Demyelination and remyelination play pivotal roles in the pathological process of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a well-established animal model of MS. Although increasing evidence shows that various stimuli can promote the activation/induction of endogenous neural stem/progenitor cells (NSPCs) in the central nervous system, the potential contributions of these cells to remyelination following inflammatory injury remain to be fully investigated. In the present study, using an adult mouse model of EAE induced by myelin oligodendrocyte glycoprotein (MOG) peptide, we investigated whether adult NSPCs in the spinal cord can lead to remyelination under inflammatory conditions. Immunohistochemistry showed that cells expressing the NSPC marker Nestin appeared after MOG peptide administration, predominantly at the sites of demyelination where abundant inflammatory cells had accumulated, whereas Nestin+ cells were rarely present in the spinal cord of PBS-treated control mice. In vitro, Nestin+ NSPCs obtained from EAE mice spinal cords could differentiate into multiple neural lineages, including neurons, astrocytes, and myelin-producing oligodendrocytes. Using the Cre-LoxP system, we established a mouse strain expressing yellow fluorescent protein (YFP) under the control of the Nestin promoter and investigated the expression patterns of YFP-expressing cells in the spinal cord after EAE induction. At the chronic phase of the disease, immunohistochemistry showed that YFP+ cells in the injured regions expressed markers for various neural lineages, including myelin-forming oligodendrocytes. These results show that adult endogenous NSPCs in the spinal cord can be subject to remyelination under inflammatory conditions, such as after EAE, suggesting that endogenous NSPCs represent a therapeutic target for MS treatment.

Stroke increases neurogenesis in the adult dentate gyrus in the short term, however, long-term effects at the cellular and functional level are poorly understood. Here we evaluated the impact of an early stroke lesion on neurogenesis and cognitive function of the aging brain. We hypothesized that a stroke disturbs dentate neurogenesis during aging correlate with impaired flexible learning. To address this issue a stroke was induced in 3-month-old C57Bl/6 mice by a middle cerebral artery occlusion (MCAO). To verify long-term changes of adult neurogenesis the thymidine analogue BrdU (5-Bromo-2'-deoxyuridine) was administrated at different time points during aging. One and half months after BrdU injections learning and memory performance were assessed with a modified version of the Morris water maze (MWM) that includes the re-learning paradigm, as well as hippocampus-dependent and -independent search strategies. After MWM performance mice were transcardially perfused. To further evaluate in detail the stroke-mediated changes on stem- and progenitor cells as well as endogenous proliferation nestin-green-fluorescent protein (GFP) mice were used. Adult nestin-GFP mice received a retroviral vector injection in the hippocampus to evaluate changes in the neuronal morphology. At an age of 20 month the nestin-GFP mice were transcardially perfused after MWM performance and BrdU application 1.5 months later. The early stroke lesion significantly decreased neurogenesis in 7.5- and 9-month-old animals and also endogenous proliferation in the latter group. Furthermore, immature doublecortin (DCX)-positive neurons were reduced in 20-month-old nestin-GFP mice after lesion. All MCAO groups showed an impaired performance in the MWM and mostly relied on hippocampal-independent search strategies. These findings indicate that an early ischemic insult leads to a dramatical decline of neurogenesis during aging that correlates with a premature development of hippocampal-dependent deficits. Our study supports the notion that an early stroke might lead to long-term cognitive deficits as observed in human patients after lesion.

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.

