Motor Neuron-like Cells Research Articles

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway.

Enterovirus 71 (EV71) causing Hand, Foot and Mouth Disease, is regarded as the most important neurotropic virus worldwide. EV71 is believed to replicate in muscles and infect motor neurons to reach the central nervous system (CNS). To further investigate the mechanisms involved, we have employed the motor neuron cell line NSC-34. NSC-34 cells were permissive to EV71 and virus production yields were strain-dependent with differential efficacy at the entry, replication and egress steps. Furthermore, unlike all the other cell lines previously reported, EV71-infected NSC-34 cells neither displayed cytopathic effect nor underwent apoptosis. Instead, autophagy was markedly up-regulated and virus-containing autophagic vacuoles were isolated from the culture supernatant, providing the first experimental evidence that EV71 can adopt a non-lytic exit pathway. Finally, the ability of EV71 to infect productively NSC-34 cells correlated with its ability to invade the CNS in vivo, supporting the relevance of NSC-34 cells to study the intrinsic neurovirulence of EV71 strains.

S-nitrosylated GAPDH mediates neuronal apoptosis induced by amyotrophic lateral sclerosis-associated mutant SOD1G93A

ABSTRACTAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with the selective loss of motor neurons in the brain, brain stem, and spinal cord. A number of the mutants of the human gene for superoxide dismutase 1 (SOD1) have been shown to cause familial ALS as a result of gain-of-function toxicity by an unknown mechanism. In this study, we show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a critical mediator of the apoptotic cell death signaling cascade induced by the ALS-associated G93A mutant of human SOD1 [SOD1(G93A)]. We observed that SOD1(G93A) induces S-nitrosylation of GAPDH and the subsequent binding of GAPDH and Siah1 in NSC34 motor neuron-like cells. Furthermore, SOD1(G93A) promoted nuclear translocation of S-nitrosylated GAPDH in the cells. In addition, SOD1(G93A)-induced apoptotic cell death was inhibited by deprenyl, a chemical inhibitor of GAPDH S-nitrosylation, in NSC34 cells. Taken together, our findings suggest that S-nitrosylation of GAPDH plays a critical role in SOD1(G93A)-induced neuronal apoptosis.

Altered mRNA Splicing in SMN-Depleted Motor Neuron-Like Cells

Spinal muscular atrophy (SMA) is an intractable neurodegenerative disease afflicting 1 in 6–10,000 live births. One of the key functions of the SMN protein is regulation of spliceosome assembly. Reduced levels of the SMN protein that are observed in SMA have been shown to result in aberrant mRNA splicing. SMN-dependent mis-spliced transcripts in motor neurons may cause stresses that are particularly harmful and may serve as potential targets for the treatment of motor neuron disease or as biomarkers in the SMA patient population. We performed deep RNA sequencing using motor neuron-like NSC-34 cells to screen for SMN-dependent mRNA processing changes that occur following acute depletion of SMN. We identified SMN-dependent splicing changes, including an intron retention event that results in the production of a truncated Rit1 transcript. This intron-retained transcript is stable and is mis-spliced in spinal cord from symptomatic SMA mice. Constitutively active Rit1 ameliorated the neurite outgrowth defect in SMN depleted NSC-34 cells, while expression of the truncated protein product of the mis-spliced Rit1 transcript inhibited neurite extension. These results reveal new insights into the biological consequence of SMN-dependent splicing in motor neuron-like cells.

Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold.

There is variety of stem cell sources but problems in ethical issues, contamination, and normal karyotype cause many limitations in obtaining and using these cells. The cells in Wharton's jelly region of umbilical cord are abundant and available stem cells with low immunological incompatibility, which could be considered for cell replacement therapy. Small molecules have been presented as less expensive biologically active compounds that can regulate different developmental process. Purmorphamine (PMA) is a small molecule that, according to some studies, possesses certain differentiation effects. In this study, we investigated the effect of the PMA on Wharton's jelly mesenchymal stem cell (WJ-MSC) differentiation into motor neuronal lineages instead of sonic hedgehog (Shh) on PCL scaffold. After exposing to induction media for 15days, the cells were characterized for expression of motor neuron markers including PAX6, NF-H, Islet1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription (PCR) and immunocytochemistry. Our results demonstrated that induced WJ-MSCs with PMA could significantly express motor neuron markers in RNA and protein levels 15days post induction. These results suggested that WJ-MSCs can differentiate to motor neuron-like cells with PMA on PCL scaffold and might provide a potential source in cell therapy for nervous system.

