Neural Crest Stem Cells Research Articles

EPO regulates neuronal differentiation of adult human neural-crest derived stem cells in a sex-specific manner

BackgroundSexual differences in the biology of human stem cells are increasingly recognized to influence their proliferation, differentiation and maturation. Especially in neurodegenerative diseases such as Alzheimers disease (AD), Parkinson’s disease (PD) or ischemic stroke, sex is a key player for disease progression and recovery of damaged tissue. Recently, the glycoprotein hormone erythropoietin (EPO) has been implicated as a regulator of neuronal differentiation and maturation in female rats.MethodsIn this study, we used adult human neural crest-derived stem cells (NCSCs) as a model system for exploring potential sex specific effects of EPO on human neuronal differentiation. We started with expression validation of the specific EPO receptor (EPOR) by performing PCR analysis in the NCSCs. Next, EPO mediated activation of nuclear factor-κB (NF-κB) via Immunocytochemistry (ICC) was performed, followed by investigating the sex-specific effects of EPO on neuronal differentiation by determining morphological changes in axonal growth and neurite formation accompanied by ICC.ResultsUndifferentiated male and female NCSCs showed a ubiquitous expression of the EPO receptor (EPOR). EPO treatment resulted in a statistically profound (male p = 0.0022, female p = 0.0012) nuclear translocation of NF-κB RELA in undifferentiated NCSCs of both sexes. But after one week of neuronal differentiation, we could show a highly significant (p = 0,0079) increase of nuclear NF-κB RELA in females only. In contrast, we observed a strong decrease (p = 0,0022) of RELA activation in male neuronal progenitors. Extending the view on the role of sex during human neuronal differentiation, here we demonstrate a significant increase of axon lengths in female NCSCs-derived neurons upon EPO-treatment (+ EPO: 167,73 (SD = 41,66) µm, w/o EPO: 77,68 (SD = 18,31) µm) compared to their male counterparts (+ EPO: 68,37 (SD = 11,97) µm, w/o EPO: 70,23 (SD = 12,89) µm).ConclusionOur present findings therefore show for the first time an EPO-driven sexual dimorphism in neuronal differentiation of human neural-crest derived stem cells and emphasize sex-specific variability as a crucial parameter in stem cell biology and for treating neurodegenerative diseases.

The Suppression Effects of Fat Mass and Obesity Associated Gene on the Hair Follicle-Derived Neural Crest Stem Cells Differentiating into Melanocyte by N6-Methyladenosine Modifying Microphthalmia-Associated Transcription Factor.

Melanocyte (MC), derived from neural crest stem cell (NCSC), are involved in the production of melanin. The mechanism by which NCSC differentiates to MC remains unclear. N6-methyladenosine (m6A) modification was applied to discuss the potential mechanism. NCSCs were isolated from hair follicles of rats, and were obtained for differentiation. Cell viability, tyrosinase secretion and activity, and transcription factors were combined to evaluated the MC differentiation. RT-qPCR was applied to determine mRNA levels, and western blot were used for protein expression detection. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RNA immunoprecipitation was used to access the protein binding relationship. In current work, NCSCs were successfully differentiated into MCs. Fat mass and obesity associated gene (FTO) was aberrant downregulated in MCs, and elevated FTO suppressed the differentiation progress of NCSCs into MCs. Furthermore, microphthalmia-associated transcription factor (Mitf), a key gene involved in MC synthesis, was enriched by FTO in a m6A modification manner and degraded by FTO. Meanwhile, the suppression functions of FTO in the differentiation of NCSCs into MCs were reversed by elevated Mitf. In short, FTO suppressed the differentiating ability of hair follicle-derived NCSCs into MCs by m6A modifying Mitf.

The potential roles of dental pulp stem cells in peripheral nerve regeneration.

