated" genes and 85% late "scar associated" genes, respectively. Comparative transcriptomic analysis of a gene level SSc compendium matrix to the iSCAR wound associated model revealed genes common in both data sets. Early scar formation genes showed biological processes of hypoxia (27.5%), vascular development (13.7%) and glycolysis (27.5), while late scar formation showed genes associated with senescence (22.6%). Next we show the effects of two different antifibrotic compounds to validate the utility of the model as a screening tool to study early and late-stagelate-stage fibrosis. An autotaxin inhibitor was used to validate the iSCAR late stage fibrotic model (iSCAR-T) and an antifibrotic tool screening compound of unknown mechanism (EX00015097) was used to study and validate both early (iSCAR-P) and late-stage (iSCAR-T) fibrosis in the iSCAR model., (© 2024. The Author(s).)

This study aimed to identify the suitable induction protocol to produce highly qualified insulin producing cells (IPCs) from human adipose tissue derived stem cells (ADSCs) and evaluate the efficacy of the most functionally IPCs in management of diabetes mellitus (DM) in rats. The ADSCs were isolated and characterized according to the standard guidelines. ADSCs were further induced to be IPCs in vitro using three different protocols. The success of trans-differentiation was assessed in vitro through analysis of pancreatic endocrine genes expression, and insulin release in response to glucose stimulation. Then, the functionalization of the generated IPCs was evaluated in vivo. The in vitro findings revealed that the laminin-coated plates in combination with insulin-transferrin-selenium, B27, N2, and nicotinamide could efficiently up-regulate the expression of pancreatic endocrine genes. The in vivo study indicated effectual homing of the PKH-26-labelled IPCs in the pancreas of treated animals. Moreover, IPCs infusion in diabetic rats induced significant improvement in the metabolic parameters and prompted considerable up-regulation in the expression of the pancreatic related genes. The regenerative effect of infused IPCs was determined through histological examination of pancreatic tissue. Conclusively, the utilization of laminin-coated plates in concomitant with extrinsic factors promoting proliferation and differentiation of ADSCs could efficiently generate functional IPCs., (© 2024. The Author(s).)

Urethral reconstruction presents a challenging issue in urology, primarily due to the limited availability of alternative materials for repair. The advancement of bioengineering technology has brought new hope to researchers, with a focus on the selection of appropriate biological scaffolds and seed cells. In order to find an ideal alternative material, we used platelet-rich fibrin as the bioscaffold and urothelial cells as the seed cells, meanwhile, we intended to investigate the effect of platelet-rich fibrin on the biological properties of urothelial cells. We transformed and characterised induced pluripotent stem cells into urothelial cells and prepared platelet-rich fibrin. Platelet-rich fibrin was cultured in a complex with urothelial cells to observe the effect of platelet-rich fibrin on the proliferation and migration ability of urothelial cells. The results showed that the induced pluripotent stem cells were successfully transformed into urothelial cells, platelet-rich fibrin was regularly arranged in cords, with platelets and other structures distributed between them, and the proliferation and migration of urothelial cells were significantly increased. These results suggested that platelet-rich fibrin is biocompatible with urothelial cells and it promotes the proliferation and migration of urothelial cells, which lays a good foundation for its use as an alternative material for urethral repair., (© 2024. The Author(s).)

As Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are easily accessible, easy to isolate, and ethically acceptable, they represent a promising source of MSCs for use in regenerative medicine. Considering decisions on WJ-MSCs collection requires extensive knowledge of the factors that impact their yield. This study's aim was to evaluate the influence of parameters related to mothers and newborns on the WJ-MSCs yield. The WJ-MSCs were isolated and expanded after being isolated from 79 umbilical cord (UC) samples. Population doubling time and cell proliferation were assessed. By flow cytometry analysis, WJ-MSCs were identified by positivity of CD105, CD90, and CD73 and negativity of CD45 and CD34. There was a statistically significant negative correlation between UC width and P1 doubling time. Maternal age and WJ-MSC yield were shown to be negatively correlated. Birth weight and gestational age showed a significant positive correlation between WJ-MSCs yield and neonatal variables. No significant correlations were detected between the WJ-MSCs and the mother parity, nor the neonatal sex, fetal presentation, or head circumference. The WJ-MSCs yield increases with younger maternal age, higher gestational age, and increased neonatal birth weight. Hence, consideration should be given to these factors when selecting the ideal donors., (© 2024. The Author(s).)

