Nerve injury converts Schwann cells in a long-term repair-like state in human neuroma tissue.

Peripheral nerve injury (PNI) induces neuroma formation at the severed nerve stump resulting in impaired nerve regeneration and functional recovery in patients. So far, molecular mechanisms and cell types present in the neuroma impeding on regeneration have only sparsely been analyzed. Herein we compare resected human neuroma tissue with intact donor nerves from the same patient. Neuroma from several post-injury timepoints (1-13 months) were included, thereby allowing for temporal correlation with molecular and cellular processes. We observed reduced axonal area and percentage of myelin producing Schwann cells (SCs) compared to intact nerves. However, total SOX10 positive SC numbers were comparable. Notably, markers for SCs in a repair mode including c-JUN, the low-affinity neurotrophin receptor (NTR) p75, SHH (sonic hedgehog) and SC proliferation (phospho-histone H3) were upregulated in neuroma, suggesting presence of SCs in repair status. In agreement, in neuroma, pro-regenerative markers such as phosphorylated i.e. activated CREB (pCREB), ATF3, GAP43 and SCG10 were upregulated. In addition, neuroma tissue was infiltrated by several types of macrophages. Finally, when taken in culture, neuroma SCs were indistinguishable from controls SCs with regard to proliferation and morphology. However, cultured neuroma SCs retained a different molecular signature from control SCs including increased inflammation and reduced gene expression for differentiation markers such as myelin genes. In summary, human neuroma tissue consists of SCs with a repair status and is infiltrated strongly by several types of macrophages.

FGF21 inhibits ferroptosis caused by mitochondrial damage to promote the repair of peripheral nerve injury.

Introduction: Ferroptosis is a new type of cell death characterized by lipid peroxidation and iron dependency, representing an emerging disease regulation mechanism. The limited understanding of ferroptosis in peripheral nerve injury (PNI) complicates the management of such injuries. Mitochondrial dysfunction, which contributes to ferroptosis, further exacerbates the challenges of peripheral nerve repair. Methods: In this study, we established an in vitro model of Schwann cells model treated with TBHP and an in vivo sciatic nerve crush injury model in rats. These models were used to investigate the effects of fibroblast growth factor 21 (FGF21) on PNI, both in vitro and in vivo, and to explore the potential mechanisms linking injury-induced ferroptosis and mitochondrial dysfunction. Results: Our findings reveal that PNI triggers abnormal accumulation of lipid reactive oxygen species (ROS) and inactivates mitochondrial respiratory chain complex III, leading to mitochondrial dysfunction. This dysfunction catalyzes the oxidation of excessive polyunsaturated fatty acids, resulting in antioxidant imbalance and loss of ferroptosis suppressor protein 1 (FSP1), which drives lipid peroxidation. Additionally, irregular iron metabolism, defective mitophagy, and other factors contribute to the induction of ferroptosis. Importantly, we found that FGF21 attenuates the abnormal accumulation of lipid ROS, restores mitochondrial function, and suppresses ferroptosis, thus promoting PNI repair. Notably, glutathione peroxidase 4 (GPX4), a downstream target of nuclear factor E2-related factor 2 (Nrf2), and the ERK/Nrf2 pathway are involved in the regulation of ferroptosis by FGF21. Conclusion: FGF21 promotes peripheral nerve repair by inhibiting ferroptosis caused by mitochondrial dysfunction. Therefore, targeting mitochondria and ferroptosis represents a promising therapeutic strategy for effective PNI repair.

Hybrid construction of tissue-engineered nerve graft using skin derived precursors induced neurons and Schwann cells to enhance peripheral neuroregeneration.

Peripheral nerve injury is a major challenge in clinical treatment due to the limited intrinsic capacity for nerve regeneration. Tissue engineering approaches offer promising solutions by providing biomimetic scaffolds and cell sources to promote nerve regeneration. In the present work, we investigated the potential role of skin-derived progenitors (SKPs), which are induced into neurons and Schwann cells (SCs), and their extracellular matrix in tissue-engineered nerve grafts (TENGs) to enhance peripheral neuroregeneration. SKPs were induced to differentiate into neurons and SCs in vitro and incorporated into nerve grafts composed of a biocompatible scaffold including chitosan neural conduit and silk fibroin filaments. In vivo experiments using a rat model of peripheral nerve injury showed that TENGs significantly enhanced nerve regeneration compared to the scaffold control group, catching up with the autograft group. Histological analysis showed improved axonal regrowth, myelination and functional recovery in animals treated with these TENGs. In addition, immunohistochemical staining confirmed the presence of induced neurons and SCs within the regenerated nerve tissue. Our results suggest that SKP-induced neurons and SCs in tissue-engineered nerve grafts have great potential for promoting peripheral nerve regeneration and represent a promising approach for clinical translation in the treatment of peripheral nerve injury. Further optimization and characterization of these engineered constructs is warranted to improve their clinical applicability and efficacy.