Traumatic brain injury usually results in neuronal loss and cognitive deficits. Promoting endogenous neurogenesis has been considered as a viable treatment option to improve functional recovery after TBI. However, neural stem/progenitor cells (NSPCs) in neurogenic regions are often unable to migrate and differentiate into mature neurons at the injury site. Transglutaminase 2 (TGM2) has been identified as a crucial component of neurogenic niche, and significantly dysregulated after TBI. Therefore, we speculate that TGM2 may play an important role in neurogenesis after TBI, and strategies targeting TGM2 to promote endogenous neural regeneration may be applied in TBI therapy. Using a tamoxifen-induced Tgm2 conditional knockout mouse line and a mouse model of stab wound injury, we investigated the role and mechanism of TGM2 in regulating hippocampal neurogenesis after TBI. We found that Tgm2 was highly expressed in adult NSPCs and up-regulated after TBI. Conditional deletion of Tgm2 resulted in the impaired proliferation and differentiation of NSPCs, while Tgm2 overexpression enhanced the abilities of self-renewal, proliferation, differentiation, and migration of NSPCs after TBI. Importantly, injection of lentivirus overexpressing TGM2 significantly promoted hippocampal neurogenesis after TBI. Therefore, TGM2 is a key regulator of hippocampal neurogenesis and a pivotal therapeutic target for intervention following TBI.

The transplantation of neural stem cells (NSCs) capable of regenerating to the cells of the central nervous system (CNS) is a promising strategy in the treatment of CNS diseases and injury. As previous studies have highlighted mesenchymal stem cells (MSCs) as a source of NSCs, this study aimed to develop a feasible, efficient, and reproducible method for the neural induction of MSCs isolated from Wharton's jelly (hWJ-MSCs). We induced neural differentiation in a monolayer culture using epidermal growth factor, basic fibroblast growth factor, N2, and B27 supplements. This resulted in a homogenous population of proliferating cells that expressed certain neural markers at both the protein and mRNA levels. Flow cytometry and immunocytochemistry confirmed the expression of neural markers: nestin, sex-determining region Y (SRY) box 1 and 2 (SOX1 and SOX2), microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP). The qRT-PCR analysis revealed significantly enhanced expression of nestin and MAP2 in differentiated cells. This study confirms that it is possible to generate NSCs-like cells from hWJ-MSCs in a 2D culture using a practical method. However, the therapeutic effectiveness of such differentiated cells should be extended to confirm the terminal differentiation ability and electrophysiological properties of neurons derived from them.

Bisphenol A (BPA) is considered as one of the most extensively synthesized and used chemicals for industrial and consumer products. Previous investigations have established that exposure to BPA has been linked to developmental, reproductive, cardiovascular, immune, and metabolic effects. Several jurisdictions have imposed restrictions and/or have banned the use of BPA in packaging material and other consumer goods. Hence, manufacturers have replaced BPA with its analogues that have a similar chemical structure. Some of these analogues have shown similar endocrine effects as BPA, while others have not been assessed. In this investigation, we compared the neurodevelopmental effects of BPA and its major replacement Bisphenol F (BPF) on rat fetal neural stem cells (rNSCs). rNSCs were exposed to cell-specific differentiation media with non-cytotoxic doses of BPA or BPF at the range of 0.05 M to 100 M concentrations and measured the degree of cell proliferation, differentiation, and morphometric parameters. Both of these compounds increased cell proliferation and impacted the differentiation rates of oligodendrocytes and neurons, in a concentration-dependent manner. Further, there were concentration-dependent decreases in the maturation of oligodendrocytes and neurons, with a concomitant increase in immature oligodendrocytes and neurons. In contrast, neither BPA nor BPF had any overall effect on cellular proliferation or the cytotoxicity of astrocytes. However, there was a concentration-dependent increase in astrocyte differentiation and morphological changes. Morphometric analysis for the astrocytes, oligodendrocytes, and neurons showed a reduction in the arborization. These data show that fetal rNSCs exposed to either BPA or BPF lead to comparable changes in the cellular differentiation, proliferation, and arborization processes.

