Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Electrophysiological Recordings from Embryonic Mouse Motoneurons Cultured on Electrospun Poly-Lactic Acid (PLA) and Polypyrrole-Coated PLA Scaffolds.

Background: Biomaterials used as cell growth stimulants should be able to provide adequate cell adhesion with no alteration in cell function. In this work, we developed a three-dimensional model of mouse spinal cord motoneurons on scaffolds composed of electrospun poly-lactic acid (PLA) fibers and plasma-polymerized polypyrrole (PPy)-coated PLA fibers. Methods: The functionality of the cultured motoneurons was assessed by evaluating both the electrophysiological response (i.e., the whole-cell Na+ and K+ currents and the firing of action potentials) and also the expression of the esicular acetylcholine transporter (VAChaT) by immunostaining techniques. While the expression of the VAChaT was confirmed on motoneurons cultured on the fibrous scaffolds, the electrophysiological responses indicated Na+ and K+ currents with lower amplitude and slower action potentials when compared to the response recorded from spinal cord motoneurons cultured on Poly-DL-Ornithine/Laminin- and plasma-polymerized PPy-coated coverslips. Results: From a morphological viewpoint, motoneurons cultured on PLA and PPy-coated PLA scaffolds did not show the development of dendritic and/or axonal processes, which were satisfactorily observed in the bidimensional cultures. Conclusion: We hypothesize that the apparently limited development of dendritic and/or axonal processes could produce a deleterious effect on the electrophysiological response of the cells, which might be due to the limited growth surface available in the fibrous scaffolds and/or to an undesired effect of the purification process.

Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel.

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

Involvement of RIG-I Pathway in Neurotropic Virus-Induced Acute Flaccid Paralysis and Subsequent Spinal Motor Neuron Death.

Poliomyelitis-like illness is a common clinical manifestation of neurotropic viral infections. Functional loss and death of motor neurons often lead to reduced muscle tone and paralysis, causing persistent motor sequelae among disease survivors. Despite several reports demonstrating the molecular basis of encephalopathy, the pathogenesis behind virus-induced flaccid paralysis remained largely unknown. The present study for the first time aims to elucidate the mechanism responsible for limb paralysis by studying clinical isolates of Japanese encephalitis virus (JEV) and Chandipura virus (CHPV) responsible for causing acute flaccid paralysis (AFP) in vast regions of Southeast Asia and the Indian subcontinent. An experimental model for studying virus-induced AFP was generated by intraperitoneal injection of 10-day-old BALB/c mice. Progressive decline in motor performance of infected animals was observed, with paralysis being correlated with death of motor neurons (MNs). Furthermore, we demonstrated that upon infection, MNs undergo an extrinsic apoptotic pathway in a RIG-I-dependent fashion via transcription factors pIRF-3 and pIRF-7. Both gene-silencing experiments using specific RIG-I-short interfering RNA and in vivo morpholino abrogated cellular apoptosis, validating the important role of pattern recognition receptor (PRR) RIG-I in MN death. Hence, from our experimental observations, we hypothesize that host innate response plays a significant role in deterioration of motor functioning upon neurotropic virus infections. IMPORTANCE Neurotropic viral infections are an increasingly common cause of immediate or delayed neuropsychiatric sequelae, cognitive impairment, and movement disorders or, in severe cases, death. Given the highest reported disability-adjusted life years and mortality rate worldwide, a better understanding of molecular mechanisms for underlying clinical manifestations like AFP will help in development of more effective tools for therapeutic solutions.

Neuronal mitochondrial dysfunction in sporadic amyotrophic lateral sclerosis is developmentally regulated.

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Mitochondria are essential for neuronal survival but the developmental timing and mechanistic importance of mitochondrial dysfunction in sporadic ALS (sALS) neurons is not fully understood. We used human induced pluripotent stem cells and generated a developmental timeline by differentiating sALS iPSCs to neural progenitors and to motor neurons and comparing mitochondrial parameters with familial ALS (fALS) and control cells at each developmental stage. We report that sALS and fALS motor neurons have elevated reactive oxygen species levels, depolarized mitochondria, impaired oxidative phosphorylation, ATP loss and defective mitochondrial protein import compared with control motor neurons. This phenotype develops with differentiation into motor neurons, the affected cell type in ALS, and does not occur in the parental undifferentiated sALS cells or sALS neural progenitors. Our work demonstrates a developmentally regulated unifying mitochondrial phenotype between patient derived sALS and fALS motor neurons. The occurrence of a unifying mitochondrial phenotype suggests that mitochondrial etiology known to SOD1-fALS may applicable to sALS. Furthermore, our findings suggest that disease-modifying treatments focused on rescue of mitochondrial function may benefit both sALS and fALS patients.