Enteric glial cell diversification is influenced by spatiotemporal factors and source of neural progenitors in mice.

Previously focused primarily on enteric neurons, studies of the enteric nervous system (ENS) in both health and disease are now broadening to recognize the equally significant role played by enteric glial cells (EGCs). Commensurate to the vast array of gastrointestinal functions they influence, EGCs exhibit considerable diversity in terms of location, morphology, molecular profiles, and functional attributes. However, the mechanisms underlying this diversification of EGCs remain largely unexplored. To begin unraveling the mechanistic complexities of EGC diversity, the current study aimed to examine its spatiotemporal aspects in greater detail, and to assess whether the various sources of enteric neural progenitors contribute differentially to this diversity. Based on established topo-morphological criteria for categorizing EGCs into four main subtypes, our detailed immunofluorescence analyses first revealed that these subtypes emerge sequentially during early postnatal development, in a coordinated manner with the structural changes that occur in the ENS. When combined with genetic cell lineage tracing experiments, our analyses then uncovered a strongly biased contribution by Schwann cell-derived enteric neural progenitors to particular topo-morphological subtypes of EGCs. Taken together, these findings provide a robust foundation for further investigations into the molecular and cellular mechanisms governing EGC diversity.

Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis.

Fibro-adipogenic progenitors (FAPs) are muscle-resident mesenchymal progenitors that can contribute to muscle tissue homeostasis and regeneration, as well as postnatal maturation and lifelong maintenance of the neuromuscular system. Recently, traumatic injury to the peripheral nerve was shown to activate FAPs, suggesting that FAPs can respond to nerve injury. However, questions of how FAPs can sense the anatomically distant peripheral nerve injury and whether FAPs can directly contribute to nerve regeneration remained unanswered. Here, utilizing single-cell transcriptomics and mouse models, we discovered that a subset of FAPs expressing GDNF receptors Ret and Gfra1 can respond to peripheral nerve injury by sensing GDNF secreted by Schwann cells. Upon GDNF sensing, this subset becomes activated and expresses Bdnf. FAP-specific inactivation of Bdnf (Prrx1Cre; Bdnffl/fl) resulted in delayed nerve regeneration owing to defective remyelination, indicating that GDNF-sensing FAPs play an important role in the remyelination process during peripheral nerve regeneration. In aged mice, significantly reduced Bdnf expression in FAPs was observed upon nerve injury, suggesting the clinical relevance of FAP-derived BDNF in the age-related delays in nerve regeneration. Collectively, our study revealed the previously unidentified role of FAPs in peripheral nerve regeneration, and the molecular mechanism behind FAPs' response to peripheral nerve injury.

Enhancement of Heme-Oxygenase 1 in the Injured Peripheral Nerve Following Sulforaphane Administration Fosters Regeneration via Proliferation and Maintenance of Repair Schwann Cells.

Nuclear erythroid 2-related factor 2 (Nrf2) and its downstream effector heme oxygenase 1 (HO-1) are commonly activated in response to cellular stresses. The elevated expression of HO-1 has been associated with markedly accelerated peripheral nerve regeneration. This study aimed to evaluate the impact of a naturally occurring dietary Nrf2/HO-1 activator-sulforaphane (SFN)-on regeneration in a murine sciatic nerve crush model. The beneficial safety profile of SFN has been thoroughly investigated and confirmed several times. Here, SFN was administered daily, starting immediately after C57BL/6 mice were subjected to sciatic nerve crush injury. Injured sciatic nerves were excised at various time points post injury for molecular, immunohistochemical and morphometric analyses. Moreover, functional assessment was performed by grip strength analysis and electrophysiology. Following SFN treatment, the early response to injury includes a modulation of autophagic pathways and marked upregulation of Nrf2/HO-1 expression. This enhancement of HO-1 expression was maintained throughout the regeneration phase and accompanied by a significant increase in repair Schwann cells. In these cells, elevated proliferation rates were observed. Significant improvements in grip strength test performance, nerve conduction velocity and remyelination were also noted following SFN treatment. Collectively, SFN modulates cytoprotective and autophagic pathways in the injured nerve, increasing the number of repair Schwann cells and contributing to effective nerve regeneration. Given the availability of SFN as a nutritional supplement, this compound might constitute a novel regenerative approach with broad patient accessibility and further studies on this topic are warranted.