Peripheral nerve diseases are significantly correlated with severe fractures or trauma and surgeries, leading to poor life quality and impairment of physical and mental health. Human dental pulp stem cells (DPSCs) are neural crest stem cells with a strong multi-directional differentiation potential and proliferation capacity that provide a novel cell source for nerve regeneration. DPSCs are easily extracted from dental pulp tissue of human permanent or deciduous teeth. DPSCs can express neurotrophic and immunomodulatory factors and, subsequently, induce blood vessel formation and nerve regeneration. Therefore, DPSCs yield valuable therapeutic potential in the management of peripheral neuropathies. With the purpose of summarizing the advances in DPSCs and their potential applications in peripheral neuropathies, this article reviews the biological characteristics of DPSCs in association with the mechanisms of peripheral nerve regeneration.

Treating peripheral nerve injury-induced spinal cord degeneration and neuropathic pain with peripherally administrated stem cells.

Treating peripheral nerve injury-induced spinal cord degeneration and neuropathic pain with peripherally administrated stem cells.

Human Olfactory Ecto-mesenchymal Stem Cells Displaying Schwann-cell-like Phenotypes and Promoting Neurite Outgrowth in Vitro.

Strategies of Schwann cell (SC) transplantation for regeneration of peripheral nerve injury involve many limitations. Stem cells can be used as alternative cell source for differentiation into Schwann cells. Given the high potential of neural crest-derived stem cells for the generation of multiple cell lineages, in this research, we considered whether olfactory ectomesenchymal stem cells (OE-MSCs) derived from neural crest can spontaneously differentiate into SC lineage. OE-MSCs were isolated from human nasal mucosa and characterized by the mesenchymal and neural crest markers. The cells were cultured in glial growth factors-free medium and further investigated in terms of the phenotypic and functional properties. Immunocytochemical staining and real-time PCR analysis indicated that the cultured OE-MSCs expressed SCs markers, SOX10, p75, S100, GFAP and MBP, differentiation indicative. It was found that the cells could secrete neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Furthermore, after co-cultured with PC12, the mean neurite length was enhanced by OE-MSCs. The findings indicated that OE-MSCs could be differentiated spontaneously into SC-like phenotypes, suggesting their applications for transplantation in peripheral nerve injuries.

Leveraging Human Oral-Derived Neural Crest-Derived Stem Cell Homing (HoNCSCs-Homing) for Therapeutic Procedures in Regenerative Implant Dentistry-A Critical Analysis

Resident stem cell pools in many tissues/organs are responsible not only for tissue maintenance during physiologic turnover but also for the process of bone healing following dental implant insertion. Recent advances have been made toward inducing stem cell mobilization and directing patients' own cells to sites of interest for treating a broad spectrum of diseases in regenerative dentistry. An evolving body of work corroborates that delivering guidance cues can mobilize stem cells from the palate and drive these cells toward a specific region. In addition, the transplantation of cell-friendly biomaterials incorporating certain biomolecules has led to the regeneration of lost/damaged tissue without the need for delivering cellular materials manipulated ex vivo. Recently, cell homing has resulted in remarkable biological discoveries in the laboratory as well as great curative successes in preclinical scenarios. Here, we review using the results of an initial clinical- and histological-controlled study the biological evidence underlying in vivo cell mobilization and homing with the aim of leveraging endogenous reparative cells for therapeutic applications in dental regenerative implantology. Considering both the promise and the obstacles of this approach, we discuss the results how oral-derived neural crest-derived stem cells of the in vivo milieu can be modified to promote the native regenerative process and inspire future tissue-engineering design.

Exosomes from Hair Follicle Epidermal Neural Crest Stem Cells Promote Acellular Nerve Allografts to Bridge Rat Facial Nerve Defects.