In vitro prion-like behaviour of TDP-43 in ALS

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and >95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

A novel amyotrophic lateral sclerosis mutation in OPTN induces ER stress and Golgi fragmentation in vitro

Mutations in the optineurin gene (OPTN) have been identified in a small proportion (<1%) of sporadic and familial ALS cases, and the exact role of optineurin in the pathogenesis of ALS remains unclear. To further examine the role of OPTN in ALS, we sought to identify novel ALS variants in OPTN and examine their potential for pathogenicity in vitro. Whole exome sequence data from 74 familial ALS cases were analysed for the presence of novel OPTN mutations. Pathogenicity was assessed by analysing effects on Golgi fragmentation, endoplasmic reticulum (ER) stress-linked CHOP activation, and cellular localization of optineurin in motor neuron-like NSC-34 cells expressing mutant optineurin. We identified a novel heterozygous missense mutation in OPTN (c.883G > T, p.Val295Phe) in a single familial ALS case. This mutation induced recognized cellular features of ALS pathogenesis including Golgi fragmentation and ER stress in NSC-34 cells. In conclusion, the identification of a novel OPTN mutation in an Australian ALS family, and its capacity to induce ALS-like pathological features in vitro, further strengthens evidence for the role of optineurin in the pathogenesis of ALS.

Induction of mice adult bone marrow mesenchymal stem cells into functional motor neuron-like cells

The differentiation of mesenchymal stem cells (MSC) into acetylcholine secreted motor neuron-like cells, followed by elongation of the cell axon, is a promising treatment for spinal cord injury and motor neuron cell dysfunction in mammals. Differentiation is induced through a pre-induction step using Beta- mercaptoethanol (BME) followed by four days of induction with retinoic acid and sonic hedgehog. This process results in a very efficient differentiation of BM-MSCs into motor neuron-like cells. Immunocytochemistry showed that these treated cells had specific motor neural markers: microtubule associated protein-2 and acetylcholine transferase. The ability of these cells to function as motor neuron cells was assessed by measuring acetylcholine levels in a culture media during differentiation. High-performance liquid chromatography (HPLC) showed that the differentiated cells were functional. Motor neuron axon elongation was then induced by adding different concentrations of a nerve growth factor (NGF) to the differentiation media. Using a collagen matrix to mimic the natural condition of neural cells in a three-dimensional model showed that the MSCs were successfully differentiated into motor neuron-like cells. This process can efficiently differentiate MSCs into functional motor neurons that can be used for autologous nervous system therapy and especially for treating spinal cord injuries.

Structural insights into the multi-determinant aggregation of TDP-43 in motor neuron-like cells

TDP-43 is aggregated in patients with ALS and FLTD through mechanisms still incompletely understood. Since aggregation in the cytosol is most probably responsible for the delocalization and loss of proper RNA-binding function of TDP-43 in the nucleus, interception of the formation of aggregates may represent a useful therapeutic option.In this study, we investigated the relative importance of the N-terminal and C-terminal moieties of TDP-43 in the aggregation process and the weight of each of the six cysteine residues in determining unfolding and aggregation of the different domains. We report that cytoplasmic inclusions formed by WT and mutant TDP-43 in motor neuron-like NSC34 cells are redox-sensitive only in part, and contain at least two components, i.e. oligomers and large aggregates, that are made of different molecular species.The two N-terminal cysteine residues contribute to the seeding for the first step in oligomerization, which is then accomplished by mechanisms depending on the four cysteines in the RNA-recognition motifs. Cysteine-independent large aggregates contain unfolded isoforms of the protein, held together by unspecific hydrophobic interactions. Interestingly, truncated isoforms are entrapped exclusively in oligomers. Ab initio modeling of TDP-43 structure, molecular dynamics and molecular docking analysis indicate a differential accessibility of cysteine residues that contributes to aggregation propensity.We propose a model of TDP-43 aggregation involving cysteine-dependent and cysteine-independent stages that may constitute a starting point to devise strategies counteracting the formation of inclusions in TDP-43 proteinopathies.

NSC-34 Motor Neuron-Like Cells Are Unsuitable as Experimental Model for Glutamate-Mediated Excitotoxicity.

