ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

Immune escape and metastasis mechanisms in melanoma: breaking down the dichotomy.

Melanoma is one of the most lethal neoplasms of the skin. Despite the revolutionary introduction of immune checkpoint inhibitors, metastatic spread, and recurrence remain critical problems in resistant cases. Melanoma employs a multitude of mechanisms to subvert the immune system and successfully metastasize to distant organs. Concerningly, recent research also shows that tumor cells can disseminate early during melanoma progression and enter dormant states, eventually leading to metastases at a future time. Immune escape and metastasis have previously been viewed as separate phenomena; however, accumulating evidence is breaking down this dichotomy. Recent research into the progressive mechanisms of melanoma provides evidence that dedifferentiation similar to classical epithelial to mesenchymal transition (EMT), genes involved in neural crest stem cell maintenance, and hypoxia/acidosis, are important factors simultaneously involved in immune escape and metastasis. The likeness between EMT and early dissemination, and differences, also become apparent in these contexts. Detailed knowledge of the mechanisms behind "dual drivers" simultaneously promoting metastatically inclined and immunosuppressive environments can yield novel strategies effective in disabling multiple facets of melanoma progression. Furthermore, understanding progression through these drivers may provide insight towards novel treatments capable of preventing recurrence arising from dormant dissemination or improving immunotherapy outcomes.

Bone Marrow Stem Cells Derived from Nerves Have Neurogenic Properties and Potential Utility for Regenerative Therapy.

Neurons and glia of the peripheral nervous system are derived from progenitor cell populations, originating from embryonic neural crest. The neural crest and vasculature are intimately associated during embryonic development and in the mature central nervous system, in which they form a neurovascular unit comprised of neurons, glia, pericytes, and vascular endothelial cells that play important roles in health and disease. Our group and others have previously reported that postnatal populations of stem cells originating from glia or Schwann cells possess neural stem cell qualities, including rapid proliferation and differentiation into mature glia and neurons. Bone marrow receives sensory and sympathetic innervation from the peripheral nervous system and is known to contain myelinating and unmyelinating Schwann cells. Herein, we describe a population of neural crest-derived Schwann cells residing in a neurovascular niche of bone marrow in association with nerve fibers. These Schwann cells can be isolated and expanded. They demonstrate plasticity in vitro, generating neural stem cells that exhibit neurogenic potential and form neural networks within the enteric nervous system in vivo following transplantation to the intestine. These cells represent a novel source of autologous neural stem cells for the treatment of neurointestinal disorders.

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.

Background and Objectives: 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. Methods and Results: 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. Conclusions: In short, FTO suppressed the differentiating ability of hair follicle-derived NCSCs into MCs by m6A modifying Mitf.

From Hair to the Brain: The Short-Term Therapeutic Potential of Human Hair Follicle-Derived Stem Cells and Their Conditioned Medium in a Rat Model of Stroke.

The short-term therapeutic impacts of stem cells and their derivatives were frequently reported in preclinical investigations of ischemic stroke (IS); however, several drawbacks including accessibility, abundancy, and ethical concerns limited their clinical application. We describe here for the first time the therapeutic potential of human hair follicle-derived stem cells (hHFSCs) and their conditioned medium (CM) in a rat model of IS. Furthermore, we hypothesized that a combination of cell therapy with repeated CM administration might enhance the restorative efficiency of this approach compared to each treatment alone. Middle cerebral artery occlusion was performed for 30 min to induce IS. Immediately after reperfusion, hHFSCs were transplanted through the intra-arterial route and/or hHFSC-CM administered intranasally. The neurological outcomes, short-term spatial working memory, and infarct size were evaluated. Furthermore, relative expression of seven target genes in three categories of neuronal markers, synaptic markers, and angiogenic markers was assessed. The hHFSCs and hHFSC-CM treatments improved neurological impairments and reduced infarct size in the IS rats. Moreover, molecular data elucidated that IS was accompanied by attenuation in the expression of neuronal and synaptic markers in the evaluated brain regions and the interventions rescued these expression changes. Although there was no considerable difference between hHFSCs and hHFSC-CM treatments in the improvement of neurological function and decrement of infarct size, combination therapy was more effective to reduce infarction and elevation of target gene expression especially in the hippocampus. These findings highlight the curative potential of hHFSCs and their CM in a rat model of IS.