Previous studies showed that acellular nerve allografts (ANAs) have been successfully utilized in repairing peripheral nerve defects, and exosomes produced by stem cells are useful in supporting axon regrowth after peripheral nerve injury. In this study, exosomes from hair follicle epidermal neural crest stem cells (EPI-NCSCs-Exos) combined with ANAs were used to bridge facial nerve defects. EPI-NCSCs-Exos were isolated by ultracentrifuge, and were identified. After coculture, EPI-NCSCs-Exos were internalized into dorsal root ganglions (DRGs) and schwann cells (SCs) in vitro, respectively. EPI-NCSCs-Exos elongate the length of axons and dendrites of DRGs, and accelerated the proliferation and migration of SCs, and increased neurotrophic factor expression of SCs as well. The next step was to assign 24 Sprague Dawley male rats randomly and equally into three groups: the autograft group, the ANA group, and the ANA + EPI-NCSCs-Exos group. Each rat manufactured a 5-mm gap of facial nerve defect and immediately bridged by the corresponding transplants, respectively. After surgery, behavioral changes and electrophysiological testing of each rat were observed and assessed. At 90 days postoperatively, the retrogradely fluorescent tracer-labeled neurons were successfully observed on the injured side in the three groups. Morphological changes of facial nerve regeneration were evaluated by transmission electron microscopy and semithin toluidine blue staining. The results showed that nerve fiber density, nerve fiber diameter, and myelin sheath thickness in the ANA group were significantly worse than those in the other two groups (P < 0.05). No significant difference in nerve fiber density and myelin sheath thickness was observed between the autograft group and the ANA + EPI-NCSCs-Exos group (P = 0.14; P = 0.23). Our data indicated that EPI-NCSCs-Exos facilitate ANAs to bridge facial nerve defects and have the potential to replace autograft therapy in clinic.

Deciphering the Role of p53 and TAp73 in Neuroblastoma: From Pathogenesis to Treatment.

Neuroblastoma (NB) is an embryonic cancer that develops from neural crest stem cells, being one of the most common malignancies in children. The clinical manifestation of this disease is highly variable, ranging from spontaneous regression to increased aggressiveness, which makes it a major therapeutic challenge in pediatric oncology. The p53 family proteins p53 and TAp73 play a key role in protecting cells against genomic instability and malignant transformation. However, in NB, their activities are commonly inhibited by interacting proteins such as murine double minute (MDM)2 and MDMX, mutant p53, ΔNp73, Itch, and Aurora kinase A. The interplay between the p53/TAp73 pathway and N-MYC, a known biomarker of poor prognosis and drug resistance in NB, also proves to be decisive in the pathogenesis of this tumor. More recently, a strong crosstalk between microRNAs (miRNAs) and p53/TAp73 has been established, which has been the focused of great attention because of its potential for developing new therapeutic strategies. Collectively, this review provides an updated overview about the critical role of the p53/TAp73 pathway in the pathogenesis of NB, highlighting encouraging clues for the advance of alternative NB targeted therapies.

Migration deficits of the neural crest caused by CXADR triplication in a human Down syndrome stem cell model

Down syndrome (DS) is the most common chromosomal abnormality in live-born infants and is caused by trisomy of chromosome 21. Most individuals with DS display craniofacial dysmorphology, including reduced sizes of the skull, maxilla, and mandible. However, the underlying pathogenesis remains largely unknown. Since the craniofacial skeleton is mainly formed by the neural crest, whether neural crest developmental defects are involved in the craniofacial anomalies of individuals with DS needs to be investigated. Here, we successfully derived DS-specific human induced pluripotent stem cells (hiPSCs) using a Sendai virus vector. When DS-hiPSCs were induced to differentiate into the neural crest, we found that trisomy 21 (T21) did not influence cell proliferation or apoptosis. However, the migratory ability of differentiated cells was significantly compromised, thus resulting in a substantially lower number of postmigratory cranial neural crest stem cells (NCSCs) in the DS group than in the control group. We further discovered that the migration defects could be partially attributed to the triplication of the coxsackievirus and adenovirus receptor gene (CXADR; an adhesion protein) in the DS group cells, since knockdown of CXADR substantially recovered the cell migratory ability and generation of postmigratory NCSCs in the DS group. Thus, the migratory deficits of neural crest cells may be an underlying cause of craniofacial dysmorphology in individuals with DS, which may suggest potential targets for therapeutic intervention to ameliorate craniofacial or other neural crest-related anomalies in DS.