Glutamate-induced excitotoxicity is a major contributor to motor neuron degeneration in the pathogenesis of amyotrophic lateral sclerosis (ALS). The spinal cord × Neuroblastoma hybrid cell line (NSC-34) is often used as a bona fide cellular model to investigate the physiopathological mechanisms of ALS. However, the physiological response of NSC-34 to glutamate remains insufficiently described. In this study, we evaluated the relevance of differentiated NSC-34 (NSC-34D) as an in vitro model for glutamate excitotoxicity studies. NSC-34D showed morphological and physiological properties of motor neuron-like cells and expressed glutamate receptor subunits GluA1–4, GluN1 and GluN2A/D. Despite these diverse characteristics, no specific effect of glutamate was observed on cultured NSC-34D survival and morphology, in contrast to what has been described in primary culture of motor neurons (MN). Moreover, a small non sustained increase in the concentration of intracellular calcium was observed in NSC-34D after exposure to glutamate compared to primary MN. Our findings, together with the inability to obtain cultures containing only differentiated cells, suggest that the motor neuron-like NSC-34 cell line is not a suitable in vitro model to study glutamate-induced excitotoxicity. We suggest that the use of primary cultures of MN is more suitable than NSC-34 cell line to explore the pathogenesis of glutamate-mediated excitotoxicity at the cellular level in ALS and other motor neuron diseases.

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

ABSTRACTGrowing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

Neuroprotective Effect of the Novel Compound ITH33/IQM9.21 Against Oxidative Stress and Na(+) and Ca(2+) Overload in Motor Neuron-like NSC-34 Cells.

Alternatives for the treatment of amyotrophic lateral sclerosis (ALS) are scarce and controversial. The etiology of neuronal vulnerability in ALS is being studied in motor neuron-like NSC-34 cells to determine the underlying mechanisms leading to selective loss of motor neurons. One such mechanism is associated with mitochondrial oxidative stress, Ca(2+) overload, and low expression of Ca(2+)-buffering proteins. Therefore, in order to elicit neuronal death in ALS, NSC-34 cells were exposed to the following cytotoxic agents: (1) a mixture of oligomycin 10µM and rotenone 30µM (O/R), or (2) phenylarsine oxide 1µM (PAO) (to mimic excess free radical production during mitochondrial dysfunction), and (3) veratridine 100µM (VTD) (to induce overload of Na(+) and Ca(2+) and to alter distribution of Ca(2+)-buffering proteins [parvalbumin and calbindin-D28k]). Thus, the aim of the study was to test the novel neuroprotective compound ITH33/IQM9.21 (ITH33) and to compare it with riluzole on in vitro models of neurotoxicity. Cell viability measured with MTT showed that only ITH33 protected against O/R at 3μM and PAO at 10μM, but not riluzole. ITH33 and riluzole were neuroprotective against VTD, blocked the maximum peak and the number of [Ca(2+)]c oscillations per cell, and restored the effect on parvalbumin. However, only riluzole reversed the effect on calbindin-D28k levels. Therefore, ITH33 was neuroprotective against oxidative stress and Na(+)/Ca(2+) overload, both of which are involved in ALS.

Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression.

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95±1.4% (mean±SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

Inhibitor of PI3K/Akt Signaling Pathway Small Molecule Promotes Motor Neuron Differentiation of Human Endometrial Stem Cells Cultured on Electrospun Biocomposite Polycaprolactone/Collagen Scaffolds.

Small molecules as useful chemical tools can affect cell differentiation and even change cell fate. It is demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can inhibit proliferation and promote neuronal differentiation of mesenchymal stem cells (MSCs). The purpose of this study was to investigate the differentiation effect of Ly294002 small molecule on the human endometrial stem cells (hEnSCs) into motor neuron-like cells on polycaprolactone (PCL)/collagen scaffolds. hEnSCs were cultured in a neurogenic inductive medium containing 1μM LY294002 on the surface of PCL/collagen electrospun fibrous scaffolds. Cell attachment and viability of cells on scaffolds were characterized by scanning electron microscope (SEM) and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The expression of neuron-specific markers was assayed by real-time PCR and immunocytochemistry analysis after 15days post induction. Results showed that attachment and differentiation of hEnSCs into motor neuron-like cells on the scaffolds with Ly294002 small molecule were higher than that of the cells on tissue culture plates as control group. In conclusion, PCL/collagen electrospun scaffolds with Ly294002 have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhances viability and differentiation of hEnSCs into neurons through inhibition of the PI3K/Akt pathway. Thus, manipulation of this pathway by small molecules can enhance neural differentiation.

