Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds： supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein （EGFP） was used to track cell division during hair cell transformation.5-Bromo-2′-deoxyuridine （BrdU） was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Inner ear hair cell regeneration A look from the past to the future

Most recent studies on regeneration of inner ear hair cells focus on use of stem cells, gene therapy and neurotrophic factors. Cochlear gene therapy has been successfully used in the treatment of neu- rosensory hearing loss. This suggests that cochlear hair cell regeneration is possible. The objective of this paper is to review research and clinical application of inner near hair cell regeneration.

Ephrin A2 protein expression in the regeneration and plasticity of cochlear hair cells in chicken following kanamycin ototoxicity

The results from this study showed that the thresholds of brainstem auditory-evoked potentials peak following 10 successive days of intramuscular injection of Roman chickens with kanamycin, starting 3 days after birth. Fluorescence immunohistochemistry analysis revealed few ganglion cells positively labeled for Ephrin A2 in the cochlea of experimental chickens from 2 days before until 7 days after the last kanamycin injection. The number of Ephrin A2-positive ganglion cell bodies was increased at 15 days after the last injection and was similar to that in normal chickens at 30 days following the cessation of kanamycin treatment. These experimental findings indicate that Ephrin A2 protein expression in the acoustic ganglia is synchronized with the connection damage and regeneration of cochlear hair cells after kanamycin exposure. Ephrin A2 may play an important role in the regeneration and plasticity of cochlear hair cells in the chick cochlea following kanamycin ototoxicity.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear

Hearing loss has become increasingly prevalent and causes considerable disability,thus gravely burdening the global economy.Irreversible loss of hair cells is a main cause of sensorineural hearing loss,and currently,the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids,but these are of limited benefit in patients.It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies.At present,how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research.Multi-ple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells,and in this article,we first review the principal mechanisms underlying hair cell reproduction.We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration,and we summarize current achievements in hair cell regeneration.Lastly,we discuss potential future approaches,such as small molecule drugs and gene therapy,which might be applied for regenerating functional hair cells in the clinic.

Three-dimensional cell-based strategies for liver regeneration

Liver regeneration and the development of effective therapies for liver failure remain formidable challenges in modern medicine.In recent years,the utilization of 3D cell-based strategies has emerged as a promising approach for addressing these urgent clinical requirements.This review provides a thorough analysis of the application of 3D cell-based approaches to liver regeneration and their potential impact on patients with end-stage liver failure.Here,we discuss various 3D culture models that incorporate hepatocytes and stem cells to restore liver function and ameliorate the consequences of liver failure.Furthermore,we explored the challenges in transitioning these innovative strategies from preclinical studies to clinical applications.The collective insights presented herein highlight the significance of 3D cell-based strategies as a transformative paradigm for liver regeneration and improved patient care.

Chemokine platelet factor 4 accelerates peripheral nerve regeneration by regulating Schwann cell activation and axon elongation

Schwann cells in peripheral nerves react to traumatic nerve injury by attempting to grow and regenerate.Howeve r,it is unclear what factors play a role in this process.In this study,we searched a GEO database and found that expression of platelet factor 4 was markedly up-regulated after sciatic nerve injury.Platelet factor is an important molecule in cell apoptosis,diffe rentiation,survival,and proliferation.Further,polymerase chain reaction and immunohistochemical staining confirmed the change in platelet factor 4 in the sciatic nerve at different time points after injury.Enzyme-linked immunosorbent assay confirmed that platelet factor 4 was secreted by Schwann cells.We also found that silencing platelet factor 4 decreased the proliferation and migration of primary cultured Schwann cells,while exogenously applied platelet factor 4 stimulated Schwann cell prolife ration and migration and neuronal axon growth.Furthermore,knocking out platelet factor 4 inhibited the prolife ration of Schwann cells in injured rat sciatic nerve.These findings suggest that Schwann cell-secreted platelet factor 4 may facilitate peripheral nerve repair and regeneration by regulating Schwann cell activation and axon growth.Thus,platelet factor 4 may be a potential therapeutic target for traumatic peripheral nerve injury.

