Search Results (22)

Neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of the central nervous system (CNS) are critical for tissue repair following injury or disease. These cells retain the capacity to proliferate, migrate, and differentiate into neurons, astrocytes, and oligodendrocytes, making them a promising therapeutic target for neurodegenerative disorders and traumatic injuries. However, the molecular mechanisms regulating their proliferation remain incompletely understood. This study investigates the role of cAMP responsive element-binding protein 5 (CREB5) in the proliferation of rat SVZ-derived NSPCs and elucidates its regulatory mechanism. Using RNA interference, we demonstrated that CREB5 knockdown significantly reduced cell viability, neurosphere formation capacity, and the number of proliferating cells (BrdU- and Ki-67-positive cells) both in vitro and in vivo. In contrast, CREB5 overexpression played opposing roles in cell proliferation. Additionally, alteration of CREB5 expression did not affect apoptosis, as assessed by TUNEL staining, indicating a specific role in proliferation rather than in cell death. Mechanistically, we identified Nuclear Factor One X (NFIX) as a transcriptional target of CREB5. CREB5 binds to the AP-1 site in the NFIX promoter, enhancing its expression. CREB5 knockdown inhibited NFIX expression, while CREB5 overexpression exerted the opposite function. ChIP and luciferase reporter assays further confirmed that CREB5 directly regulates NFIX promoter activity. More importantly, alteration of NFIX expression could reverse the effect of CREB5 on NSPC proliferation. These findings highlight CREB5 as a key regulator of NSPC proliferation through its interaction with NFIX, providing a potential therapeutic target for stem cell-based treatments of CNS disorders. Full article

Human induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic, universal cells that could be transplanted to any recipient without requiring a matching donor could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells, we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T-cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However, loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack, limiting graft survival. To counter this negative effect, we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF), an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression, combined with concurrent B2M and CIITA deletion, generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders. Full article

Amniotic fluid is a complex and dynamic biological matrix that surrounds the fetus during the pregnancy. From this fluid, is possible to isolate various cell types with particular interest directed towards stem cells (AF-SCs). These cells are highly appealing due to their numerous potential applications in the field of regenerative medicine for tissues and organs as well as for treating conditions such as traumatic or ischemic injuries to the nervous system, myocardial infarction, or cancer. AF-SCs, when subcultured in the presence of basic Fibroblast Growth Factor (bFGF), have been shown to survive and migrate when transplanted into the striatum of the rat brain, exhibiting behavior characteristics of neuronal/glial progenitor cells. In this work, we performed an electrophysiological characterization to ascertain the propensity of AF-SCs to differentiate into glial and neuronal cells by bFGF. By using patch clamp technique we characterized a fibroblast-like morphology that display a barium-sensitive inward-rectifying potassium current (Kir) and calcium-activated potassium currents (KCa). The electrophysiological and calcium dynamics of histamine, a marker of undifferentiated neural progenitors, was further studied. Histamine promoted intracellular calcium increase by Fura-2 recording and calcium-activated potassium current activation with a similar temporal profile in AF-SC. The data presented in this paper ultimately confirm the expression in AF-SCs of the Kir and KCa currents, also showing regulation by endogenous stimuli such as histamine for the latter. Full article

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation. Full article

Astrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFβ pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by “milking of the brain”, it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal. Full article

Neural stem cells (NSCs) have been recently identified in the neonatal rat medial geniculate body (MGB). NSCs are characterized by three cardinal features: mitotic self-renewal, formation of progenitors, and differentiation into all neuroectodermal cell lineages. NSCs and the molecular factors affecting them are particularly interesting, as they present a potential target for treating neurologically based hearing disorders. It is unclear whether an NSC niche exists in the rat MGB up to the adult stage and which neurogenic factors are essential during maturation. The rat MGB was examined on postnatal days 8, 12, and 16, and at the adult stadium. The cardinal features of NSCs were detected in MGB cells of all age groups examined by neurosphere, passage, and differentiation assays. In addition, real-time quantitative polymerase chain reaction arrays were used to compare the mRNA levels of 84 genes relevant to NSCs and neurogenesis. In summary, cells of the MGB display the cardinal features of NSCs up to the adult stage with a decreasing NSC potential over time. Neurogenic factors with high importance for MGB neurogenesis were identified on the mRNA level. These findings should contribute to a better understanding of MGB neurogenesis and its regenerative capacity. Full article

Tissue engineering for spinal cord injury (SCI) remains a complex and challenging task. Biomaterial scaffolds have been suggested as a potential solution for supporting cell survival and differentiation at the injury site. However, different biomaterials display multiple properties that significantly impact neural tissue at a cellular level. Here, we evaluated the behavior of different cell lines seeded on chitosan (CHI), poly (ε-caprolactone) (PCL), and poly (L-lactic acid) (PLLA) scaffolds. We demonstrated that the surface properties of a material play a crucial role in cell morphology and differentiation. While the direct contact of a polymer with the cells did not cause cytotoxicity or inhibit the spread of neural progenitor cells derived from neurospheres (NPCdn), neonatal rat spinal cord cells (SCC) and NPCdn only attached and matured on PCL and PLLA surfaces. Scanning electron microscopy and computational analysis suggested that cells attached to the material’s surface emerged into distinct morphological populations. Flow cytometry revealed a higher differentiation of neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) into glial cells on all biomaterials. Immunofluorescence assays demonstrated that PCL and PLLA guided neuronal differentiation and network development in SCC. Our data emphasize the importance of selecting appropriate biomaterials for tissue engineering in SCI treatment. Full article