Calcium phosphate-based materials (CaP) are introduced as potential dental pulp capping materials for deciduous teeth. The present study investigated the influence of inorganic phosphate (P i ) on regulating stem cells isolated from human exfoliated deciduous teeth (SHED). SHEDs were treated with P i . Cell cycle progression and apoptosis were examined using flow cytometry analysis. Osteo/odontogenic and adipogenic differentiation were analyzed using alizarin red S and oil red O staining, respectively. The mRNA expression profile was investigated using a high-throughput RNA sequencing technique. P i increased the late apoptotic cell population while cell cycle progression was not altered. P i upregulated osteo/odontoblastic gene expression and enhanced calcium deposition. P i -induced mineralization was reversed by pretreatment of cells with Foscarnet, or p38 inhibitor. P i treatment inhibited adipogenic differentiation as determined by decreased PPARγ expression and reduced intracellular lipid accumulation. Bioinformatic analysis of gene expression profiles demonstrated several involved pathways, including PI3K/AKT, MAPK, EGFR, and VEGF signaling. In conclusion, P i enhanced osteo/odontogenic but inhibited adipogenic differentiation in SHED., (© 2024. The Author(s).)

Natural Killer (NK) cells are pivotal in immune responses to viral infections, malignancies, autoimmune diseases, and transplantation. Assessment of NK cell adhesion, migration, and cytotoxicity is fundamental for in vitro studies. We propose a novel live-cell tracking method that addresses these three major aspects of NK cell function using human NK cells and primary porcine aortic endothelial cells (PAECs) in two-dimensional (2D) static assays and an in-house cylindrical 3D microfluidic system. The results showed a significant increase of NK cytotoxicity against pTNF-activated PAECs, with apoptotic cell death observed in the majority of dead cells, while no difference was observed in the conventional Delfia assay. Computed analysis of NK cell trajectories revealed distinct migratory behaviors, including trajectory length, diameter, average speed, and arrest coefficient. In 3D microfluidic experiments, NK cell attachment to pTNF-activated PAECs substantially increased, accompanied by more dead PAECs compared to control conditions. NK cell trajectories showed versatile migration in various directions and interactions with PAECs. This study uniquely demonstrates NK attachment and killing in a 3D system that mimics blood vessel conditions. Our microscope method offers sensitive single-cell level results, addressing diverse aspects of NK functions. It is adaptable for studying other immune and target cells, providing insights into various biological questions., (© 2024. The Author(s).)

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives, particularly extracellular vesicles (EVs), for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs, alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction, indicating a potential mechanism for their immunomodulatory effects. Furthermore, iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally, iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably, priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics, given their potent immunomodulatory and regenerative properties., (© 2024. The Author(s).)