Leveraging Human Oral-Derived Neural Crest-Derived Stem Cell Homing (hoNCSC-Homing) for Therapeutic Procedures in Regenerative Implant Dentistry-A Critical Analysis

Resident stem cell pools in many tissues/organs are responsible not only for tissue maintenance during physiologic turnover but also for the process of bone healing following dental implant insertion. Recent advances have been made toward inducing stem cell mobilization and directing patients’ own cells to sites of interest for treating a broad spectrum of diseases in regenerative dentistry. An evolving body of work corroborates that delivering guidance cues can mobilize stem cells from the palate and drive these cells toward a specific region. In addition, the transplantation of cell-friendly biomaterials incorporating certain biomolecules has led to the regeneration of lost/damaged tissue without the need for delivering cellular materials manipulated ex vivo. Recently, cell homing has resulted in remarkable biological discoveries in the laboratory as well as great curative successes in preclinical scenarios. Here, we review using the results of an initial clinical- and histological-controlled study the biological evidence underlying in vivo cell mobilization and homing with the aim of leveraging endogenous reparative cells for therapeutic applications in dental regenerative implantology. Considering both the promise and the obstacles of this approach, we discuss the results how oral-derived neural crest-derived stem cells of the in vivo milieu can be modified to promote the native regenerative process and inspire future tissue-engineering design.

Human Pluripotent Stem Cell-Based Models for Hirschsprung Disease: From 2-D Cell to 3-D Organoid Model.

Hirschsprung disease (HSCR) is a complex congenital disorder caused by defects in the development of the enteric nervous system (ENS). It is attributed to failures of the enteric neural crest stem cells (ENCCs) to proliferate, differentiate and/or migrate, leading to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. Due to the oligogenic nature of the disease, some HSCR conditions could not be phenocopied in animal models. Building the patient-based disease model using human induced pluripotent stem cells (hPSC) has opened up a new opportunity to untangle the unknowns of the disease. The expanding armamentarium of hPSC-based therapies provides needed new tools for developing cell-replacement therapy for HSCR. Here we summarize the recent studies of hPSC-based models of ENS in 2-D and 3-D culture systems. These studies have highlighted how hPSC-based models complement the population-based genetic screens and bioinformatic approaches for the discovery of new HSCR susceptibility genes and provide a human model for the close-to-physiological functional studies. We will also discuss the potential applications of these hPSC-based models in translational medicines and their advantages and limitations. The use of these hPSC-based models for drug discovery or cell replacement therapy likely leads to new treatment strategies for HSCR in the future. Further improvements in incorporating hPSC-based models with the human-mouse chimera model and organ-on-a-chip system for establishing a better disease model of HSCR and for drug discovery will further propel us to success in the development of an efficacious treatment for HSCR.

Development of Multi-target therapy for Alzheimer’s disease using human neural crest-derived stem cells

Development of Multi-target therapy for Alzheimer’s disease using human neural crest-derived stem cells

Hepatocyte Growth Factor Promotes Differentiation Potential and Stress Response of Human Stem Cells from Apical Papilla

Harsh local microenvironment, such as hypoxia and lack of instructive clues for transplanted stem cells, presents the serious obstacle for stem cell therapies’ efficacy. Therefore, continued efforts have been taken to improve stem cells’ viability and plasticity. Hepatocyte growth factor (HGF) has previously been reported to mitigate the complications of various human diseases in animal model studies and in some clinical trials. Besides, human stem cells from the root apical papilla (SCAP) are deemed a better resource of mesenchymal stem cells due to derived stem cells holding greater amplification ability in vitro compared with those from other dental resources. To move forward, evaluating effects and understanding underlying molecular mechanisms of HGF on SCAP for periodontal regeneration are needed. In this study, HGF was transgenically expressed in SCAP, and it was found that HGF enhanced osteo/dentinogenic differentiation capacity of SCAP compared with those of non-treated control in an ectopic mineralization model. Moreover, HGF reduced the apoptosis of SCAP under both normoxic and hypoxic conditions, whereas the combination of HGF and hypoxia exposure had inhibitory effects on cell proliferation during an 8-day in vitro culture period. Transcriptome analysis further revealed that suppressed cell cycle progression and activated BMP/TGFβ, Hedgehog, WNT, FGF, HOX, and other morphogen family members result upon HGF overexpression, which may render SCAP recapitulate part of neural crest stem cell characteristics. Moreover, strengthened stress response modulation such as unfolded protein response, macroautophagy, and anti-apoptotic molecules might explain the increased viability of SCAP. In all, our results imply that these potential mechanisms underlying HGF-promoting SCAP differentiation could be further elucidated and harnessed to improve periodontal tissue regeneration.

