Expression Of Neuronal Markers Research Articles

Transferrin Enhances Neuronal Differentiation.

Although transferrin (Tf) is a glycoprotein best known for its role in iron delivery, iron-independent functions have also been reported. Here, we assessed apoTf (aTf) treatment effects on Neuro-2a (N2a) cells, a mouse neuroblastoma cell line which, once differentiated, shares many properties with neurons, including process outgrowth, expression of selective neuronal markers, and electrical activity. We first examined the binding of Tf to its receptor (TfR) in our model and verified that, like neurons, N2a cells can internalize Tf from the culture medium. Next, studies on neuronal developmental parameters showed that Tf increases N2a survival through a decrease in apoptosis. Additionally, Tf accelerated the morphological development of N2a cells by promoting neurite outgrowth. These pro-differentiating effects were also observed in primary cultures of mouse cortical neurons treated with aTf, as neurons matured at a higher rate than controls and showed a decrease in the expression of early neuronal markers. Further experiments in iron-enriched and iron-deficient media showed that Tf preserved its pro-differentiation properties in N2a cells, with results hinting at a modulatory role for iron. Moreover, N2a-microglia co-cultures revealed an increase in IL-10 upon aTf treatment, which may be thought to favor N2a differentiation. Taken together, these findings suggest that Tf reduces cell death and favors the neuronal differentiation process, thus making Tf a promising candidate to be used in regenerative strategies for neurodegenerative diseases.

Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization

The critical requirements in developing clinical-grade human-induced pluripotent stem cells–derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

The Effect of Rosmarinic Acid on Neural Differentiation of Wartons Jelly-derived Mesenchymal Stem Cells in Two-dimensional and Three-dimensional Cultures using Chitosan-based Hydrogel.

Numerous studies have shown the positive effects of rosmarinic acid on the nervous system. Rosmarinic acid as a herbal compound with anti-inflammatory effects can prevent thedestructive effect of inflammation on the nervous system. Furthermore, various studies haveemphasized the advantages of three-dimensional (3D) culture over the two-dimensional (2D) culture of cells. In this study, thermosensitive chitosan (CH)-based hydrogel as a 3D scaffoldwith the combination of chitosan, beta-glycerol phosphate and hydroxyl ethyl cellulose (CH-GP-HEC) loaded with rosmarinic acid was used to induce neuronal differentiation in humanWharton jelly stem cells. Also, cells were divided into eight groups to evaluate the effect of 3Dcell culture and to compare gene expression in different induction conditions. The results ofgene expression analysis showed the highest expression of neuronal markers in Whartons jelly derived mesenchymal stem cells (WJMSCs) cultured in chitosan, beta-glycerol phosphate and hydroxyl ethyl cellulose (ch-gp-hec) loaded with differentiation medium androsmarinic acid. According to the results of gene expression, rosmarinic acid alone has a positiveeffect on the induction of expression of neural markers. This positive effect is enhanced by cellculture in 3D conditions. This study shows that rosmarinic acid can be considered an inexpensiveand available compound for use in neural tissue engineering. The results of this study indicatethat rosmarinic acid can be considered a cheap and available compound for use in neural tissueengineering.

Enhancing Neurological Competence of Nanoencapsulated Cordyceps/Turmeric Extracts in Human Neuroblastoma SH-SY5Y Cells.

Neurological diseases, including Alzheimer's, Parkinson's diseases, and brain cancers, are reportedly caused by genetic aberration and cellular malfunction. Herbs with bioactive compounds that have anti-oxidant effects such as cordyceps and turmeric, are of interest to clinical applications due to their minimal adverse effects. The aim of study is to develop the nanoencapsulated cordyceps and turmeric extracts and investigate their capability to enhance the biological activity and improve neuronal function. Human neuroblastoma SH-SY5Y cells were utilized as a neuronal model to investigate the properties of nanoencapsulated cordyceps or turmeric extracts, called CMP and TEP, respectively. SH-SY5Y cells were treated with either CMP or TEP and examined the biological consequences, including neuronal maturation and neuronal function. The results showed that both CMP and TEP improved cellular uptake efficiency within 6h by 2.3 and 2.8 times, respectively. Besides, they were able to inhibit cellular proliferation of SH-SY5Y cells up to 153- and 218-fold changes, and increase the expression of mature neuronal markers (TUJ1, PAX6, and NESTIN). Upon the treatment of CMP and TEP, the expression of dopaminergic-specific genes (LMX1B, FOXA2, EN1, and NURR1), and the secretion level of dopamine were significantly improved up to 3.3-fold and 3.0-fold, respectively, while the expression of Alzheimer genes (PSEN1, PSEN2, and APP), and the secretion of amyloid precursor protein were significantly reduced by 32-fold and 108-fold, respectively. Importantly, the autophagy activity was upregulated by CMP and TEP at 6.3- and 5.5-fold changes, respectively. This finding suggested that the nanoencapsulated cordyceps and turmeric extracts accelerated neuronal maturation and alleviated neuronal pathology in human neural cells. This paves the way for nanotechnology-driven drug delivery systems that could potentially be used as an alternative medicine in the future for neurological diseases.