Cystine-glutamate antiporter deletion accelerates motor recovery and improves histological outcomes following spinal cord injury in mice.

xCT is the specific subunit of System xc-, an antiporter importing cystine while releasing glutamate. Although xCT expression has been found in the spinal cord, its expression and role after spinal cord injury (SCI) remain unknown. The aim of this study was to characterize the role of xCT on functional and histological outcomes following SCI induced in wild-type (xCT+/+) and in xCT-deficient mice (xCT-/-). In the normal mouse spinal cord, slc7a11/xCT mRNA was detected in meningeal fibroblasts, vascular mural cells, astrocytes, motor neurons and to a lesser extent in microglia. slc7a11/xCT gene and protein were upregulated within two weeks post-SCI. xCT-/- mice recovered muscular grip strength as well as pre-SCI weight faster than xCT+/+ mice. Histology of xCT-/- spinal cords revealed significantly more spared motor neurons and a higher number of quiescent microglia. In xCT-/- mice, inflammatory polarization shifted towards higher mRNA expression of ym1 and igf1 (anti-inflammatory) while lower levels of nox2 and tnf-a (pro-inflammatory). Although astrocyte polarization did not differ, we quantified an increased expression of lcn2 mRNA. Our results show that slc7a11/xCT is overexpressed early following SCI and is detrimental to motor neuron survival. xCT deletion modulates intraspinal glial activation by shifting towards an anti-inflammatory profile.

Involvement of ferroptosis in human motor neuron cell death.

Ferroptosis was recently defined as a novel type of programmed cell death depending on iron and lipid peroxidation. It is biologically different from other types of cell death such as apoptosis. While the involvement of ferroptosis in cancer, patient and animal model have been intensely studied, ferroptosis in human motor neuron model is still clearly unknown. Here we carefully assessed ferroptosis using human iPS cell-derived motor neuron (hiMNs). We found that almost all hiMNs died by the treatment of glutathione peroxidase 4 (GPX4) inhibitors. Importantly, the cell death was rescued by one antioxidant, vitamin E acetate, iron chelators and lipid peroxidase inhibitors with high dynamic ranges. Finally, these data clearly indicated that ferroptosis constitutively occurs in hiMNs, suggesting the possibility that it might play a biologically and pathologically important roles in motor neuron death such as motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS).

Differentiation of human dental pulp stem cells into functional motor neuron: In vitro and ex vivo study.

There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and ; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slices.

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation.

BACKGROUND: Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. RESULTS: Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-ß signaling pathway and consistent activation of sonic hedgehog, Wnt/ß-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. CONCLUSIONS: Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

Distinct subtypes of proprioceptive dorsal root ganglion neurons regulate adaptive proprioception in mice.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.

Intravenous infusion of mesenchymal stem cells delays disease progression in the SOD1G93A transgenic amyotrophic lateral sclerosis rat model.

ALS is a devastating neurodegenerative disease with few curative strategies. Both sporadic and familial ALS display common clinical features that show progressive paralysis. The pathogenesis remains unclear, but disruption of the blood-spinal cord barrier (BSCB) may contribute to the degeneration of motor neurons. Thus, restoration of the disrupted BSCB and neuroprotection for degenerating motor neurons could be therapeutic targets. We tested the hypothesis that an intravenous infusion of MSCs would delay disease progression through the preservation of BSCB function and increased expression of a neurotrophic factor, neurturin, in SOD1G93A ALS rats. When the open-field locomotor function was under 16 on the Basso, Beattie, and Bresnahan (BBB) scoring scale, the rats were randomized into two groups; one received an intravenous infusion of MSCs, while the other received vehicle alone. Locomotor function was recorded using BBB scoring and rotarod testing. Histological analyses, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), were performed. The MSC group exhibited reduced deterioration of locomotor activity compared to the vehicle group, which displayed progressive deterioration of hind limb function. We observed the protection of motor neuron loss and preservation of microvasculature using Evans blue leakage and immunohistochemical analyses in the MSC group. Confocal microscopy revealed infused green fluorescent protein+ (GFP+) MSCs in the spinal cord, and the GFP gene was detected by nested PCR. Neurturin expression levels were significantly higher in the MSC group. Thus, restoration of the BSCB and the protection of motor neurons might be contributing mechanisms to delay disease progression in SOD1G93A ALS rats.