The failure of brain microglia to clear excess amyloid β (Aβ) is considered a leading cause of the progression of Alzheimer's disease pathology. Resident brain neural precursor cells (NPCs) possess immune-modulatory and neuro-protective properties, which are thought to maintain brain homeostasis. We have recently showed that resident mouse brain NPCs exhibit an acquired decline in their trophic properties in the Alzheimer's disease brain environment. Therefore, we hypothesized that functional NPCs may support microglial phagocytic activity, and that NPCs derived from the adult AD mouse brain may fail to support the clearance of Aβ by microglia. We first identified in the AD brain, in vivo and ex vivo, a subpopulation of microglia that express high Aβ phagocytic activity. Time-lapse microscopy showed that co-culturing newborn NPCs with microglia induced a significant increase in the fraction of microglia with high Aβ phagocytic activity. Freshly isolated NPCs from adult wild type, but not AD, mouse brain, induced an increase in the fraction of microglia with high Aβ phagocytic activity. Finally, we showed that NPCs also possess the ability to promote Aβ degradation within the microglia with high Aβ phagocytic activity. Thus, resident brain NPCs support microglial function to clear Aβ, but NPCs derived from the AD environment fail to do so. We suggest that the failure of AD brain NPCs to support Aβ clearance from the brain by microglia may accelerate disease pathology.

Astrocytes act as neural stem cells (NSCs) that have the potential to self-renew and differentiate into other neuronal cells. The protein expression of these astrocytes depends on the stage of differentiation, showing sequential expression of multiple proteins such as octamer-binding transcription factor 4 (Oct4), nestin, glial fibrillary acidic protein (GFAP), and aldehyde dehydrogenase 1 family member L1 (aldh1L1). Photobiomodulation (PBM) affects cell apoptosis, proliferation, migration, and adhesion. We hypothesized that astrocyte proliferation and differentiation would be modulated by PBM. We used an optimized astrocyte culture method and a 660-nanometer light-emitting diode (LED) to enhance the biological actions of many kinds of cells. We determined that the 660-nanometer LED promoted the biological actions of cultured astrocytes by increasing the reactive oxygen species levels. The overall viability of the cultured cells, which included various cells other than astrocytes, did not change after LED exposure; however, astrocyte-specific proliferation was observed by the increased co-expression of GFAP and bromodeoxyuridine (BrdU)/Ki67. Furthermore, the 660-nanometer LED provides evidence of differentiation, as shown by the decreased Oct4 and GFAP co-expression and increased nestin and aldh1L1 expression. These results demonstrate that a 660-nanometer LED can modify astrocyte proliferation, which suggests the efficacy of the therapeutic application of LED in various pathological states of the central nervous system.

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases.

Hepatocytes are the main parenchymal cells of the liver and play important roles in liver homeostasis and disease process. The heterogeneity of normal hepatocytes has been reported, but there is little knowledge about hepatocyte subtype and distinctive functions during liver cholestatic injury. Bile duct ligation (BDL)-induced mouse liver injury model was employed, and single-cell RNA sequencing was performed. Western blot and qPCR were used to study gene expression. Immunofluoresence was employed to detect the expressions of marker genes in hepatocytes. We detected a specific hepatocyte cluster (BDL-6) expressing extracellular matrix genes, indicating these hepatocytes might undergo epithelia-mesenchymal transition. Hepatocytes of BDL-6 also performed tissue repair functions (such as angiogenesis) during cholestatic injury. We also found that four clusters of cholestatic hepatocytes (BDL-2, BDL-3, BDL-4, and BDL-5) were involved in inflammatory process in different ways. To be specific, BDL-2/3/5 were inflammation-regulated hepatocytes, while BDL-4 played a role in cell chemotaxis. Among these four clusters, BDL-5 was special. because the hepatocytes of BDL-5 were proliferating hepatocytes. Our analysis provided more knowledge of hepatocyte distinctive functions in injured liver and gave rise to future treatment aiming at hepatocytes.