Hyperactivation of TRPV4 causes the hippocampal pyroptosis pathway and results in cognitive impairment in LPS-treated mice

Pyroptosis, a newly discovered proinflammatory programmed cell death, is involved in the regulation of cognitive dysfunction, such as Alzheimer's disease. Exploring potential drug targets that prevent pyroptotic procedures might benefit the development of a cure for these diseases. In the present study, we explored whether the transient receptor potential vanilloid 4 (TRPV4) blocker HC067047 and knockdown of TRPV4 in the hippocampus could improve cognitive behavior through the inhibition of pyroptosis in a mouse model developed using systemic administration of lipopolysaccharide (LPS). We found that systemic administration of HC067047 or knockdown of hippocampal TRPV4 prevented the activation of canonical and noncanonical pyroptosis in the hippocampus of LPS-treated mice. Consistent with the inhibition of the hippocampal pyroptosis pathway, a knockdown of hippocampal TRPV4 lowered expression of TNF-α, IL-1β, IL-18, and IL-6. Furthermore, we verified that the main pyroptosis cell type might be a neuron, indicated by reduced neuronal marker expression. Mechanically, we also found that knockdown of hippocampal TRPV4 might inhibit phosphorylation of CamkⅡα which results in NFκb mediated inflammasome reduction in the hippocampus of LPS-treated mice. More interestingly, mice intraperitoneally injected with HC067047 or the hippocampus injected with TRPV4 shRNA showed improved cognitive behavior, as indicated by the enhanced discrimination ratio in the NORT, NOPT, and SNPT. Collectively, we consider that HC067047 might be a small molecular drug that prevents pyroptosis, and TRPV4 could be an effective therapeutic target for preventing pyroptosis-induced cognitive dysfunction.

Restoration of functional PAX3 transcriptional factor enhanced neuronal differentiation in PAX3b isoform-depleted neuroblastoma cells.

Reexpressed PAX3 transcription factor is believed to be responsible for the differentiation defects observed in neuroblastoma. Although the importance of PAX3 in neuronal differentiation is documented how it is involved in the defective differentiation remains unexplored particularly with its isoforms. Here, first we have analyzed PAX3 expression, its functional status, and its correlation with the neuronal marker expression in SH-SY5Y and its parental SK-N-SH cells. We have found that SH-SY5Y cells which expressed more PAX3 showed increased expression of neuronal marker genes (TUBB, MAP2, NEFL, NEUROG2, SYP) and reported PAX3 target genes (MET, TGFA, and NCAM1) than the SK-N-SH cells that had low PAX3 level. Retinoic acid treatment is unable to induce neuronal differentiation in cells (SK-N-SH) with low PAX3 level/activity. Moreover, ectopic expression of PAX3 in SK-N-SH cells neither induces neuronal marker genes nor its target genes. PAX3 isoform expression analysis revealed the expression of PAX3b isoform that contains only paired domain in SK-N-SH cells, whereas in SH-SY5Y cells, we could also observe PAX3c isoform that contains all functional domains. Further, PAX3b depletion in SK-N-SH cells is not induced PAX3 target genes, and the cells remain poorly differentiated. Interestingly, ectopic PAX3 expression in PAX3b-depleted SK-N-SH cells enhanced neuronal outgrowth along with neuronal marker gene induction. Collectively, these results showed that the PAX3b isoform may be responsible for the differentiation defect observed in SK-N-SH cells and restoration of functional PAX3 in the absence of PAX3b can induce neurogenesis in these cells.