Derivation of motor neuron-like cells from neonatal mouse testis in a simple culture condition.

Embryonic stem cell (ESC) therapy is an exciting way to treat neurodegenerative disease and central nervous system injury. However, many ethical and immunological problems surround the use of embryonic stem cells. Finding an alternative source of stem cells is therefore pertinent. In this study, spermatogonia stem cells (SSCs) were used to generate mature motor neurons. SSCs were extracted from neonatal testes and cultured in DMED/F12 medium for 3weeks. Characterisation of SSC-derived ESC-like cells was confirmed by RT-qPCR, immunostaining, alkaline phosphatase activity and their ability to form embryoid bodies (EBs). The EBs were induced by retinoic acid and Sonic hedgehog and trypsinised to obtain single induced cells. The single cells were cultured in neural medium for 18days. Characterisation of neural precursors and motor neuron-like cells was confirmed by RT-qPCR and immunocytochemical analysis at the 7th day (early stage) and 18th day (late stage), respectively, of culturing. The neural precursors were found to be positive for nestin and Sox2, and a small fraction of cells expressed β-tubulin III. Upon further differentiation, multipolar neurons were detected that expressed β-tubulin III and MAP2 markers. Moreover, the expression levels of Olig2 and PAX6 were significantly lower, while HB9, Isl1 and Isl2 expression levels were higher at the late stage when compared to the early stage. These results show that SSCs have the potential to differentiate to motor neuron-like cells and express markers specific for mature motor neurons. However, the functional ability of these cells remains to be evaluated in future studies.

Motor neuron-like NSC-34 cells as a new model for the study of vitamin D metabolism in the brain

Vitamin D3 is a pro-hormone, which is sequentially activated by 25- and 1α-hydroxylation to form 25-hydroxyvitamin D3 [25(OH)D3] and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], respectively. Subsequent inactivation is performed by 24-hydroxylation. These reactions are carried out by a series of CYP450 enzymes. The 25-hydroxylation involves mainly CYP2R1 and CYP27A1, whereas 1α-hydroxylation and 24-hydroxylation are catalyzed by CYP27B1 and CYP24A1, respectively, and are tightly regulated to maintain adequate levels of the active vitamin D hormone, 1α,25(OH)2D3. Altered circulating vitamin D levels, in particular 25(OH)D3, have been linked to several disorders of the nervous system, e.g., schizophrenia and Parkinson disease. However, little is known about the mechanisms of vitamin D actions in the neurons. In this study, we examined vitamin D metabolism and its regulation in a murine motor neuron-like hybrid cell line, NSC-34. We found that these cells express mRNAs for the four major CYP450 enzymes involved in vitamin D activation and inactivation, and vitamin D receptor (VDR) that mediates vitamin D actions. We also found high levels of CYP24A1-dependent 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] production, that was inhibited by the well-known CYP enzyme inhibitor ketoconazole and by several inhibitors that are more specific for CYP24A1. Furthermore, CYP24A1 mRNA levels in NSC-34 cells were up-regulated by 1α,25(OH)2D3 and its synthetic analogs, EB1089 and tacalcitol. Our results suggest that NSC-34 cells could be a novel model for the studies of neuronal vitamin D metabolism and its mechanism of actions.

Disruption of TCA Cycle and Glutamate Metabolism Identified by Metabolomics in an In Vitro Model of Amyotrophic Lateral Sclerosis.

This study aims to develop a cellular metabolomics model that reproduces the pathophysiological conditions found in amyotrophic lateral sclerosis in order to improve knowledge of disease physiology. We used a co-culture model combining the motor neuron-like cell line NSC-34 and the astrocyte clone C8-D1A, with each over-expressing wild-type or G93C mutant human SOD1, to examine amyotrophic lateral sclerosis (ALS) physiology. We focused on the effects of mutant human SOD1 as well as oxidative stress induced by menadione on intracellular metabolism using a metabolomics approach through gas chromatography coupled with mass spectrometry (GC-MS) analysis. Preliminary non-supervised analysis by Principal Component Analysis (PCA) revealed that cell type, genetic environment, and time of culture influenced the metabolomics profiles. Supervised analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) on data from intracellular metabolomics profiles of SOD1G93C co-cultures produced metabolites involved in glutamate metabolism and the tricarboxylic acid cycle (TCA) cycle. This study revealed the feasibility of using a metabolomics approach in a cellular model of ALS. We identified potential disruption of the TCA cycle and glutamate metabolism under oxidative stress, which is consistent with prior research in the disease. Analysis of metabolic alterations in an in vitro model is a novel approach to investigation of disease physiology.