Hydrogel loaded with bone marrow stromal cell-derived exosomes promotes bone regeneration by inhibiting inflammatory responses and angiogenesis

BACKGROUND Bone healing is a complex process involving early inflammatory immune regu-lation,angiogenesis,osteogenic differentiation,and biomineralization.Fracture repair poses challenges for orthopedic surgeons,necessitating the search for efficient healing methods.AIM To investigate the underlying mechanism by which hydrogel-loaded exosomes derived from bone marrow mesenchymal stem cells(BMSCs)facilitate the process of fracture healing.METHODS Hydrogels and loaded BMSC-derived exosome(BMSC-exo)gels were charac-terized to validate their properties.In vitro evaluations were conducted to assess the impact of hydrogels on various stages of the healing process.Hydrogels could recruit macrophages and inhibit inflammatory responses,enhance of human umbilical vein endothelial cell angiogenesis,and promote the osteogenic differen-tiation of primary cranial osteoblasts.Furthermore,the effect of hydrogel on fracture healing was confirmed using a mouse fracture model.RESULTS The hydrogel effectively attenuated the inflammatory response during the initial repair stage and subsequently facilitated vascular migration,promoted the formation of large vessels,and enabled functional vascularization during bone repair.These effects were further validated in fracture models.CONCLUSION We successfully fabricated a hydrogel loaded with BMSC-exo that modulates macrophage polarization and angiogenesis to influence bone regeneration.

Bhlhe40 protects cochlear hair cell-like HEI-OC1 cells against H_(2)O_(2) ‑triggered oxidative injury

Background:Cochlear hair cell injury is a common pathological feature of hearing loss.The basic helix-loop-helix family,member e40(Bhlhe40),a gene belonging to the basic helix-loop-helix(bHLH)family,exhibits strong transcriptional repression activity.Methods:Oxidative damage,in House Ear Institute-Organ of Corti 1(HEI-OC1)cells,was caused using hydrogen peroxide(H2O2).The Ad-Bhlhe40 particles were constructed to overexpress Bhlhe40 in HEI-OC1 cells.Various assays including cell counting kit-8(CCK-8),terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay(TUNEL),flow cytometry,immunofluorescence,and corresponding commercial kits were employed to investigate the impacts of Bhlhe40 on cell viability,apoptosis,oxidative stress levels,mitochondrial membrane potential and cellular senescence.Additionally,a dual-luciferase reporter assay was performed to confirm the targeting of the histone deacetylases 2(Hdac2)by Bhlhe40.Results:The results revealed that Bhlhe40 was downregulated in H_(2)O_(2)-treated HEI-OC1 cells,but its overexpression improved cell viability and mitigated H_(2)O_(2)-induced oxidative injury in HEI-OC1 cells with increase of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPx)activities and decrease of reactive oxygen species(ROS)levels.Besides,overexpression of Bhlhe40 suppressed H_(2)O_(2)-triggered cell senescence,as evidenced by the fact that the upregulation of P53,P21,and P16 in HEI-OC1 cells treated with H2O2 were all alleviated by Bhlhe40 overexpression.And we further verified that overexpression of Bhlhe40 could inhibit the expression of Hdac2,which may be related to the repression of Hdac2 transcription.Conclusion:This study suggests that Bhlhe40 plays a protective role against senescence and oxidative damage in cochlear hair cells exposed to H2O2.

Regulatory T cells in skin regeneration and wound healing

As the body’s integumentary system,the skin is vulnerable to injuries.The subsequent wound healing processes aim to restore dermal and epidermal integrity and functionality.To this end,multiple tissue-resident cells and recruited immune cells cooperate to efficiently repair the injured tissue.Such temporally-and spatially-coordinated interplay necessitates tight regulation to prevent collateral damage such as overshooting immune responses and excessive inflammation.In this context,regulatory T cells(Tregs)hold a key role in balancing immune homeostasis and mediating cutaneous wound healing.A comprehensive understanding of Tregs’multifaceted field of activity may help decipher wound pathologies and,ultimately,establish new treatment modalities.Herein,we review the role of Tregs in orchestrating the regeneration of skin adnexa and catalyzing healthy wound repair.Further,we discuss how Tregs operate during fibrosis,keloidosis,and scarring.

Oscillatory Dynamics of Heterogeneous Stem Cell Regeneration

Stem cell regeneration is an essential biological process in the maintenance of tissue homeostasis;dysregulation of stem cell regeneration may result in dynamic diseases that show oscillations in cell numbers.Cell heterogeneity and plasticity are necessary for the dynamic equilibrium of tissue homeostasis;however,how these features may affect the oscillatory dynamics of the stem cell regeneration process remains poorly understood.Here,based on a mathematical model of heterogeneous stem cell regeneration that includes cell heterogeneity and random transition of epigenetic states,we study the conditions to have oscillation solutions through bifurcation analysis and numerical simulations.Our results show various model system dynamics with changes in different parameters associated with kinetic rates,cellular heterogeneity,and plasticity.We show that introducing heterogeneity and plasticity to cells can result in oscillation dynamics,as we have seen in the homogeneous stem cell regeneration system.However,increasing the cell heterogeneity and plasticity intends to maintain tissue homeostasis under certain conditions.The current study is an initiatory investigation of how cell heterogeneity and plasticity may affect stem cell regeneration dynamics,and many questions remain to be further studied both biologically and mathematically.