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

Loss-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.

Therapeutic potential of hair follicle-derived stem cell intranasal transplantation in a rat model of ischemic stroke.

BACKGROUND: Stem cell-based therapy has received considerable attention as a potential candidate in the treatment of ischemic stroke; however, employing an appropriate type of stem cells and an effective delivery route are still challenging. In the present study, we investigated the therapeutic effect of safe, noninvasive, and brain-targeted intranasal administration of hair follicle-derived stem cells (HFSCs) in a rat model of ischemic stroke. METHODS: Stem cells were obtained from the adult rat hair follicles. In experiment 1, stroke was induced by 30 min middle cerebral artery occlusion (MCAO) and stem cells were intranasally transplanted immediately after ischemia. In experiment 2, stroke was induced by 120 min MCAO and stem cells were administered 24 h after cerebral ischemia. In all experimental groups, neurological performance, short-term spatial working memory and infarct volume were assessed. Moreover, relative expression of major trophic factors in the striatum and cortex was evaluated by the quantitative PCR technique. The end point of experiment 1 was day 3 and the end point of experiment 2 was day 15. RESULTS: In both experiments, intranasal administration of HFSCs improved functional performance and decreased infarct volume compared to the MCAO rats. Furthermore, NeuN and VEGF expression were higher in the transplanted group and stem cell therapy partially prevented BDNF and neurotrophin-3 over-expression induced by cerebral ischemia. CONCLUSIONS: These findings highlight the curative potential of HFSCs following intranasal transplantation in a rat model of ischemic stroke.

Intramuscular delivery of neural crest stem cell spheroids enhances neuromuscular regeneration after denervation injury.

BACKGROUND: Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical. METHODS: We established a streamlined protocol to derive, isolate, and characterize functional p75+ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro. RESULTS: NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids. CONCLUSIONS: We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration.

Neural Crest Stem Cells in Juvenile Angiofibromas.

The etiology of juvenile angiofibroma (JA) has been a controversial topic for more than 160 years. Numerous theories have been proposed to explain this rare benign neoplasm arising predominately in adolescent males, focusing mainly on either the vascular or fibrous component. To assess our hypothesis of JA's being a malformation arising from neural crest cells/remnants of the first branchial arch plexus, we performed immunohistochemical analyses of neural crest stem cells (NCSC) and epithelial-mesenchymal transition (EMT) candidates. Immunoexpression of the NCSC marker CD271p75 was observed in all investigated JA's (n = 22), mainly around the pathological vessels. Close to CD271p75-positive cells, high MMP3-staining was also observed. Additionally, from one JA with sufficient material, RT-qPCR identified differences in the expression pattern of PDGFRß, MMP2 and MMP3 in MACS®-separated CD271p75positive vs. CD271p75 negative cell fractions. Our results, together with the consideration of the literature, provide evidence that JA's represent a malformation within the first branchial arch artery/plexus remnants deriving from NCSC. This theory would explain the typical site of tumor origin as well as the characteristic tumor blood supply, whereas the process of EMT provides an explanation for the vascular and fibrous tumor component.

Enhanced Neural Differentiation of Epidermal Neural Crest Stem Cell by Synergistic Effect of Lithium carbonate and Crocin on BDNF and GDNF Expression as Neurotrophic Factors.

Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration of nerve cells. Due to the complexity of conditions in neurodegenerative diseases, combination therapy, including cell and drug therapy is important as a new therapeutic strategy. Epidermal neural crest stem cells (EPI-NCSCs) are among the best choices in cell therapy for various neurological diseases. In this study, the effect of Lithium carbonate and Crocin, considering their effects on cellular signaling pathways and neuroprotective properties were investigated on the expression of neurotrophic factors BDNF and GDNF in EPI-NCSCs. EPI-NCSCs were isolated from the hair follicle and treated with different concentrations of drugs [Lithium, Crocin, and lithium + Crocin] for 72h. Then, trial concentrations were selected by MTT assay. The cells were treated with selected concentrations (Lithium 1 mM, Crocin 1.5 mM, and for co-treatment Lithium 1 mM and Crocin 1 mM) for 7 days. The Real-Time PCR results indicated an increasing in expression of BDNF and GDNF in treated cells as compared with control (* p < 0.05, ** p < 0.01 and *** p < 0.001). The results in this study confirmed and supported the neuroprotective/neurogenesis effects of Lithium and Crocin. It also showed that the proposed protocol could be used to increase EPI-NCSCs differentiation potential into neural cells in cell therapy and combination therapy of neurodegenerative diseases.

Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells.

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer. Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50-70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11. Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2. Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Effect of scaffold properties on adhesion and maintenance of boundary cap neural crest stem cells in vitro.

Optimal combination of stem cells and biocompatible support material is a promising strategy for successful tissue engineering. The required differentiation of stem cells is crucial for functionality of engineered tissues and can be regulated by chemical and physical cues. Here we examined how boundary cap neural crest stem cells (bNCSCs) are affected when cultured in the same medium, but on collagen- or laminin-polyacrylamide (PAA) scaffolds of different stiffness (0.5, 1, or ~7 kPa). bNCSCs displayed marked differences in their ability to attach, maintain a large cell population and differentiate, depending on scaffold stiffness. These findings show that the design of physical cues is an important parameter to achieve optimal stem cell properties for tissue repair and engineering.

Substrate stiffness affects the morphology and gene expression of epidermal neural crest stem cells in a short term culture.

According to the intrinsic plasticity of stem cells, controlling their fate is a critical issue in cell-based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect the fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting a high level of plasticity. This study was conducted to define the influence of substrate stiffness on the lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modulus in the range of 0.7-30 kPa were synthesized and coated with collagen and stem cells were seeded on them for 24 hr. The obtained data showed that cells can attach faster to hydrogels compared with culture plate and cells on <1 kPa stiffness show more neuronal-like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin, and PDGFR-α increased on all of them and SOX-10 and doublecortin gene expression were higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of glial cell line-derived neurotrophic factor, neurotrophin-3, and vascular endothelial growth factor in these stem cells. On the basis of the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profiles similar to the nervous system and superior usefulness in therapeutic applications.

Co-transplantation of Epidermal Neural Crest Stem Cells and Olfactory Ensheathing Cells Repairs Sciatic Nerve Defects in Rats.

Cell-based therapy is an alternative strategy to improve outcomes of peripheral nerve injury (PNI). Epidermal neural crest stem cell (EPI-NCSC) is obtained from autologous tissue without immunological rejection, which could expand quickly in vitro and is suitable candidate for cell-based therapy. Olfactory ensheathing cell (OEC) could secrete multiple neurotrophic factors (NTFs), which is often used to repair PNI individually. However, whether the combination of EPI-NCSC and OEC have better effects on PNI repair remains unclear. Here we use EPI-NCSC and OEC co-transplantation in a rat sciatic nerve defect model to ascertain the effects and potential mechanisms of cells co-transplantation on PNI. The effect of EPI-NCSC and OEC co-transplantation on PNI is assessed by using a combination of immunohistochemistry (IHC), electrophysiological recording and neural function test. Co-transplantation of EPI-NCSC and OEC exerts a beneficial effect upon PNI such as better organized structure, nerve function recovery, and lower motoneuron apoptosis. IHC and enzyme-linked immuno sorbent assay (ELISA) further demonstrate that cells co-transplantation may improve PNI via the expression of brain derived growth factor (BDNF) and nerve growth factor (NGF) up-regulated by EPI-NCSC and OEC synergistically. Eventually, the results from this study reveal that EPI-NCSC and OEC co-transplantation effectively repairs PNI through enhancing the level of BDNF and NGF, indicating that cells co-transplantation may serve as a fruitful avenue for PNI in clinic treatment.

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(-/-) cells, descendants from the tumor originating cell. These NF1(-/-)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(-/-) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

Spatial Genomic Analysis: A Multiplexed Transcriptional Profiling Method that Reveals Subpopulations of Cells Within Intact Tissues.