Nuclear Magnetic Resonance Treatment Induces ßNGF Release from Schwann Cells and Enhances the Neurite Growth of Dorsal Root Ganglion Neurons In Vitro.

Peripheral nerve regeneration depends on close interaction between neurons and Schwann cells (SCs). After nerve injury, SCs produce growth factors and cytokines that are crucial for axon re-growth. Previous studies revealed the supernatant of SCs exposed to nuclear magnetic resonance therapy (NMRT) treatment to increase survival and neurite formation of rat dorsal root ganglion (DRG) neurons in vitro. The aim of this study was to identify factors involved in transferring the observed NMRT-induced effects to SCs and consequently to DRG neurons. Conditioned media of NMRT-treated (CM NMRT) and untreated SCs (CM CTRL) were tested by beta-nerve growth factor (ßNGF) ELISA and multiplex cytokine panels to profile secreted factors. The expression of nociceptive transient receptor potential vanilloid 1 (TRPV1) channels was assessed and the intracellular calcium response in DRG neurons to high-potassium solution, capsaicin or adenosine triphosphate was measured mimicking noxious stimuli. NMRT induced the secretion of ßNGF and pro-regenerative-signaling factors. Blocking antibody experiments confirmed ßNGF as the main factor responsible for neurotrophic/neuritogenic effects of CM NMRT. The TRPV1 expression or sensitivity to specific stimuli was not altered, whereas the viability of cultured DRG neurons was increased. Positive effects of CM NMRT supernatant on DRG neurons are primarily mediated by increased ßNGF levels.

Inter Arytenoid Granular Cell Tumor of Larynx-A Rare Case Report.

Granular cell tumors (GCTs) are uncommon neoplasms, often originating from Schwann cells, with granular cytoplasm being a hallmark feature. Laryngeal GCTs, comprising 3% to 10% of cases, present diagnostic challenges due to their resemblance to squamous cell carcinoma. We present a case of a 46-year-old male with throat pain, diagnosed with a laryngeal GCT. Histopathological examination and immunohistochemistry confirmed the diagnosis. Laryngeal GCTs typically manifest as small, firm submucosal nodules, posing challenges in differentiation from vocal fold polyps. Diagnosis relies on histological examination, with characteristic features including eosinophilic granular cytoplasm and positive staining for specific markers. Malignant transformation, though rare, necessitates vigilant monitoring and accurate diagnosis. Treatment involves complete surgical excision with long-term follow-up to detect recurrence. This case underscores the importance of awareness and accurate diagnosis in managing laryngeal GCTs, ensuring timely intervention and optimal patient outcomes.

Vestibular schwannoma causing normal pressure hydrocephalus.

Vestibular schwannoma is a common benign tumour that may cause local complications. However, vestibular schwannoma has a known association with communicating hydrocephalus presenting with symptoms of normal pressure hydrocephalus and requiring treatment by ventricular shunting or tumour resection. We report a 79-year-old woman who presented with subacute gait apraxia, cognitive impairment and urinary incontinence. CT and MR imaging identified a 20 mm vestibular schwannoma and communicating hydrocephalus; her cerebrospinal fluid (CSF) protein was elevated. Her symptoms improved following ventriculoperitoneal shunt insertion. The mechanism by which non-obstructing vestibular schwannoma causes hydrocephalus is unclear, but hyperproteinorrachia is probably important, likely by impeding CSF resorption.

Melittin Alleviates Oxidative Stress Injury in Schwann Cells by Targeting Interleukin-1 Receptor Type 1 to Downregulate Nuclear Factor Kappa B-Mediated Inflammatory Response In Vitro.