This study explores methods to isolate high-pure monocytes and optimize the best growth factor concentration to generate monocytes-derived dendritic cells (mo-DCs), subset DC1, which is crucial in immune responses. Three protocols for monocyte isolation from peripheral blood mononuclear cells (PBMCs) were evaluated: three-hour incubation on FBS-coated flasks; an overnight incubation on FBS-coated flasks; and Magnetic Activated Cell Sorting (MACS). Additionally, five different concentrations of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and human recombinant interleukin-4 (hrIL-4) were compared. We used Flow cytometry to assess the isolation, purification, and generation of pure monocytes characterized as CD14 + , and expression of mo-DC classical markers (HLA-DR, CD80, CD83, and CD86). The obtained results show that monocytes isolated with the second method (overnight incubation) had the highest purity (P < 0.0001) but the lowest yield (P > 0.05), balancing purity and cost-effectiveness. A combination of hrGM-CSF and hrIL-4 at 400 U/mL produced the most favorable outcomes, leading to the highest rate of mo-DC generation (P < 0.05). Notably, this concentration resulted in increasing expression of HLA-DR, CD80, and CD86 surface markers in the generated DCs (P < 0.0001), with no changes in CD83 expression levels. In conclusion, this study offers valuable insights into selecting the optimal approach for monocyte isolation and mo-DC generation in various research contexts, providing a foundation for more effective immunological studies., (© 2024. The Author(s).)

The cytotoxic effects of three root canal sealers with different bases on human dental pulp stem cells were assessed in this study using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The cytotoxic effects of three root canal sealers with different bases on human dental pulp stem cells (DPSCs) were assessed in this study using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The cytotoxicity of the sealers was tested after one, 4, and 7 d. Human dental pulp stem cell proliferation was concluded using an MTT assay. Cells not treated with sealer extract were used as controls. The absorption levels were measured using an Eliza spectrophotometer. P was set at 0.05 when the percentage of cell proliferation was matched between groups and observation times using one-way analysis of variance (ANOVA).During the second passage (P2), human dental pulp stem cells displayed a single morphological and phenotypic trait, with fibroblast morphology being the most common. There were no appreciable variations between the four groups after a day. There was a notable variation in the average percentage of cell proliferation between the groups after 4 and 7 days. The control group had the highest percentage, followed by the GuttaFlow Bioseal group, the Well Root St group, and the AH-Plus group, which had the lowest percentage. For every sealing group, after one day, the highest mean percentage of cell proliferation was recorded, followed by day four, and after day seven, the lowest mean percentage. The observation periods showed minimal cytotoxic effects of GuttaFlow Bioseal, whereas AH-Plus was the most cytotoxic to human dental pulp stem cells. The highest mean percentage of cell proliferation for all sealers was recorded on day one., (© 2024. The Author(s).)

Oxytocin has various effects ranging from promoting labor in pregnant women to alleviating stress. Recently, we reported the hair growth-promoting effects of oxytocin in hair follicle organoids. However, its clinical application faces challenges such as rapid degradation in vivo and poor permeability due to its large molecular weight. Therefore, in this study, we investigated the effects of the oxytocin receptor (OXTR) agonists WAY267464 and LIT001 as alternatives to oxytocin on hair growth. Human dermal papilla (DP) cells were cultured in WAY267464 or LIT001-supplemented medium. The addition of WAY267464 and LIT001 increased the expression of hair growth-related genes in DP cells. We tested the hair growth-promoting effects of WAY267464 and LIT001 using hair follicle organoids in vitro and found that they significantly promoted hair follicle sprouting. Thus, our findings indicate that WAY267464 and LIT001 are potential hair growth agents and may encourage further research on the development of novel hair growth agents targeting OXTR in patients with alopecia., (© 2024. The Author(s).)