Here, we present Spatial Genomic Analysis (SGA), a quantitative single-cell transcriptional profiling method that takes advantage of single-molecule imaging of individual transcripts for up to a hundred genes. SGA relies on a machine learning-based image analysis pipeline that performs cell segmentation and transcript counting in a robust way. SGA is suitable for various in situ applications and was originally developed to address heterogeneity in the neural crest, which is a transient embryonic stem cell population important for formation of various vertebrate body structures. After being specified as multipotent neural crest stem cells in the dorsal neural tube, they go through an epithelial to mesenchymal transition in order to migrate to different destinations around the body, and gradually turn from stem cells to progenitors prior to final commitment. The molecular details of this process remain largely unknown, and upon their emergence, the neural crest cells have been considered as a single homogeneous population. Technical limitations have restricted the possibility to parse the neural crest cell pool into subgroups according to multiplex gene expression properties. By using SGA, we were able to identify subgroups inside the neural crest niche in the dorsal neural tube. The high sensitivity of the method allows detection of low expression levels and we were able to determine factors not previously shown to be present in neural crest stem cells, such as pluripotency or lineage markers. Finally, SGA analysis also provides prediction of gene relationships within individual cells, and thus has broad utility for powerful transcriptome analyses in original biological contexts.

Optimal electrical stimulation boosts stem cell therapy in nerve regeneration.

Peripheral nerve injuries often lead to incomplete recovery and contribute to significant disability to approximately 360,000 people in the USA each year. Stem cell therapy holds significant promise for peripheral nerve regeneration, but maintenance of stem cell viability and differentiation potential in vivo are still major obstacles for translation. Using a made-in-house 96-well vertical electrical stimulation (ES) platform, we investigated the effects of different stimulating pulse frequency, duration and field direction on human neural crest stem cell (NCSC) differentiation. We observed dendritic morphology with enhanced neuronal differentiation for NCSCs cultured on cathodes subject to 20 Hz, 100µs pulse at a potential gradient of 200 mV/mm. We further evaluated the effect of a novel cell-based therapy featuring optimized pulsatile ES of NCSCs for in vivo transplantation following peripheral nerve regeneration. 15 mm critical-sized sciatic nerve injuries were generated with subsequent surgical repair in sixty athymic nude rats. Injured animals were randomly assigned into five groups (N = 12 per group): blank control, ES, NCSC, NCSC + ES, and autologous nerve graft. The optimized ES was applied immediately after surgical repair for 1 h in ES and NCSC + ES groups. Recovery was assessed by behavioral (CatWalk gait analysis), wet muscle-mass, histomorphometric, and immunohistochemical analyses at either 6 or 12 weeks after surgery (N = 6 per group). Gastrocnemius muscle wet mass measurements in ES + NCSC group were comparable to autologous nerve transplantation and significantly higher than other groups (p < 0.05). Quantitative histomorphometric analysis and catwalk gait analysis showed similar improvements by ES on NCSCs (p < 0.05). A higher number of viable NCSCs was shown via immunochemical analysis, with higher Schwann cell (SC) differentiation in the NCSC + ES group compared to the NCSC group (p < 0.05). Overall, ES on NCSC transplantation significantly enhanced nerve regeneration after injury and repair, and was comparable to autograft treatment. Thus, ES can be a potent alternative to biochemical and physical cues for modulating stem cell survival and differentiation. This novel cell-based intervention presents an effective and safe approach for improved outcomes after peripheral nerve repair.

Epidermal neural crest stem cell-derived glia enhance neurotrophic elements in an ex vivo model of spinal cord injury.