BACKGROUND AND OBJECTIVES: In ancient China, bee venom was widely used to treat various diseases. Although using bee venom is not currently a mainstream medical method, some have applied it to treat certain conditions, including idiopathic facial paralysis (IFP). Recently, melittin (Mel), the main active component of bee venom, has been shown strong anti-inflammatory and analgesic effects. However, how bee venom improves neurological dysfunction in facial paralysis remains unknown. This study aimed to investigate the anti-neurotraumatic effect of Mel on Schwann cells (SCs), the main cells of the neuron sheath, injured by oxidative stress. METHODS: A model of hypoxic SCs was established, and CCK-8 assay, siRNA transfection, enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, western blot, immunofluorescence, and cell ultrastructure analyses were conducted to investigate the mitigation of hypoxia-induced damage to SCs in vitro, revealing the effects of Mel on oxidative stress injury in SCs. RESULTS: The overexpression of HIF-1α in CoCl2-induced SCs (p < 0.05) indicated the establishment of an SCs hypoxia model. The proliferation and regeneration process of the hypoxic SCs enhanced in the Mel-treated group compared to the CoCl2 group has been proven through the CCK-8 experiment (p < 0.0001) and S-100 mRNA expression detection (p < 0.0001). The increased level of reactive oxygen species (ROS) (p < 0.001) and decreased superoxide dismutase (SOD) levels (p < 0.05) in the CoCl2-induced SCs indicated that Mel can alleviate the oxidative stress damage to SCs induced by CoCl2. Mel alleviated oxidative stress and inflammation in hypoxic SCs by reducing pro-inflammatory cytokines IL-1ß (p < 0.0001) and TNF-α (p < 0.0001). In addition, Mel augmented cellular vitality and regulated indicators related to oxygen metabolism, cell repair, neurometabolism, and vascular endothelial formation after hypoxia, such as C-JUN (p < 0.05), glial cell line-derived neurotrophic factor (GDNF; p < 0.001), vascular endothelial growth factor (VEGF; p < 0.05), hypoxia-inducible factor 1-alpha (HIF-1α; p < 0.05), interleukin-1 receptor type 1 (IL-1R1; p < 0.05), enolase1 (ENO1; p < 0.05), aldose reductase (AR; p < 0.01), SOD (p < 0.05), nerve growth factor (NGF; p < 0.05), and inducible nitric oxide synthase (iNOS; p < 0.05). In terms of its mechanism, Mel inhibited the expression of proteins associated with the NF-κB pathway such as IKK (p < 0.01), p65 (p < 0.05), p60 (p < 0.001), IRAK1 (p < 0.05), and increased IKB-α (p < 0.0001). Moreover, knocking out of IL-1R1 in the si-IL-1R1 group enhanced the therapeutic effect of Mel compared to the Mel-treated group (all of which p < 0.05). CONCLUSION: This research provided evidence of the substantial involvement of IL-1R1 in oxidative stress damage caused by hypoxia in SCs and proved that Mel alleviated oxidative stress injury in SCs by targeting IL-1R1 to downregulate the NF-κB-mediated inflammatory response. Mel could potentially serve as an innovative therapeutic approach for the treatment of IFP.

Reduced PTPRS expression promotes epithelial-mesenchymal transition of Schwann cells in NF1-related plexiform neurofibromas.

Plexiform neurofibromas (PNFs) are a prevalent and severe phenotype associated with NF1, characterized by a high teratogenic rate and potential for malignant transformation. The growth and recurrence of PNFs are attributed to aberrant proliferation and migration of Nf1-deficient Schwann cells. Protein tyrosine phosphatase receptor S (PTPRS) is believed to modulate cell migration and invasion by inhibiting the EMT process in NF1-derived malignant peripheral nerve sheath tumors. Nevertheless, the specific role of PTPRS in NF1-derived PNFs remains to be elucidated. The study utilized the GEO database and tissue microarray to illustrate a decrease in PTPRS expression in PNF tissues, linked to tumor recurrence. Furthermore, the down- and over-expression of PTPRS in Nf1-deficient Schwann cell lines resulted in the changes of cell migration and EMT processes. Additionally, RTK assay and WB showed that PTPRS knockdown can promote EGFR expression and phosphorylation. The restoration of EMT processes disrupted by alterations in PTPRS levels in Schwann cells can be achieved through EGFR knockdown and EGFR inhibitor. Moreover, high EGFR expression has been significantly correlated with poor prognosis. These findings underscore the potential role of PTPRS as a tumor suppressor in the recurrence of PNF via the regulation of EGFR-mediated EMT processes, suggesting potential targets for future clinical interventions.