Zonisamide Enhances Neurite Elongation of Primary Motor Neurons and Facilitates Peripheral Nerve Regeneration In Vitro and in a Mouse Model.

No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies.

Cellular activity of Wharton's Jelly-derived mesenchymal stem cells on electrospun fibrous and solvent-cast film scaffolds.

It was shown that topography and surface chemistry of materials influence cell behaviors. In this study, the effects of chemistry and topography of scaffold surface on adhesion, proliferation and differentiation of Wharton's Jelly mesenchymal (WJMSCs) stem cells into motor neurons were investigated. WJMSCs were cultivated in an neurogenic inductive medium on the surface of modified and unmodified polycaprolactone (PCL) electrospun fibrous and solvent-cast film scaffolds. All the scaffolds were characterized according to their ability to support cell attachment and viability by SEM and MTT assay. The expression of motor neuron-specific markers was assayed by real-time PCR after 15days post induction. Results showed that attachment, proliferation and differentiation of WJMSCs into motor neuron-like cells on the nanotopographic surface was higher than that of the cells on the solvent-cast scaffolds. In addition, regardless of their topography, WJMSCs cultured on collagen-coated PCL nanofibrous showed results similar to collagen-coated PCL films. Results suggested that surface chemistry has more impact on WJMSCs behaviour rather than topography. In conclusion, collagen-coated electrospun PCL have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhance viability and differentiation of WJMSCs.

Evaluation of the role of an antioxidant gene in NSC-34 motor neuron-like cells as a model of a motor neuron disease.

Spinal muscular atrophy is a rare genetic disease, which primarily affects motor neurons and predominantly occurs in children. To date, alternatives for the treatment of the disease have been controversial. Spinal muscular atrophy has a multi-factorial aetiology, with mitochondrial oxidative stress considered as the crucial pathogenic mechanism. To determine the mechanisms underlying the loss of motor neurons, NSC-34 motor neuron-like cells are often used as an in vitro model of spinal muscular atrophy. As plastin 3 (PLS3) has been demonstrated as a modifier of spinal muscular atrophy, the aim of the current study was to evaluate the neuroprotective effect of PLS3 in NSC-34 cells. Plastin 3 was overexpressed in human embryonic kidney 293T cells and NSC-34 cells via lentiviral transduction. NSC-34 cells transduced with a lentiviral vector carrying the gene for LacZ β-galactosidase served as a control. Oxidative stress was then induced by depriving cells of serum, and the protective effect of PLS3 was assessed using a cellular reactive oxygen species detection assay. While PLS3 was successfully overexpressed in human embryonic kidney 293T cells and NSC-34 cells, upregulation of this protein did not significantly decrease oxidative stress in serum-deprived NSC-34 cells relative to controls. Plastin 3 overexpression in NSC-34 cells did not elicit an antioxidative effect following serum deprivation.

Induction of human umbilical Wharton's jelly-derived mesenchymal stem cells toward motor neuron-like cells.

The most important property of stem cells from different sources is the capacity to differentiate into various cells and tissue types. However, problems including contamination, normal karyotype, and ethical issues cause many limitations in obtaining and using these cells from different sources. The cells in Wharton's jelly region of umbilical cord represent a pool source of primitive cells with properties of mesenchymal stem cells (MSCs). The aim of this study was to determine the potential of human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) for differentiation to motor neuron cells. WJMSCs were induced to differentiate into motor neuron-like cells by using different signaling molecules and neurotrophic factors in vitro. Differentiated neurons were then characterized for expression of motor neuron markers including nestin, PAX6, NF-H, Islet 1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription PCR and immunocytochemistry. Our results showed that differentiated WJMSCs could significantly express motor neuron biomarkers in RNA and protein levels 15 d post induction. These results suggested that WJMSCs can differentiate to motor neuron-like cells and might provide a potential source in cell therapy for neurodegenerative disease.