Engineering Nanofiber Scaffolds with Biomimetic Cues for Differentiation of Skin-Derived Neural Crest-like Stem Cells to Schwann Cells.

Our laboratory reported the derivation of neural crest stem cell (NCSC)-like cells from the interfollicular epidermis of the neonatal and adult epidermis. These keratinocyte (KC)-derived Neural Crest (NC)-like cells (KC-NC) could differentiate into functional neurons, Schwann cells (SC), melanocytes, and smooth muscle cells in vitro. Most notably, KC-NC migrated along stereotypical pathways and gave rise to multiple NC derivatives upon transplantation into chicken embryos, corroborating their NC phenotype. Here, we present an innovative design concept for developing anisotropically aligned scaffolds with chemically immobilized biological cues to promote differentiation of the KC-NC towards the SC. Specifically, we designed electrospun nanofibers and examined the effect of bioactive cues in guiding KC-NC differentiation into SC. KC-NC attached to nanofibers and adopted a spindle-like morphology, similar to the native extracellular matrix (ECM) microarchitecture of the peripheral nerves. Immobilization of biological cues, especially Neuregulin1 (NRG1) promoted the differentiation of KC-NC into the SC lineage. This study suggests that poly-ε-caprolactone (PCL) nanofibers decorated with topographical and cell-instructive cues may be a potential platform for enhancing KC-NC differentiation toward SC.

Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor.

SUMMARYLoss-of-function mutations in the polycomb repressive complex 2 (PRC2) occur frequently in malignant peripheral nerve sheath tumor, an aggressive sarcoma that arises from NF1-deficient Schwann cells. To define the oncogenic mechanisms underlying PRC2 loss, we use engineered cells that dynamically reassemble a competent PRC2 coupled with single-cell sequencing from clinical samples. We discover a two-pronged oncogenic process: first, PRC2 loss leads to remodeling of the bivalent chromatin and enhancer landscape, causing the upregulation of developmentally regulated transcription factors that enforce a transcriptional circuit serving as the cell’s core vulnerability. Second, PRC2 loss reduces type I interferon signaling and antigen presentation as downstream consequences of hyperactivated Ras and its cross talk with STAT/IRF transcription factors. Mapping of the transcriptional program of these PRC2-deficient tumor cells onto a constructed developmental trajectory of normal Schwann cells reveals that changes induced by PRC2 loss enforce a cellular profile characteristic of a primitive mesenchymal neural crest stem cell.

Role of dual specificity phosphatases (DUSPs) in melanoma cellular plasticity and drug resistance

