Riluzole treatment modulates KCC2 and EAAT-2 receptor expression and Ca2+ accumulation following ventral root avulsion injury.

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltage-activated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2.

Restoration of Motor Function through Delayed Intraspinal Delivery of Human IL-10-Encoding Nucleoside-Modified mRNA after Spinal Cord Injury.

Efficient in vivo delivery of anti-inflammatory proteins to modulate the microenvironment of an injured spinal cord and promote neuroprotection and functional recovery is a great challenge. Nucleoside-modified messenger RNA (mRNA) has become a promising new modality that can be utilized for the safe and efficient delivery of therapeutic proteins. Here, we used lipid nanoparticle (LNP)-encapsulated human interleukin-10 (hIL-10)-encoding nucleoside-modified mRNA to induce neuroprotection and functional recovery following rat spinal cord contusion injury. Intralesional administration of hIL-10 mRNA-LNP to rats led to a remarkable reduction of the microglia/macrophage reaction in the injured spinal segment and induced significant functional recovery compared to controls. Furthermore, hIL-10 mRNA treatment induced increased expression in tissue inhibitor of matrix metalloproteinase 1 and ciliary neurotrophic factor levels in the affected spinal segment indicating a time-delayed secondary effect of IL-10 5 d after injection. Our results suggest that treatment with nucleoside-modified mRNAs encoding neuroprotective factors is an effective strategy for spinal cord injury repair.

Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter.

Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy's success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

Stem Cell Secretome for Spinal Cord Repair: Is It More than Just a Random Baseline Set of Factors?

Hundreds of thousands of people suffer spinal cord injuries each year. The experimental application of stem cells following spinal cord injury has opened a new era to promote neuroprotection and neuroregeneration of damaged tissue. Currently, there is great interest in the intravenous administration of the secretome produced by mesenchymal stem cells in acute or subacute spinal cord injuries. However, it is important to highlight that undifferentiated neural stem cells and induced pluripotent stem cells are able to adapt to the damaged environment and produce the so-called lesion-induced secretome. This review article focuses on current research related to the secretome and the lesion-induced secretome and their roles in modulating spinal cord injury symptoms and functional recovery, emphasizing different compositions of the lesion-induced secretome in various models of spinal cord injury.

Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.

Grafted human induced pluripotent stem cells improve the outcome of spinal cord injury: modulation of the lesion microenvironment.

Spinal cord injury results in irreversible tissue damage followed by a very limited recovery of function. In this study we investigated whether transplantation of undifferentiated human induced pluripotent stem cells (hiPSCs) into the injured rat spinal cord is able to induce morphological and functional improvement. hiPSCs were grafted intraspinally or intravenously one week after a thoracic (T11) spinal cord contusion injury performed in Fischer 344 rats. Grafted animals showed significantly better functional recovery than the control rats which received only contusion injury. Morphologically, the contusion cavity was significantly smaller, and the amount of spared tissue was significantly greater in grafted animals than in controls. Retrograde tracing studies showed a statistically significant increase in the number of FB-labeled neurons in different segments of the spinal cord, the brainstem and the sensorimotor cortex. The extent of functional improvement was inversely related to the amount of chondroitin-sulphate around the cavity and the astrocytic and microglial reactions in the injured segment. The grafts produced GDNF, IL-10 and MIP1-alpha for at least one week. These data suggest that grafted undifferentiated hiPSCs are able to induce morphological and functional recovery after spinal cord contusion injury.

Presence of Titanium and Toxic Effects Observed in Rat Lungs, Kidneys, and Central Nervous System in vivo and in Cultured Astrocytes in vitro on Exposure by Titanium Dioxide Nanorods.

BACKGROUND: Non-spherical titanium dioxide (TiO2) nanoparticles have been increasingly applied in various biomedical and technological fields. Their toxicological characterization is, however, less complete than that of roundish nanoparticles. MATERIALS AND METHODS: Anatase form TiO2 nanorods, ca. 15x65 nm in size, were applied to cultured astrocytes in vitro and to the airways of young adult Wistar rats in vivo in 5, 10, and 8 mg/kg BW dose for altogether 28 days. Presence of nanorods and cellular damage was investigated in the astrocytes and in rat lungs and kidneys. Functional damage of the nervous system was studied by electrophysiological methods. RESULTS: The treated astrocytes showed loss of viability without detectable apoptosis. In rats, TiO2 nanorods applied to the airways reached the blood and various organs including the lungs, kidneys, and the central nervous system. In lung and kidney samples, nanorods were observed within (partly damaged) phagolysosomes and attached to organelles, and apoptotic cell death was also detected. In cortical and peripheral electrophysiological activity, alterations corresponding to energy shortage (resulting possibly from mitochondrial damage) and astrocytic dysfunction were detected. Local titanium levels and relative weight of the investigated organs, apoptotic cell death in the lungs and kidneys, and changes in the central and peripheral nervous activity were mostly proportional to the applied doses, and viability loss of the cultured astrocytes was also dose-dependent, suggesting causal relationship of treatments and effects. CONCLUSION: Based on localization of the visualized nanorods, on neuro-functional changes, and on literature data, the toxic mechanism involved mitochondrial damage, oxidative stress, and apoptotic cell death. These indicate potential human toxicity and occupational risk in case of exposure to rod-shaped TiO2 nanoparticles.

Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres are Suitable for Neurotoxicity Screening.

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.

Neuroectodermal Stem Cells Grafted into the Injured Spinal Cord Induce Both Axonal Regeneration and Morphological Restoration via Multiple Mechanisms.

Spinal cord contusion injury leads to severe loss of gray and white matter and subsequent deficit of motor and sensory functions below the lesion. In this study, we investigated whether application of murine clonal embryonic neuroectodermal stem cells can prevent the spinal cord secondary damage and induce functional recovery. Stem cells (NE-GFP-4C cell line) were grafted intraspinally or intravenously immediately or one week after thoracic spinal cord contusion injury. Control animals received cell culture medium or fibrin intraspinally one week after injury. Functional tests (Basso, Beattie, Bresnahan, CatWalk®) and detailed morphological analysis were performed to evaluate the effects of grafted cells. Stem cells applied either locally or intravenously induced significantly improved functional recovery compared with their controls. Morphologically, stem cell grafting prevented the formation of secondary injury and promoted sparing of the gray and white matters. The transplanted cells integrated into the host tissue and differentiated into neurons, astrocytes, and oligodendrocytes. In intraspinally grafted animals, the corticospinal tract axons regenerated along the ventral border of the cavity and have grown several millimeters, even beyond the caudal end of the lesion. The extent of regeneration and functional improvement was inversely related to the amounts of chondroitin sulphate and ephrin-B2 molecules around the cavity and to the microglial and astrocytic reactions in the injured segment early after injury. The grafts produced glial cell derived neurotrophic factor, macrophage inflammatory protein-1a, interleukin (IL)-6 and IL-10 in a paracrine fashion for at least one week. Treating the grafted cords with neutralizing antibodies against these four factors through the use of osmotic pumps nearly completely abolished the effect of the graft. The non-significant functional improvement after function blocking is likely because the stem cell derivatives settled in the injured cord. These data suggest that grafted neuroectodermal stem cells are able to prevent the secondary spinal cord damage and induce significant regeneration via multiple mechanisms.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.