Mechanism of mesenchymal stem cells in liver regeneration:Insights and future directions

Mesenchymal stem cells(MSCs)are a prevalent source for stem cell therapy and play a crucial role in modulating both innate and adaptive immune responses.Non-alcoholic fatty liver disease(NAFLD)is characterized by the accumulation of triglycerides in liver cells and involves immune system activation,leading to histological changes,tissue damage,and clinical symptoms.A recent publication by Jiang et al,highlighted the potential of MSCs to mitigate in NAFLD progression by targeting various molecular pathways,including glycolipid metabolism,inflammation,oxidative stress,endoplasmic reticulum stress,and fibrosis.In this editorial,we comment on their research and discuss the efficacy of MSC therapy in treating NAFLD.

Subcellular distribution of prohibitin 1 in rat liver during liver regeneration and its cellular implication

BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.

Potential plausible role of Wharton’s jelly mesenchymal stem cells for diabetic bone regeneration

This letter addresses the review titled“Wharton’s jelly mesenchymal stem cells:Future regenerative medicine for clinical applications in mitigation of radiation injury”.The review highlights the regenerative potential of Wharton’s jelly mesenchymal stem cells(WJ-MSCs)and describes why WJ-MSCs will become one of the most probable stem cells for future regenerative medicine.The potential plausible role of WJ-MSCs for diabetic bone regeneration should be noticeable,which will provide a new strategy for improving bone regeneration under diabetic conditions.

Runx2 regulates peripheral nerve regeneration to promote Schwann cell migration and re-myelination

Runx2 is a major regulator of osteoblast differentiation and function;however,the role of Runx2 in peripheral nerve repair is unclea r.Here,we analyzed Runx2expression following injury and found that it was specifically up-regulated in Schwann cells.Furthermore,using Schwann cell-specific Runx2 knocko ut mice,we studied peripheral nerve development and regeneration and found that multiple steps in the regeneration process following sciatic nerve injury were Runx2-dependent.Changes observed in Runx2 knoc kout mice include increased prolife ration of Schwann cells,impaired Schwann cell migration and axonal regrowth,reduced re-myelination of axo ns,and a block in macrophage clearance in the late stage of regeneration.Taken together,our findings indicate that Runx2 is a key regulator of Schwann cell plasticity,and therefore peripheral nerve repair.Thus,our study shows that Runx2 plays a major role in Schwann cell migration,re-myelination,and peripheral nerve functional recovery following injury.

Therapeutic Approach for Hair Growth and Regeneration Using Bioactive Formulation Containing Mesenchymal Stromal Cell-Derived Conditioned Medium

Background and Aims: Androgenetic alopecia (AGA) is a common form of hair loss in both men and women. Despite its high prevalence and associated patient morbidity, the approved therapeutic options are limited to finasteride and minoxidil. The present study is aimed at assessing the efficacy of hair serum formulation, Trichosera®containing Bone marrow-derived mesenchymal stromal cells conditioned media as an active ingredient, for hair fall control and hair regrowth in healthy Indian human volunteers. Methods: The product was made using a 20% concentration of 10X Conditioned Media along with excipients. The final product was tested for physicochemical parameters, biomarkers, total protein content and microbial limits as per our in-house specifications. Results: The primary irritation patch test showed that the product is non-irritant and dermatologically safe. A clinical study on 40 subjects was conducted to evaluate the effectiveness of the bioactive formulation in hair fall control and hair regrowth in healthy volunteers. Phototrichogram measurement showed hair density and hair growth rate increased significantly by 11.54% and 18.66% at week 24. Hair tensile strength also increased significantly by 41.10% at 12 weeks follow-up. Hair pull test, to see a reduction in pulled hair and comb’s test to show a decrease in hair fall significantly improved from week 4 onwards. There were no significant adverse events in response to the product application. Conclusion: It is concluded that the hair serum product is completely safe on direct application to the scalp and showed significant improvement in the hair growth rate, hair density, scalp condition and reduction in hair fall. .