Hyperglycemia affects neuronal differentiation and Nestin, FOXO1, and LMO3 mRNA expression of human Wharton's jelly mesenchymal stem cells of children from diabetic mothers

Pregestational Diabetes Mellitus (PDM) during pregnancy constitutes an unfavorable embryonic and fetal development environment, with a high incidence of congenital malformations (CM). Neural tube defects are the second most common type of CM in children of diabetic mothers (CDM), who also have an elevated risk of developing neurodevelopmental disorders. The mechanisms that lead to these neuronal disorders in CDM are not yet fully understood. The present study aimed to know the effect of hyperglycemia on proliferation, neuronal differentiation percentage, and expression of neuronal differentiation mRNA markers in human umbilical cord Wharton's jelly mesenchymal stem cells (hUCWJMSC) of children from normoglycemic pregnancies (NGP) and PDM. We isolated and characterized hUCWJMSC by flow cytometry, immunofluorescence, RT-PCR and were induced to differentiate into adipocytes, osteocytes, and neurons. Proliferation assays were performed to determine the doubling time, and Nestin, TUBB3, FOXO1, KCNK2, LMO3, and MAP2 mRNA gene expression was assessed by semiquantitative RT-PCR. Hyperglycemia significantly decreased proliferation and neuronal differentiation percentage in NGP and PDM cells treated with 40 mM d-glucose. Nestin mRNA expression decreased under control glycemic conditions, while FOXO1, KCNK2, LMO3, and MAP2 mRNA expression increased during neuronal differentiation in both NGP and PDM cells. On the other hand, under hyperglycemic conditions, Nestin was significantly decreased in cells from NGP but not in cells from PDM, while mRNA expression of FOXO1 and LMO3 was significantly increased in cells from NGP, but not in cells from PDM. We found evidence that maternal PDM, with hyperglycemia in culture, affects the biological properties of fetal cells. All these results could be part of fetal programming.

Clinicopathological and molecular features of multinodular and vacuolating neuronal tumors of the cerebrum

Objective: To investigate clinicopathological features of multinodular and vacuolar neurodegenerative tumor (MVNT) of the cerebrum, and to investigate its immunophenotype, molecular characteristics and prognosis. Methods: Four cases were collected at the General Hospital of Southern Theater Command, Guangzhou, China and one case was collected at the First People's Hospital of Huizhou, China from 2013 to 2021. Clinical, histological, immunohistochemical and molecular characteristics of these five cases were analyzed. Follow-up was carried out to evaluate their prognoses. Results: There were four females and one male, with an average age of 42 years (range, 17 to 51 years). Four patients presented with seizures, while one presented with discomfort on the head. Pre-operative imaging demonstrated non-enhancing, T2-hyperintense multinodular lesions in the deep cortex and superficial white matter of the frontal (n=1) or temporal lobes (n=4). Microscopically, the tumor cells were mostly arranged in discrete and coalescent nodules primarily within the deep cortical ribbon and superficial subcortical white matter. The tumors were composed of large cells with ganglionic morphology, vesicular nuclei, prominent nucleoli and amphophilic or lightly basophilic cytoplasm. They exhibited varying degrees of matrix vacuolization. Vacuolated tumor cells did not show overt cellular atypia or any mitotic activities. Immunohistochemically, tumor cells exhibited widespread nuclear staining for the HuC/HuD neuronal antigens, SOX10 and Olig2. Expression of other neuronal markers, including synaptophysin, neurofilament and MAP2, was patchy to absent. The tumor cells were negative for NeuN, GFAP, p53, H3K27M, IDH1 R132H, ATRX, BRG1, INI1 and BRAF V600E. No aberrant molecular changes were identified in case 3 and case 5 using next-generation sequencing (including 131 genes related to diagnosis and prognosis of central nervous system tumors). All patients underwent complete or substantial tumor excision without adjuvant chemoradiotherapy. Post-operative follow-up information over intervals of 6 months to 8 years was available for five patients. All patients were free of recurrence. Conclusions: MVNT is an indolent tumor, mostly affecting adults, which supports classifying MVNT as WHO grade 1. There is no tumor recurrence even in the patients treated with subtotal surgical excision. MVNTs may be considered for observation or non-surgical treatments if they are asymptomatic.