Toxoplasma gondii infection damages the perineuronal nets in a murine model.

BACKGROUND: Behavioral and neurochemical alterations associated with toxoplasmosis may be influenced by the persistence of tissue cysts and activation of an immune response in the brain of Toxoplasma gondii-infected hosts. The cerebral extracellular matrix is organised as perineuronal nets (PNNs) that are both released and ensheath by some neurons and glial cells. There is evidences to suggest that PNNs impairment is a pathophysiological mechanism associated with neuropsychiatric conditions. However, there is a lack of information regarding the impact of parasitic infections on the PNNs integrity and how this could affect the host's behavior. OBJECTIVES: In this context, we aimed to analyse the impact of T. gondii infection on cyst burden, PNNs integrity, and possible effects in the locomotor activity of chronically infected mice. METHODS: We infected mice with T. gondii ME-49 strain. After thirty days, we assessed locomotor performance of animals using the open field test, followed by evaluation of cysts burden and PNNs integrity in four brain regions (primary and secondary motor cortices, prefrontal and somesthetic cortex) to assess the PNNs integrity using Wisteria floribunda agglutinin (WFA) labeling by immunohistochemical analyses. FINDINGS AND MAIN CONCLUSIONS: Our findings revealed a random distribution of cysts in the brain, the disruption of PNNs surrounding neurons in four areas of the cerebral cortex and hyperlocomotor behavior in T. gondii-infected mice. These results can contribute to elucidate the link toxoplasmosis with the establishment of neuroinflammatory response in neuropsychiatric disorders and to raise a discussion about the mechanisms related to changes in brain connectivity, with possible behavioral repercussions during chronic T. gondii infection.

Highly Efficient Conversion of Motor Neuron-Like NSC-34 Cells into Functional Motor Neurons by Prostaglandin E2.

Motor neuron diseases are a group of progressive neurological disorders that degenerate motor neurons. The neuroblastoma × spinal cord hybrid cell line NSC-34 is widely used as an experimental model in studies of motor neuron diseases. However, the differentiation efficiency of NSC-34 cells to neurons is not always sufficient. We have found that prostaglandin E2 (PGE2) induces morphological differentiation in NSC-34 cells. The present study investigated the functional properties of PGE2-differentiated NSC-34 cells. Retinoic acid (RA), a widely-used agent inducing cell differentiation, facilitated neuritogenesis, which peaked on day 7, whereas PGE2-induced neuritogenesis took only 2 days to reach the same level. Whole-cell patch-clamp recordings showed that the current threshold of PGE2-treated cell action potentials was lower than that of RA-treated cells. PGE2 and RA increased the protein expression levels of neuronal differentiation markers, microtubule-associated protein 2c and synaptophysin, and to the same extent, motor neuron-specific markers HB9 and Islet-1. On the other hand, protein levels of choline acetyltransferase and basal release of acetylcholine in PGE2-treated cells were higher than in RA-treated cells. These results suggest that PGE2 is a rapid and efficient differentiation-inducing factor for the preparation of functionally mature motor neurons from NSC-34 cells.

Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases.