The medial geniculate body (MGB) is a nucleus of the diencephalon representing a relevant segment of the auditory pathway and is part of the metathalamus. It receives afferent information via the inferior brachium of the inferior colliculus and transmits efferent fibers via acoustic radiations to the auditory cortex. Neural stem cells (NSCs) have been detected in certain areas along the auditory pathway. They are of great importance as the induction of an adult stem cell niche might open a regenerative approach to a causal treatment of hearing disorders. Up to now, the existence of NSCs in the MGB has not been determined. Therefore, this study investigated whether the MGB has a neural stem cell potential. For this purpose, cells were extracted from the MGB of PND 8 Sprague-Dawley rats and cultured in a free-floating cell culture assay, which showed mitotic activity and positive staining for stem cell and progenitor markers. In differentiation assays, the markers β-III-tubulin, GFAP, and MBP demonstrated the capacity of single cells to differentiate into neuronal and glial cells. In conclusion, cells from the MGB exhibited the cardinal features of NSCs: self-renewal, the formation of progenitor cells, and differentiation into all neuronal lineage cells. These findings may contribute to a better understanding of the development of the auditory pathway. Full article

A growing body of evidence suggests that hyperbaric oxygenation (HBO) may affect the activity of adult neural stem cells (NSCs). Since the role of NSCs in recovery from brain injury is still unclear, the purpose of this study was to investigate the effects of sensorimotor cortex ablation (SCA) and HBO treatment (HBOT) on the processes of neurogenesis in the adult dentate gyrus (DG), a region of the hippocampus that is the site of adult neurogenesis. Ten-week-old Wistar rats were divided into groups: Control (C, intact animals), Sham control (S, animals that underwent the surgical procedure without opening the skull), SCA (animals in whom the right sensorimotor cortex was removed via suction ablation), and SCA + HBO (operated animals that passed HBOT). HBOT protocol: pressure applied at 2.5 absolute atmospheres for 60 min, once daily for 10 days. Using immunohistochemistry and double immunofluorescence labeling, we show that SCA causes significant loss of neurons in the DG. Newborn neurons in the subgranular zone (SGZ), inner-third, and partially mid-third of the granule cell layer are predominantly affected by SCA. HBOT decreases the SCA-caused loss of immature neurons, prevents reduction of dendritic arborization, and increases proliferation of progenitor cells. Our results suggest a protective effect of HBO by reducing the vulnerability of immature neurons in the adult DG to SCA injury. Full article

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and β-tubulin 3 through the Wnt3A signaling pathway regulation markers (β-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation. Full article

Locomotor function after spinal cord injury (SCI) is critical for assessing recovery. Currently, available means to improve locomotor function include surgery, physical therapy rehabilitation and exoskeleton. Stem cell therapy with neural progenitor cells (NPCs) transplantation is a promising reparative strategy. Along this line, patient-specific induced pluripotent stem cells (iPSCs) are a remarkable autologous cell source, which offer many advantages including: great potential to generate isografts avoiding immunosuppression; the availability of a variety of somatic cells without ethical controversy related to embryo use; and vast differentiation. In this current work, to realize the therapeutic potential of iPSC-NPCs for the treatment of SCI, we transplanted purified iPSCs-derived NPCs into a cervical contusion SCI rat model. Our results showed that the iPSC-NPCs were able to survive and differentiate into both neurons and astrocytes and, importantly, improve forelimb locomotor function as assessed by the grooming task and horizontal ladder test. Purified iPSC-NPCs represent a promising cell type that could be further tested and developed into a clinically useful cell source for targeted cell therapy for cervical SCI patients. Full article

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells. Full article

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration. Full article

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1^G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1^G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs). Full article

Despite medical advances, neurological recovery after severe traumatic brain injury (TBI) remains poor. Elevated levels of high mobility group box protein-1 (HMGB1) are associated with poor outcomes; likely via interaction with receptors for advanced-glycation-end-products (RAGE). We examined the hypothesis that HMGB1 post-TBI is anti-neurogenic and whether this is pharmacologically reversible. Post-natal rat cortical mixed neuro-glial cell cultures were subjected to needle-scratch injury and examined for HMGB1-activation/neuroinflammation. HMGB1-related genes/networks were examined using genome-wide RNA-seq studies in cortical perilesional tissue samples from adult mice. Post-natal rat cortical neural stem/progenitor cell cultures were generated to quantify effects of injury-condition medium (ICM) on neurogenesis with/without RAGE antagonist glycyrrhizin. Needle-injury upregulated TNF-α/NOS-2 mRNA-expressions at 6 h, increased proportions of activated microglia, and caused neuronal loss at 24 h. Transcriptome analysis revealed activation of HMGB1 pathway genes/canonical pathways in vivo at 24 h. A 50% increase in HMGB1 protein expression, and nuclear-to-cytoplasmic translocation of HMGB1 in neurons and microglia at 24 h post-injury was demonstrated in vitro. ICM reduced total numbers/proportions of neuronal cells, but reversed by 0.5 μM glycyrrhizin. HMGB1 is activated following in vivo post mechanical injury, and glycyrrhizin alleviates detrimental effects of ICM on cortical neurogenesis. Our findings highlight glycyrrhizin as a potential therapeutic agent post-TBI. Full article