Abstract 13344: Endothelin B Receptor Agonist, Sovateltide, Promotes Neural Regeneration by Improving Mitochondria Function in a Neonatal Rat Model of Hypoxic-Ischemic Encephalopathy

Introduction: Our preliminary studies have indicated the role of Endothelin B receptors (ETBR) stimulation in decreasing oxidative stress and cell death after sovateltide treatment following hypoxic-ischemic encephalopathy (HIE) in rat pups, which suggests the involvement of ETBR in neonatal hypoxic-ischemic injury. However, its mechanism of action is not yet established. Therefore, this study investigated the effect of sovateltide on mitochondrial dysfunction and neurogenesis in a neonatal rat model of HIE. Hypothesis : Sovateltide protects neuronal survival via preserving mitochondrial activity. Methods: Sprague-Dawley male and female rat pups grouped separately, were divided into 5 different subgroups (1) Control; (2) HIE + Vehicle; (3) HIE + Hypothermia; (4) HIE + sovateltide; and (5) HIE + sovateltide + hypothermia. HIE was induced by ligating the right carotid artery on a postnatal day (PND) 7, followed by hypoxia for 120 min and hypothermia (32-34°C) for 5 h. On PND 7, sovateltide (5 μg/kg, ICV) was injected after hypoxic-ischemic injury. Pups were euthanized on PND 10. Brains were isolated for analysis of markers for mitochondrial fission and fusion as well as neurogenesis in rat brain tissues using western blots. Results : Animals receiving sovateltide showed a significantly increased expression of neuronal differentiation marker, NeuroD1 (p<0.0001) and DoubleCortin (p<0.001) along with neural marker for mature neurons, NeuN (p<0.001) compared to vehicle. Additionally, an upregulation (p<0.0001) of mitochondrial fusion-related proteins, Mfn2, and downregulation of fission proteins, Drp1, in the sovateltide group compared to the vehicle was observed. Higher neuronal progenitor cell differentiation along with better mitochondrial biogenesis in the brain of sovateltide alone or as an adjunct to hypothermia in rat pups was noted. Similar findings were obtained in both male and female rats. Conclusion : Stimulation of ETBR by sovateltide promotes neural regeneration via maintaining mitochondrial dynamics, indicating a potential neuroprotective effect and may, therefore, offer a legitimate therapeutic target for the treatment of neonatal HI brain injury.

Inhibition of Pim Kinase Supports the Neural Stem Cell Phenotype in Neuroblastoma

INTRODUCTION: Relapse rates for high-risk neuroblastoma exceed 50%. Expression of the serine-threonine protein kinase, PIM, is associated with cancer progression and is negatively correlated with neural stemness markers in neural progenitor cells. Because promoting cellular differentiation is a component of neuroblastoma therapy, and PIM kinase negatively affects neural stemness, we aimed to determine whether PIM affected the neural stem cell phenotype in neuroblastoma. METHODS: We queried the Kocak (R2) gene database to compare PIM1 and the neural stemness marker, Nanog, in neuroblastoma. For in vitro studies, SK-N-BE(2), human neuroblastoma cells and human neuroblastoma patient–derived xenograft (PDX06) cells were treated with siRNA to achieve PIM1 kinase knockdown or with AZD1208 for PIM inhibition. Abundance of mRNA for the neural stemness marker, Nanog, was detected with quantitative reverse transcription polymerase chain reaction, and protein expression was confirmed with immunoblotting. Flow cytometry detected cell surface expression of CD133 and CXCR4, 2 additional neural stemness markers. Data were reported as mean ± SEM, with p ≤ 0.05 considered significant. RESULTS: R2 database revealed a negative correlation between PIM1 and Nanog (Fig. 1A). Protein expression of Nanog was increased following siRNA knockdown of PIM1 (Fig. 1B). AZD1208 increased the abundance of Nanog mRNA and resultant protein expression (Fig. 1C). AZD1208 treatment significantly increased cell surface expression of CD133 and CXCR4 (Fig. 1D).Figure 1CONCLUSION: Neuroblastoma cells treated with PIM inhibition dedifferentiated to a more neuronal stem cell phenotype as demonstrated by increased expression of neuronal stemness markers. These findings support further studies exploring the clinical implications of PIM kinase inhibition as an adjunct to neuroblastoma therapy.