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Mechanism by which Rab5 promotes regeneration and functional recovery of zebrafish Mauthner axons

Rab5 is a GTPase protein that is involved in intracellular membrane trafficking. It functions by binding to various effector proteins and regulating cellular responses, including the formation of transport vesicles and their fusion with the cellular membrane. Rab5 has been reported to play an important role in the development of the zebrafish embryo;however, its role in axonal regeneration in the central nervous system remains unclear. In this study, we established a zebrafish Mauthner cell model of axonal injury using single-cell electroporation and two-photon axotomy techniques. We found that overexpression of Rab5 in single Mauthner cells promoted marked axonal regeneration and increased the number of intra-axonal transport vesicles. In contrast, treatment of zebrafish larvae with the Rab kinase inhibitor CID-1067700markedly inhibited axonal regeneration in Mauthner cells. We also found that Rab5 activated phosphatidylinositol 3-kinase(PI3K) during axonal repair of Mauthner cells and promoted the recovery of zebrafish locomotor function. Additionally, rapamycin, an inhibitor of the mechanistic target of rapamycin downstream of PI3K, markedly hindered axonal regeneration. These findings suggest that Rab5 promotes the axonal regeneration of injured zebrafish Mauthner cells by activating the PI3K signaling pathway.

Low-temperature 3D-printed collagen/chitosan scaffolds loaded with exosomes derived from neural stem cells pretreated with insulin growth factor-1 enhance neural regeneration after traumatic brain injury

There are various clinical treatments for traumatic brain injury,including surgery,drug therapy,and rehabilitation therapy;howeve r,the therapeutic effects are limited.Scaffolds combined with exosomes represent a promising but challenging method for improving the repair of traumatic brain injury.In this study,we determined the ability of a novel 3D-printed collagen/chitosan scaffold loaded with exosomes derived from neural stem cells pretreated with insulin-like growth factor-1(3D-CC-INEXOS) to improve traumatic brain injury repair and functional recove ry after traumatic brain injury in rats.Composite scaffolds comprising collagen,chitosan,and exosomes derived from neural stem cells pretreated with insulin-like growth fa ctor-1(INEXOS) continuously released exosomes for 2weeks.Transplantation of 3D-CC-INExos scaffolds significantly improved motor and cognitive functions in a rat traumatic brain injury model,as assessed by the Morris water maze test and modified neurological seve rity scores.In addition,immunofluorescence staining and transmission electron microscopy showed that3D-CC-INExos implantation significantly improved the recove ry of damaged nerve tissue in the injured area.In conclusion,this study suggests that transplanted3D-CC-INExos scaffolds might provide a potential strategy for the treatment of traumatic brain injury and lay a solid foundation for clinical translation.

DUSP2 deletion with CRISPR/Cas9 promotes Mauthner cell axonal regeneration at the early stage of zebrafish

Axon regeneration of central neurons is a complex process that is tightly regulated by multiple extrinsic and intrinsic factors.The expression levels of distinct genes are changed after central neural system(CNS)injury and affect axon regeneration.A previous study identified dusp2 as an upregulated gene in zebrafish with spinal cord injury.Here,we found that dual specificity phosphatase 2(DUSP2)is a negative regulator of axon regeneration of the Mauthner cell(M-cell).DUSP2 is a phosphatase that mediates the dephosphorylation of JNK.In this study,we knocked out dusp2 by CRISPR/Cas9 and found that M-cell axons of dusp2(-/-)zebrafish had a better regeneration at the early stage after birth(within 8 days after birth),while those of dusp2^(+/-)zebrafish did not.Overexpression of DUSP2 in Tg(Tol 056)zebrafish by single-cell electroporation retarded the regeneration of M-cell axons.Western blotting results showed that DUSP2 knockout slightly increased the levels of phosphorylated JNK.These findings suggest that knocking out DUSP2 promoted the regeneration of zebrafish M-cell axons,possibly through enhancing JNK phosphorylation.

Role of transforming growth factor-βin peripheral nerve regeneration

Injuries caused by trauma and neurodegenerative diseases can damage the peripheral nervous system and cause functional deficits.Unlike in the central nervous system,damaged axons in peripheral nerves can be induced to regenerate in response to intrinsic cues after reprogramming or in a growth-promoting microenvironment created by Schwann cells.However,axon regeneration and repair do not automatically result in the restoration of function,which is the ultimate therapeutic goal but also a major clinical challenge.Transforming growth factor(TGF)is a multifunctional cytokine that regulates various biological processes including tissue repair,embryo development,and cell growth and differentiation.There is accumulating evidence that TGF-βfamily proteins participate in peripheral nerve repair through various factors and signaling pathways by regulating the growth and transformation of Schwann cells;recruiting specific immune cells;controlling the permeability of the blood-nerve barrier,thereby stimulating axon growth;and inhibiting remyelination of regenerated axons.TGF-βhas been applied to the treatment of peripheral nerve injury in animal models.In this context,we review the functions of TGF-βin peripheral nerve regeneration and potential clinical applications.