Cellular retinoic acid-binding protein 1 (CRABP1) is highly expressed in motor neurons. Degenerated motor neuron-like MN1 cells are engineered by introducing SODG93A or AR-65Q to model degenerated amyotrophic lateral sclerosis (ALS) or spinal bulbar muscular atrophy neurons. Retinoic acid (RA)/sonic hedgehog (Shh)-induced embryonic stem cells differentiation into motor neurons are employed to study up-regulation of Crabp1 by Shh. In SODG93A or AR-65Q MN1 neurons, CRABP1 level is reduced, revealing a correlation of motor neuron degeneration with Crabp1 down-regulation. Up-regulation of Crabp1 by Shh is mediated by glioma-associated oncogene homolog 1 (Gli1) that binds the Gli target sequence in Crabp1's neuron-specific regulatory region upstream of minimal promoter. Gli1 binding triggers chromatin juxtaposition with minimal promoter, activating transcription. Motor neuron differentiation and Crabp1 up-regulation are both inhibited by blunting Shh with Gli inhibitor GANT61. Expression data mining of ALS and spinal muscular atrophy (SMA) motor neurons shows reduced CRABP1, coincided with reduction in Shh-Gli1 signaling components. This study reports motor neuron degeneration correlated with down-regulation in Crabp1 and Shh-Gli signaling. Shh-Gli up-regulation of Crabp1 involves specific chromatin remodeling. The physiological and pathological implication of this regulatory pathway in motor neuron degeneration is supported by gene expression data of ALS and SMA patients.

Differentiation of Intrafusal Fibers from Human Induced Pluripotent Stem Cells.

Human-based "body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers, sensory neurons) and efferent (extrafusal fibers, motoneurons) cells, in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling.

Microtubule stabilizer epothilone B as a motor neuron differentiation agent for human endometrial stem cells.

Microtubule-stabilizing agents (MSAs), until now, have primarily been considered for their anti-proliferative effects in the setting of cancer. However, recent studies have revealed that one particular MSA, epothilone B (EpoB), can promote axonal regeneration after traumatic spinal cord injuries (SCI) even in the presence of inhibitor molecules such as neurite outgrowth inhibitor-A (Nogo-A). On the basis of the importance of having an efficient motor neuron (MN) differentiation protocol for stem cell therapy and the attention of MSAs for SCI treatment, our study investigated the effect of EpoB on human endometrial stem cells (hEnSCs) differentiation into MN-like cells. hEnSCs were isolated and characterized by flow cytometry. The hEnSC cell viability was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To mimic the in vivo inhibitory environment, hEnSCs were also differentiated in the presence of Nogo-A. After 15 days of differentiation, the expressions of MN-markers were evaluated by real-time reverse-transcriptase polymerase chain reaction and immunofluorescence. According to the MTT assay results, three doses (1, 5, and 10 nM) of EpoB were selected to evaluate their effect on MN-differentiation. All selected doses can increase the efficacy of hEnSCs differentiation into MN-like cells. In particular, the 10 nM EpoB dosage was shown to increase the axon elongation, cell alignment, and upregulation of these MN-markers compared with other doses. EpoB can improve MN differentiation from hEnSC and potentially provide a unique route for neuronal replacement in the setting of SCI.

Toxoplasma gondii infection damages the perineuronal nets in a murine model

BACKGROUND Behavioral and neurochemical alterations associated with toxoplasmosis may be influenced by the persistence of tissue cysts and activation of an immune response in the brain of Toxoplasma gondii-infected hosts. The cerebral extracellular matrix is organised as perineuronal nets (PNNs) that are both released and ensheath by some neurons and glial cells. There is evidences to suggest that PNNs impairment is a pathophysiological mechanism associated with neuropsychiatric conditions. However, there is a lack of information regarding the impact of parasitic infections on the PNNs integrity and how this could affect the host's behavior. OBJECTIVES In this context, we aimed to analyse the impact of T. gondii infection on cyst burden, PNNs integrity, and possible effects in the locomotor activity of chronically infected mice. METHODS We infected mice with T. gondii ME-49 strain. After thirty days, we assessed locomotor performance of animals using the open field test, followed by evaluation of cysts burden and PNNs integrity in four brain regions (primary and secondary motor cortices, prefrontal and somesthetic cortex) to assess the PNNs integrity using Wisteria floribunda agglutinin (WFA) labeling by immunohistochemical analyses. FINDINGS AND MAIN CONCLUSIONS Our findings revealed a random distribution of cysts in the brain, the disruption of PNNs surrounding neurons in four areas of the cerebral cortex and hyperlocomotor behavior in T. gondii-infected mice. These results can contribute to elucidate the link toxoplasmosis with the establishment of neuroinflammatory response in neuropsychiatric disorders and to raise a discussion about the mechanisms related to changes in brain connectivity, with possible behavioral repercussions during chronic T. gondii infection.