Passage number affects differentiation of sensory neurons from human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are a valuable resource for neurological disease-modeling and drug discovery due to their ability to differentiate into neurons reflecting the genetics of the patient from which they are derived. iPSC-derived cultures, however, are highly variable due to heterogeneity in culture conditions. We investigated the effect of passage number on iPSC differentiation to optimize the generation of sensory neurons (iPSC-dSNs). Three iPSC lines reprogrammed from the peripheral blood of three donors were differentiated into iPSC-dSNs at passage numbers within each of the following ranges: low (5–10), intermediate (20–26), and high (30–38). Morphology and pluripotency of the parent iPSCs were assessed prior to differentiation. iPSC-dSNs were evaluated based on electrophysiological properties and expression of key neuronal markers. All iPSC lines displayed similar morphology and were similarly pluripotent across passage numbers. However, the expression levels of neuronal markers and sodium channel function analyses indicated that iPSC-dSNs differentiated from low passage numbers better recapitulated the sensory neuron phenotype than those differentiated from intermediate or high passage numbers. Our results demonstrate that lower passage numbers may be better suited for differentiation into peripheral sensory neurons.

Neuroregenerative Potential of Stem-Cells-from-Apical-Papilla–Derived Neuronal Cell Spheroids Regulated by Stem Cells from Apical Papillae Under Various Microenvironments in a Pulp-On-Chip System

IntroductionStem cells from apical papillae (SCAPs) display potent neuroregenerative potential by secreting neurotrophic factors and differentiating into neuronal-like cells, which are regulated by local niches. This study aimed to explore the interactions between SCAP-derived neuronal cell spheroids and SCAPs under various microenvironments in a pulp-on-a-chip system. MethodsSCAPs were induced into neuronal cells by a cocktail of chemical molecules. The expression of neuronal markers was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. SCAP-derived neuronal cell spheroids were generated with micromolded agarose gels and embedded in collagen hydrogel. After characterization, the SCAP-derived neuronal cell spheroids were cocultured with SCAPs in a pulp-on-chip system under various microenvironments: α-Minimum Essential Medium (MEM)/neuronal maturation medium (NMM) (neurosphere only); undifferentiated SCAPs with α-MEM/NMM (neurosphere + SCAP); osteogenic-induced SCAPs (Oi-SCAPs) with osteogenic medium/NMM containing 0.02% hydroxyapatite nanoparticles (neurosphere + Oi-SCAP/hydroxyapatite nanoparticle); and Oi-SCAPs with α-MEM/NMM (neurosphere + Oi-SCAP). Neurite outgrowth and MAP2 and TUJ1 expression in SCAP-derived neuronal cell spheroids were assessed by immunostaining. ResultsSCAPs were efficiently chemically induced into neuronal-like cells. The expression levels of MAP2 and TUJ1 were upregulated in SCAP-derived neuronal cell spheroids when cocultured with undifferentiated SCAPs, followed by Oi-SCAPs with α-MEM and neurospheres cultured alone, while coculturing with Oi-SACPs in osteogenic medium/NMM with hydroxyapatite nanoparticles displayed the lowest expression. Neurite spreading was more prominent in the spheroids cocultured with undifferentiated SCAPs than in those of the other 3 groups. ConclusionThis work revealed that local microenvironments critically regulate the neuroregenerative potential of SCAP-derived neuronal cell spheroids.

Cerebral Organoids to Study Central Mechanisms of Pain: The Effect of Stem Cell Secretome on Opioid Receptors and Neuroplasticity

Over 90% of chronic pain (CP) patients receive opioids-based treatments, which led to a public health crisis with lasting impacts on social and economic wellbeing based on opioid addiction. Opioids act through activation of μ (MOR), δ (DOR), and κ (KOR) opioid receptors, which are broadly and differentially distributed throughout the brain. Chronic opioid consumption leads to brain changes such as alterations on neurotransmission, dendritic branching, and spine density, as well as an increase in apoptosis. To overcome opioid-related issues, extensive efforts have been made to search for an alternative treatment. Bioactive molecules secreted by stem cells, collectively known as secretome, have shown a positive impact in different pain models. However, there is a lack of studies on the role of secretome in modulating opioid receptors. By using cerebral organoids (CeO), a self-organized, functional, and multicellular 3D structure that resemble the brain, we were able to identify MOR, DOR, and KOR at different stages of maturation. Treatment with secretome increased MOR expression highlighting a possible role in pain signaling mechanisms. Opioid treatments did not impact the expression of neuronal maturation markers but together with secretome, they increased astrogliogenesis. Opioid-treated organoids presented higher dopamine secretion recapitulating an important physiological event after opioid exposure. This work demonstrates that CeO is an important model system for the study of opioid signaling with potential implications to the understanding of basic mechanisms related to pain physiology.