Joint single-cell genetic and transcriptomic analysis reveal pre-malignant SCP-like subclones in human neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a heterogeneous embryonal malignancy and the deadliest tumor of infancy. It is a complex disease that can result in diverse clinical outcomes. In some children, tumors regress spontaneously. Others respond well to existing treatments. But for the high-risk group, which constitutes approximately 40% of all patients, the prognosis remains dire despite collaborative efforts in basic and clinical research. While its exact cellular origin is still under debate, NB is assumed to arise from the neural crest cell lineage including multipotent Schwann cell precursors (SCPs), which differentiate into sympatho-adrenal cell states eventually producing chromaffin cells and sympathoblasts. METHODS: To investigate clonal development of neuroblastoma cell states, we performed haplotype-specific analysis of human tumor samples using single-cell multi-omics, including joint DNA/RNA sequencing of sorted single cells (DNTR-seq). Samples were also assessed using immunofluorescence stainings and fluorescence in-situ hybridization (FISH). RESULTS: Beyond adrenergic tumor cells, we identify subpopulations of aneuploid SCP-like cells, characterized by clonal expansion, whole-chromosome 17 gains, as well as expression programs of proliferation, apoptosis, and a non-immunomodulatory phenotype. CONCLUSION: Aneuploid pre-malignant SCP-like cells represent a novel feature of NB. Genetic evidence and tumor phylogeny suggest that these clones and malignant adrenergic populations originate from aneuploidy-prone cells of migrating neural crest or SCP origin, before lineage commitment to sympatho-adrenal cell states. Our findings expand the phenotypic spectrum of NB cell states. Considering the multipotency of SCPs in development, we suggest that the transformation of fetal SCPs may represent one possible mechanism of tumor initiation in NB with chromosome 17 aberrations as a characteristic element.

Melanocyte lineage dynamics in development, growth and disease.

Melanocytes evolved to produce the melanin that gives colour to our hair, eyes and skin. The melanocyte lineage also gives rise to melanoma, the most lethal form of skin cancer. The melanocyte lineage differentiates from neural crest cells during development, and most melanocytes reside in the skin and hair, where they are replenished by melanocyte stem cells. Because the molecular mechanisms necessary for melanocyte specification, migration, proliferation and differentiation are co-opted during melanoma initiation and progression, studying melanocyte development is directly relevant to human disease. Here, through the lens of advances in cellular omic and genomic technologies, we review the latest findings in melanocyte development and differentiation, and how these developmental pathways become dysregulated in disease.

Piezo1 promotes peripheral nerve fibrotic scar formation through Schwann cell senescence.

After peripheral nerve injury (PNI), the long-term healing process at the injury site involves a progressive accumulation of collagen fibers and the development of localized scar tissue. Excessive formation of scar tissue within nerves hinders the process of nerve repair. In this study, we demonstrate that scar formation following nerve injury induces alterations in the local physical microenvironment, specifically an increase in nerve stiffness. Recent research has indicated heightened expression of Piezo1 in Schwann cells (SCs). Our findings also indicate Piezo1 expression in SCs and its association with suppressed proliferation and migration. Transcriptomic data suggests that activation of Piezo1 results in elevated expression of senescence-associated genes. GO enrichment analysis reveals upregulation of the TGF-ß pathway. Overall, our study highlights the potential for Piezo1-induced signaling to regulate SC senescence and its potential significance in the pathophysiology of fibrotic scar formation surrounding peripheral nerves.

EGR3 Inhibits Tumor Progression by Inducing Schwann Cell-Like Differentiation.

The mechanism and function of the expression of Schwann characteristics by nevus cells in the mature zone of the dermis are unknown. Early growth response 3 (EGR3) induces Schwann cell-like differentiation of melanoma cells by simulating the process of nevus maturation, which leads to a strong phenotypic transformation of the cells, including the formation of long protrusions and a decrease in cell motility, proliferation, and melanin production. Meanwhile, EGR3 regulates the levels of myelin protein zero (MPZ) and collagen type I alpha 1 chain (COL1A1) through SRY-box transcription factor 10 (SOX10)-dependent and independent mechanisms, by binding to non-strictly conserved motifs, respectively. Schwann cell-like differentiation demonstrates significant benefits in both in vivo and clinical studies. Finally, a CD86-P2A-EGR3 recombinant mRNA vaccine is developed which leads to tumor control through forced cell differentiation and enhanced immune infiltration. Together, these data support further development of the recombinant mRNA as a treatment for cancer.