Melanoma cells exhibit phenotypic plasticity that allows transition from a proliferative and differentiated phenotype to a more invasive and undifferentiated or transdifferentiated phenotype often associated with drug resistance. The mechanisms that control melanoma phenotype plasticity and its role in drug resistance are not fully understood. We previously demonstrated that emergence of MAPK inhibitor (MAPKi)-resistance phenotype is associated with decreased expression of stem cell proliferation genes and increased expression of MAPK inactivation genes, including dual specificity phosphatases (DUSPs). Several members of the DUSP family genes, specifically DUSP1, -3, -8 and -9, are expressed in primary and metastatic melanoma cell lines and pre-and post BRAFi treated melanoma cells. Here, we show that knockdown of DUSP1 or DUSP8 or treatment with BCI, a pharmacological inhibitor of DUSP1/6 decrease the survival of MAPKi-resistant cells and sensitizes them to BRAFi and MEKi. Pharmacological inhibition of DUSP1/6 upregulated nestin, a neural crest stem cell marker, in both MAPKi-sensitive cells and cells with acquired MAPKi-resistance. In contrast, treatment with BCI resulted in upregulation of MAP2, a neuronal differentiation marker, only in MAPKi-sensitive cells but caused downregulation of both MAP2 and GFAP, a glial marker, in all MAPKi-resistant cell lines. These data suggest that DUSP proteins are involved in the regulation of cellular plasticity cells and melanoma drug resistance and are potential targets for treatment of MAPKi-resistant melanoma.

PEDOT: PSS promotes neurogenic commitment of neural crest-derived stem cells.

Poly (3,4-ethylendioxythiophene) polystyrene sulphonate (PEDOT:PSS) is the workhorse of organic bioelectronics and is steadily gaining interest also in tissue engineering due to the opportunity to endow traditional biomaterials for scaffolds with conductive properties. Biomaterials capable of promoting neural stem cell differentiation by application of suitable electrical stimulation protocols are highly desirable in neural tissue engineering. In this study, we evaluated the adhesion, proliferation, maintenance of neural crest stemness markers and neurogenic commitment of neural crest-derived human dental pulp stem cells (hDPSCs) cultured on PEDOT:PSS nanostructured thin films deposited either by spin coating (SC-PEDOT) or by electropolymerization (ED-PEDOT). In addition, we evaluated the immunomodulatory properties of hDPSCs on PEDOT:PSS by investigating the expression and maintenance of the Fas ligand (FasL). We found that both SC-PEDOT and ED-PEDOT thin films supported hDPSCs adhesion and proliferation; however, the number of cells on the ED-PEDOT after 1 week of culture was significantly higher than that on SC-PEDOT. To be noted, both PEDOT:PSS films did not affect the stemness phenotype of hDPSCs, as indicated by the maintenance of the neural crest markers Nestin and SOX10. Interestingly, neurogenic induction was clearly promoted on ED-PEDOT, as indicated by the strong expression of MAP-2 and —Tubulin-III as well as evident cytoskeletal reorganisation and appreciable morphology shift towards a neuronal-like shape. In addition, strong FasL expression was detected on both undifferentiated or undergoing neurogenic commitment hDPSCs, suggesting that ED-PEDOT supports the expression and maintenance of FasL under both expansion and differentiation conditions.

Stem cells from human exfoliated deciduous teeth relieves Alzheimer's disease symptoms in SAMP8 mice by up-regulating the PPARγ pathway.

The pathology of Alzheimer's disease (AD) is complex and heterogeneous, and there are currently no drugs that can stop its progression. The failure of traditional chemical small-molecule drug development showed the weakness of single target and made researchers look to cell therapy with multiple regulatory effects. Stem cells from human exfoliated deciduous teeth (SHED) are a kind of neural crest-derived mesenchymal stem cells which have broad prospects in the treatment of neurodegenerative diseases. In this study, we demonstrated the therapeutic effects of SHED in AD mice, including behavioral improvement, neuronal protection, and alleviation of neuroinflammation. Tracking experiments on SHED showed that some of the transplanted cells could enter the brain. To elucidate the role played by the majority of cells transplanted into veins, blood proteomic assays were performed. Data are available via ProteomeXchange with identifier PXD030313. Among the altered proteins, the PPAR pathway related to energy metabolism was considered to be an important signaling pathway involved in regulation through gene ontology analysis and pathway analysis. Western blot showed that the transplantation of SHED improved the glucose metabolism in AD mice by increasing the PPARγ signaling pathway. These results suggested that SHED have a potential in relieving AD pathological symptoms and improving behavioral cognition. The therapeutic mechanism of SHED is related to up-regulating PPARγ signaling pathway and reducing neuronal damage.