Soy yogurt using Lactobacillus plantarum 200655 and fructooligosaccharides: neuroprotective effects against oxidative stress.

This study aimed to evaluate the effect of Lactobacillus plantarum 200655 and fructooligosaccharides (FOS) on soymilk fermentation and the neuroprotective effects of fermented soymilk (FS). The addition of FOS did not affect the physicochemical properties during fermentation. It helped that L. plantarum 200655 survive for 21days of storage at 4°C. FOS increased the β-glucosidase activity of L. plantarum 200655, total phenolic content, and antioxidant activities, such as radical scavenging and reducing power of FS. In addition, FS with FOS exerted neuroprotective effects in SH-SY5Y cells against H2O2-induced oxidative stress. FS with 3% and 5% FOS (FS3 and FS5) significantly increased cell viability and gene expression of neuronal markers, such as brain-derived neurotrophic factor and tyrosine hydroxylase. Moreover, FS3 and FS5 significantly reduced lactate dehydrogenase release and the gene expression of Bax/Bcl-2 ratio, caspase-9, and caspase-3. These results indicated that FS3 and FS5, with enhanced antioxidant properties, could protect SH-SY5Y cells against H2O2-induced damage. Therefore, soymilk fermented with L. plantarum 200655 and FOS can be used as a prophylactic functional food with neuroprotective effects against oxidative stress.

32912 Reciprocal control of epidermis and sensory neurons in juvenile and adult human skin in a re-innervated epidermis model

Our objective was to characterize epidermal and neuronal interaction in juvenile or adult using a new model of re-innervated epidermis. Human sensory neurons were obtained from iPS cells, their differentiation and functionality were validated by observation of neuronal morphology, neuronal markers expression, and increase of intracellular calcium following lactic acid (LA) stimulation. Human sensory neurons were cultured in presence of epidermis obtained from juvenile and adult donors. The reinnervation of the epidermis was confirmed by PGP9.5 immunostaining revealing the presence of nerve fibres in the epidermis.

Methacrylic acid-based biomaterials promote peripheral innervation in the subcutaneous space of mice

Peripheral nerve innervation is essential for regulating tissue repair and regeneration. MAA-based biomaterials have been previously shown to promote angiogenesis. Here we show a new role for MAA-based biomaterials in promoting terminal axon nerve growth. Our results demonstrate that MAA-based biomaterials promote peripheral nerve growth in an Igf-1 and Shh dependent manner. The resulting nerves increased the sensitivity of treated mice paws to nociception. iDISCO clearing showed that MAA increased the presence of peripheral nerve structures in whole explants. MAA was also able to increase the expression of key neuronal markers and growth factors in a peripheral neuropathy model, the diabetic db/db mouse, suggesting that MAA-based biomaterials may be relevant to treatment of peripheral neuropathy. Moreover, in a peripheral neuropathy model, MAA was able to up-regulate the expression of growth factors for an extended duration suggesting MAA may prevent degeneration through an effect on factors that promote survival. As all tissues are innervated, MAA-based biomaterials could have broad applications in the promoting regeneration and preventing degeneration of peripheral nerves.

Graphene-collagen cryogel controls neuroinflammation and fosters accelerated axonal regeneration in spinal cord injury.