Growing evidence that cell-based therapies can improve recovery outcome in spinal cord injury (SCI) models substantiates their application for treatment of human with SCI. To address the effectiveness of these stem cells, potential candidates should be evaluated in proper SCI platform that allows direct real-time monitoring. In this study, the role of epidermal neural crest stem cells (EPI-NCSCs) was elucidated in an ex vivo model of SCI, and valproic acid (VPA) was administered to ameliorate the inhospitable context of injury for grafted EPI-NCSCs. Here the contusion was induced in organotypic spinal cord slice culture at day seven in vitro using a weight drop device and one hour post injury the GFP- expressing EPI-NCSCs were grafted followed by VPA administration. The evaluation of treated slices seven days after injury revealed that grafted stem cells survived on the injured slices and expressed GFAP, whereas they did not express any detectable levels of the neural progenitor marker doublecortin (DCX), which was expressed prior to transplantation. Immunoblotting data demonstrated that the expression of GFAP, BDNF, neurotrophin-3 (NT3), and Bcl2 increased significantly in stem cell treated slices. This study illustrated that the fate of transplanted stem cells has been directed to the glial lineage in the ex vivo context of injury and EPI-NCSCs may ameliorate the SCI condition through releasing neurotrophic factors directly and/or via inducing resident spinal cord cells.

Quantitative Multimodal Evaluation of Passaging Human Neural Crest Stem Cells for Peripheral Nerve Regeneration.

Peripheral nerve injury is a major burden to societies worldwide, however, current therapy options (e.g. autologous nerve grafts) are unable to produce satisfactory outcomes. Many studies have shown that stem cell transplantation holds great potential for peripheral nerve repair, and human neural crest stem cells (hNCSCs), which give rise to a variety of tissues in the peripheral nervous system, are particularly promising. NCSCs are one of the best candidates for clinical translation, however, to ensure the viability and quality of NCSCs for research and clinical use, the effect of in vitro cell passaging on therapeutic effects needs be evaluated given that passaging is required to expand NCSCs to meet the demands of transplantation in preclinical research and clinical trials. To date, no study has investigated the quality of NCSCs past the 5th passage in vivo. In this study, we employed a multimodal evaluation system to investigate changes in outcomes between transplantation with 5th (p5) and 6th passage (p6) NCSCs in a 15 mm rat sciatic nerve injury and repair model. Using CatWalk gait analysis, gastrocnemius muscle index, electrophysiology, immunohistochemistry, and histomorphometric analysis, we showed that p6 NCSCs demonstrated decreased cell survival, Schwann-cell differentiation, axonal growth, and functional outcomes compared to p5 NCSCs (all p < 0.05). In conclusion, p6 NCSCs showed significantly reduced therapeutic efficacy compared to p5 NCSCs for peripheral nerve regeneration.

Protective Effects of Neural Crest-Derived Stem Cell-Conditioned Media against Ischemia-Reperfusion-Induced Lung Injury in Rats.

Current treatments for ischemia-reperfusion (IR)-induced acute lung injury are limited. Mesenchymal stem cell-conditioned medium (CM) has been reported to attenuate lung injury. Neural crest stem cells (NCSCs), a type of multipotent stem cells, are more easily obtained than mesenchymal stem cells. We hypothesize that NCSC-CM has anti-inflammatory properties that could protect against IR-induced lung injury in rats. In this study, NCSC-CM was derived from rat NCSCs. Typical acute lung injury was induced by 30-min ischemia followed by 90-min reperfusion in adult male Sprague-Dawley rats. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected to analyze the degree of lung injury after the experiment. NCSC-CM was administered before ischemia and after reperfusion. NCSC-CM treatment significantly attenuated IR-induced lung edema, as indicated by decreases in pulmonary vascular permeability, lung weight gain, wet to dry weight ratio, lung weight to body weight ratio, pulmonary arterial pressure, and protein level in BALF. The levels of tumor necrosis factor-α and interleukin-6 in the BALF were also significantly decreased. Additionally, NCSC-CM improved lung pathology and neutrophil infiltration in the lung tissue, and significantly suppressed nuclear factor (NF)-κB activity and IκB-α degradation in the lung. However, heating NCSC-CM eliminated these protective effects. Our experiment demonstrates that NCSC-CM treatment decreases IR-induced acute lung injury and that the protective mechanism may be attributable to the inhibition of NF-κB activation and the inflammatory response. Therefore, NCSC-CM may be a novel approach for treating IR-induced lung injury.