With the increasing demand for exercise, the population of patients with ankle sprain to anterior talofibular ligament injury has the characteristics of a large base and high requirements for returning to sports, and how to promote the repair of damaged ligaments from a microscopic perspective is an urgent problem to be solved. In many studies, human amniotic mesenchymal stem cells have strong differentiation ability, and can be induced to continuously differentiate into ligament cells to achieve the purpose of repairing damaged ligaments. Human amniotic stem cells were extracted and cultured from human amniotic tissues, evaluated by cell identification and other techniques, and evaluated into ligament differentiation by toluidine blue, alizarin red, oil red O staining and detection of ligament cell differentiation, protein detection by Western blot, mRNA level by qPCR, and finally, the targeted binding relationship between miR-16a-5p and mRNA FGF2 was verified by double luciferase reporter assay. The expression of collagen type 1 (COL 1), collagen type 3 (COL3), SCX and MKX was increased by overexpression of mRNA FGF2, respectively, and miR-16a-5p had a targeted effect on FGF2 and regulated the ligamentous differentiation of human amniotic mesenchymal stem cells. We found that the regulatory effect of overexpressed mRNA FGF2 on mesenchymal stem cells could be inhibited by up-regulation of miR-16a-5p, while the knockdown of FGF2 could reverse the regulatory effect of miR-16a-5p inhibition on ligament-forming differentiation of human amniotic mesenchymal stem cells. In this study, we discovered the existence of the miR-16a-5p-FGF2 axis in human amniotic mesenchymal stem cells, and the differentiation of human amniotic mesenchymal stem cells into ligamentous cells can be regulated by regulating various links in this axis., (© 2024. The Author(s).)

Human mesenchymal stem cells (hMSCs) with extended lifespan and differentiation potential that can recapitulate in vivo characteristics could significantly contribute to basic research, drug development, and cell therapy. Specifically, they could ensure a stable supply of specific cellular resources, and possibly extracellular vesicles. Here, we established a technology for extending the lifespan while maintaining differentiation potential, termed "rejuvenation," of hMSCs (rej-hMSCs) using nonintegrative and conditionally removable temperature-sensitive Sendai virus (SeV) vectors. Various immortalizing factors (i.e., Bmi-1, hTERT, SV40T, and/or HPV E6/E7) were first introduced by the SeV vector into the cells. A combination of three SeVs with Bmi-1, hTERT, or SV40T conferred markedly improved cell proliferation and cloning ability while maintaining differentiation potential and a normal karyotype. An extended lifespan was also demonstrated in other cell types. The rejuvenation of long-passaged or aged hMSCs was also confirmed. SeV vectors were rapidly removed as a function of cell doubling by increasing the temperature from 35 °C to 37 °C or higher, while proliferative ability was maintained. Following FACS sorting, the complete removal of SeV vectors was confirmed by qPCR analyses. Therefore, our cell rejuvenation technology could contribute to research and clinical applications by enabling the supply of modified cells without damaging host chromosomes., (© 2024. The Author(s).)

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by β-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis., (© 2024. The Author(s).)

Treatment of Limbal Stem Cell Deficiency (LSCD), based on autologous transplantation of the patient's stem cells, is one of the few medical stem cell therapies approved by the European Medicines Agency (EMA). It relies on isolating and culturing in vivo Limbal Epithelial Stem Cells (LESC) and then populating them on the fibrin substrate, creating a scaffold for corneal epithelial regeneration. Such a solution is then implanted into the patient's eye. The epithelial cell culture process is specific, and its results strongly depend on the initial cell seeding density. Achieving control of the density and repeatability of the process is a desirable aim and can contribute to the success of the therapy. The study aimed to test bioprinting as a potential technique to increase the control over LESCs seeding on a scaffold and improve process reproducibility. Cells were applied to 0.5 mm thick, flat, transparent fibrin substrates using extrusion bioprinting; the control was the traditional manual application of cells using a pipette. The use of 3D printer enabled uniform coverage of the scaffold surface, and LESCs density in printed lines was close to the targeted value. Moreover, printed cells had higher cell viability than those seeded traditionally (91.1 ± 8.2% vs 82.6 ± 12.8%). The growth rate of the epithelium was higher in bioprinted samples. In both methods, the epithelium had favorable phenotypic features (p63 + and CK14 +). 3D printing constitutes a promising approach in LSCD therapy. It provides favorable conditions for LESCs growth and process reproducibility. Its application may lead to reduced cell requirements, thereby to using fewer cells on lower passages, which will contribute to preserving LESCs proliferative potential., (© 2024. The Author(s).)