Spinal cord injury (SCI) is a devastating condition resulting in loss of motor function. The pathology of SCI is multifaceted and involves a cascade of events, including neuroinflammation and neuronal degeneration at the epicenter, limiting repair process. We developed a supermacroporous, mechanically elastic, electro-conductive, graphene crosslinked collagen (Gr-Col) cryogels for the regeneration of the spinal cord post-injury. The effects of graphene in controlling astrocytes reactivity and microglia polarization are evaluated in spinal cord slice organotypic culture and rat spinal cord lateral hemisection model of SCI. In our work, the application of external electric stimulus results in the enhanced expression of neuronal markers in an organotypic culture. The implantation of Gr-Col cryogels in rat thoracic T9-T11 hemisection model demonstrates an improved functional recovery within 14days post-injury (DPI), promoted myelination, and decreases the lesion volume at the injury site. Decrease in the expression of STAT3 in the implanted Gr-Col cryogels may be responsible for the decrease in astrocytes reactivity. Microglia cells within the implanted cryogels shows higher anti-inflammatory phenotype (M2) than inflammatory (M1) phenotype. The higher expression of mature axonal markers like β-tubulin III, GAP43, doublecortin, and neurofilament 200 in the implanted Gr-Col cryogel confirms the axonal regeneration after 28 DPI. Gr-Col cryogels also modulate the production of ECM matrix, favouring the axonal regeneration. This study shows that Gr-Col cryogels decreases neuroinflammation and accelerate axonal regeneration.

Basic Fibroblast Growth Factor Induces Cholinergic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are considered a potential tool for regenerating damaged tissues due to their great multipotency into various cell types. Here, we attempted to find the appropriate conditions for neuronal differentiation of tonsil-derived MSCs (TMSCs) and expand the potential application of TMSCs for treating neurological diseases. The TMSCs were differentiated in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) supplemented with various neurotrophic factors for 7-28days to determine the optimal neuronal differentiation condition for the TMSCs. The morphologies as well as the levels of the neural markers and neurotransmitters were assessed to determine neuronal differentiation potentials and the neuronal lineages of the differentiated TMSCs. Our initial study demonstrated that DMEM/F12 supplemented with 50ng/mL basic fibroblast growth factor with 10μM forskolin was the optimal condition for neuronal differentiation for the TMSCs. TMSCs had higher protein expression of neuronal markers, including neuron-specific enolase (NSE), GAP43, postsynaptic density protein 95 (PSD95), and synaptosomal-associated protein of 25 kDa (SNAP25) compared to the undifferentiated TMSCs. Immunofluorescence staining also validated the increased mature neuron markers, NeuN and synaptophysin, in the differentiated TMSCs. The expression of glial fibrillar acidic protein and ionized calcium-binding adaptor molecule 1 the markers of astrocytes and microglia, were also slightly increased. Additionally, the differentiated TMSCs released a significantly higher level of acetylcholine, the cholinergic neurotransmitter, as analyzed by the liquid chromatography-tandem mass spectrometry and showed an enhanced choline acetyltransferase immunoreactivity compared to the undifferentiated cells. Our study suggests that the optimized condition favors the TMSCs to differentiate into cholinergic neuron-like phenotype, which could be used as a possible therapeutic tool in treating certain neurological disorders such as Alzheimer's disease.

Expression and activation of nuclear hormone receptors result in neuronal differentiation and favorable prognosis in neuroblastoma

BackgroundNeuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR).MethodsCell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo.ResultsHere, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome.ConclusionTogether, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

Prussian Blue Nanoparticle-Mediated Scalable Thermal Stimulation for In Vitro Neuronal Differentiation.

Heating has recently been applied as an alternative to electrical stimulation to modulate excitability and to induce neuritogenesis and the expression of neuronal markers; however, a long-term functional differentiation has not been described so far. Here, we present the results obtained by a new approach for scalable thermal stimulation on the behavior of a model of dorsal root ganglion neurons, the F-11 cell line. Initially, we performed experiments of bulk stimulation in an incubator for different time intervals and temperatures, and significant differences in neurite elongation and in electrophysiological properties were observed in cultures exposed at 41.5 °C for 30 min. Thus, we exposed the cultures to the same temperature increase using a near-infrared laser to irradiate a disc of Prussian blue nanoparticles and poly-vinyl alcohol that we had adhered to the outer surface of the petri dish. In irradiated cells, neurites were significantly longer, and the electrophysiological properties (action potential firing frequency and spontaneous activity) were significantly increased compared to the control. These results show for the first time that a targeted thermal stimulation could induce morphological and functional neuronal differentiation and support the future application of this method as a strategy to modify neuronal behavior in vivo.