TFEB/3 Govern Repair Schwann Cell Generation and Function Following Peripheral Nerve Injury.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

Loss of YAP in Schwann cells improves HNPP pathophysiology.

Rapid nerve conduction in the peripheral nervous system (PNS) is facilitated by the multilamellar myelin sheath encasing many axons of peripheral nerves. Charcot-Marie-Tooth type 1A (CMT1A), and hereditary neuropathy with liability to pressure palsy (HNPP) are common demyelinating inherited peripheral neuropathies and are caused by mutations in the peripheral myelin protein 22 (PMP22) gene. Duplication of PMP22 leads to its overexpression and causes CMT1A, while its deletion results in PMP22 under expression and causes HNPP. Here, we investigated novel targets for modulating the protein level of PMP22 in HNPP. We found that genetic attenuation of the transcriptional coactivator Yap in Schwann cells reduces p-TAZ levels, increased TAZ activity, and increases PMP22 in peripheral nerves. Based on these findings, we ablated Yap alleles in Schwann cells of the Pmp22-haploinsufficient mouse model of HNPP and identified fewer tomacula on morphological assessment and improved nerve conduction in peripheral nerves. These findings suggest YAP modulation may be a new avenue for treatment of HNPP.

Role of Piezo2 in Schwann Cell Volume Regulation and Its Impact on Neurotrophic Release Regulation.

BACKGROUND/AIMS: Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aß-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aß and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. METHODS: Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. RESULTS: Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. CONCLUSION: Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.

Hyaluronic acid hybrid formulations optimised for 3D printing of nerve conduits and the delivery of the novel neurotrophic-like compound tyrosol to enhance peripheral nerve regeneration via Schwann cell proliferation.

Peripheral nerve injuries, predominantly affecting individuals aged 20-40, pose significant healthcare challenges, with current surgical methods often failing to achieve complete functional recovery. This study focuses on the development of 3D printed hydrogel nerve conduits using modified hyaluronic acid (HA) for potentially enhancing peripheral nerve regeneration. Hyaluronic acid was chemically altered with cysteamine HCl and methacrylic anhydride to create thiolated HA (HA-SH) and methacrylated HA (HA-MA), achieving a modification degree of approximately 20 %. This modification was crucial to maintain the receptor interaction of HA. The modified HA was rigorously tested to ensure cytocompatibility in neuronal and glial cell lines. Subsequently, various 3D printed HA formulations were evaluated, focusing on improving HA's inherent mechanical weaknesses. These formulations were assessed for cytotoxicity through direct contact and elution extract testing, confirming their safety over a 24-h period. Among the neurotrophic compounds tested, Tyrosol emerged as the most effective in promoting Schwann cell proliferation in vitro. The 3D printed HA system demonstrated proficiency in loading and releasing Tyrosol at physiological pH. The findings from this research highlight the promising role of 3D printed HA and Tyrosol in the field of nerve tissue engineering, offering a novel approach to peripheral nerve regeneration.

Robust differentiation of human pluripotent stem cells into Schwann cells.

Research into Schwann cell (SC)-related diseases has been hampered by the difficulty of obtaining human-derived SCs, which have limited proliferative capacity. This has resulted in a delay in progress in drug discovery and cell therapy targeting SCs. To overcome these limitations, we developed a robust method for inducing the differentiation of human induced pluripotent stem cells (hiPSCs) into SCs. We established hiPSC lines and successfully generated high-purity Schwann cell precursors (SCPs) from size-controlled hiPSC aggregates by precisely timed treatment with our proprietary enzyme solution. Such SCPs were successfully expanded and further differentiated into myelin basic protein (MBP) expressing SC populations when treated with an appropriate medium containing dibutyryl-cAMP (db-cAMP). These differentiated cells secreted factors that induced neurite outgrowth in vitro. Our method allows for the efficient and stable production of SCPs and SCs from hiPSCs. This robust induction and maturation method has the potential to be a valuable tool in drug discovery and cell therapy targeting SC-related diseases.