The Role of SIBLING Proteins in Dental, Periodontal, and Craniofacial Development

The majority of dental, periodontal, and craniofacial tissues are derived from the neural crest cells and ectoderm. Neural crest stem cells are pluripotent, capable of differentiating into a variety of cells. These cells can include osteoblasts, odontoblasts, cementoblasts, chondroblasts, and fibroblasts, which are responsible for forming some of the tissues of the oral and craniofacial complex. The hard tissue forming cells deposit a matrix composed of collagen and non-collagenous proteins (NCPs) that later undergoes mineralization. The NCPs play a role in the mineralization of collagen. One such category of NCPs is the small integrin-binding ligand, the N-linked glycoprotein (SIBLING) family of proteins. This family is composed of dentin sialophosphosprotein (DSPP), osteopontin (OPN), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and matrix extracellular phosphoglycoprotein (MEPE). The SIBLING family is known to have regulatory effects in the mineralization process of collagen fibers and the maturation of hydroxyapatite crystals. It is well established that SIBLING proteins have critical roles in tooth development. Recent literature has described the expression and role of SIBLING proteins in other areas of the oral and craniofacial complex as well. The objective of the present literature review is to summarize and discuss the different roles the SIBLING proteins play in the development of dental, periodontal, and craniofacial tissues.

Implantation of a nerve protector embedded with human GMSC-derived Schwann-like cells accelerates regeneration of crush-injured rat sciatic nerves

BackgroundPeripheral nerve injuries (PNIs) remain one of the great clinical challenges because of their considerable long-term disability potential. Postnatal neural crest-derived multipotent stem cells, including gingiva-derived mesenchymal stem cells (GMSCs), represent a promising source of seed cells for tissue engineering and regenerative therapy of various disorders, including PNIs. Here, we generated GMSC-repopulated nerve protectors and evaluated their therapeutic effects in a crush injury model of rat sciatic nerves.MethodsGMSCs were mixed in methacrylated collagen and cultured for 48 h, allowing the conversion of GMSCs into Schwann-like cells (GiSCs). The phenotype of GiSCs was verified by fluorescence studies on the expression of Schwann cell markers. GMSCs encapsulated in the methacrylated 3D-collagen hydrogel were co-cultured with THP-1-derived macrophages, and the secretion of anti-inflammatory cytokine IL-10 or inflammatory cytokines TNF-α and IL-1β in the supernatant was determined by ELISA. In addition, GMSCs mixed in the methacrylated collagen were filled into a nerve protector made from the decellularized small intestine submucosal extracellular matrix (SIS-ECM) and cultured for 24 h, allowing the generation of functionalized nerve protectors repopulated with GiSCs. We implanted the nerve protector to wrap the injury site of rat sciatic nerves and performed functional and histological assessments 4 weeks post-surgery.ResultsGMSCs encapsulated in the methacrylated 3D-collagen hydrogel were directly converted into Schwann-like cells (GiSCs) characterized by the expression of S-100β, p75NTR, BDNF, and GDNF. In vitro, co-culture of GMSCs encapsulated in the 3D-collagen hydrogel with macrophages remarkably increased the secretion of IL-10, an anti-inflammatory cytokine characteristic of pro-regenerative (M2) macrophages, but robustly reduced LPS-stimulated secretion of TNF-1α and IL-1β, two cytokines characteristic of pro-inflammatory (M1) macrophages. In addition, our results indicate that implantation of functionalized nerve protectors repopulated with GiSCs significantly accelerated functional recovery and axonal regeneration of crush-injured rat sciatic nerves accompanied by increased infiltration of pro-regenerative (M2) macrophages while a decreased infiltration of pro-inflammatory (M1) macrophages.ConclusionsCollectively, these findings suggest that Schwann-like cells converted from GMSCs represent a promising source of supportive cells for regenerative therapy of PNI through their dual functions, neurotrophic effects, and immunomodulation of pro-inflammatory (M1)/pro-regenerative (M2) macrophages.