Glioblastoma is characterized by extensive necrotic areas with surrounding hypoxia. The cancer cell response to hypoxia in these areas is well-described; it involves a metabolic shift and an increase in stem cell-like characteristics. Less is known about the hypoxic response of tumor-associated astrocytes, a major component of the glioma tumor microenvironment. Here, we used primary human astrocytes and a genetically engineered glioma mouse model to investigate the response of this stromal cell type to hypoxia. We found that astrocytes became reactive in response to intermediate and severe hypoxia, similarly to irradiated and temozolomide-treated astrocytes. Hypoxic astrocytes displayed a potent hypoxia response that appeared to be driven primarily by hypoxia-inducible factor 2-alpha (HIF-2α). This response involved the activation of classical HIF target genes and the increased production of hypoxia-associated cytokines such as TGF-β1, IL-3, angiogenin, VEGF-A, and IL-1 alpha. In vivo, astrocytes were present in proximity to perinecrotic areas surrounding HIF-2α expressing cells, suggesting that hypoxic astrocytes contribute to the glioma microenvironment. Extracellular matrix derived from hypoxic astrocytes increased the proliferation and drug efflux capability of glioma cells. Together, our findings suggest that hypoxic astrocytes are implicated in tumor growth and potentially stemness maintenance by remodeling the tumor microenvironment.

Induced pluripotent stem cells (iPSCs) have revolutionized the study of human diseases as they can renew indefinitely, undergo multi-lineage differentiation, and generate disease-specific models. However, the difficulty of working with iPSCs is that they are prone to genetic instability. Furthermore, genetically unstable iPSCs are often discarded, as they can have unforeseen consequences on pathophysiological or therapeutic read-outs. We generated iPSCs from two brothers of a previously unstudied family affected with the inherited retinal dystrophy choroideremia. We detected complex rearrangements involving chromosomes 12, 20 and/or 5 in the generated iPSCs. Suspecting an underlying chromosomal aberration, we performed karyotype analysis of the original fibroblasts, and of blood cells from additional family members. We identified a novel chromosomal translocation t(12;20)(q24.3;q11.2) segregating in this family. We determined that the translocation was balanced and did not impact subsequent retinal differentiation. We show for the first time that an undetected genetic instability in somatic cells can breed further instability upon reprogramming. Therefore, the detection of chromosomal aberrations in iPSCs should not be disregarded, as they may reveal rearrangements segregating in families. Furthermore, as such rearrangements are often associated with reproductive failure or birth defects, this in turn has important consequences for genetic counseling of family members.

Human mesenchymal stem cells (hMSC) can be derived from various tissue sources and differentiated into dopaminergic (DAergic) neurons using various types of inducers. There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible. However, this area is still under extensive research. In this study, we aim to compare hMSCs derived from bone marrow (BM), adipose tissue (AD) and dental pulp (DP) to generate functional dopaminergic neurons, using FGF2 and forskolin. Post-differentiation, multiple factors were used to characterize the cells at morphological, morphometric, ultra-structural, mRNA and protein levels for various markers (Nestin, NF, MAP2, Tuj1, TH, DAT, PitX3, Ngn2, Kv4.2, SCN5A). Cells' functionality was studied by calcium ion imaging, along with the amount of dopamine secreted by the cells in the culture medium.

Regenerative endodontic procedures (REPs) are promising for dental pulp tissue regeneration; however, their application in permanent teeth remains challenging. We assessed the potential combination of an REP and local dental pulp cell (DPC) transplantation in the mature molars of C57BL/6 mice with (REP + DPC group) or without (REP group) transplantation of DPCs from green fluorescent protein (GFP) transgenic mice. After 4 weeks, the regenerated tissue was evaluated by micro-computed tomography and histological analyses to detect odontoblasts, vasculogenesis, and neurogenesis. DPCs were assessed for mesenchymal and pluripotency markers. Four weeks after the REP, the molars showed no signs of periapical lesions, and both the REP and REP + DPC groups exhibited a pulp-like tissue composed of a cellular matrix with vessels surrounded by an eosin-stained acellular matrix that resembled hard tissue. However, the REP + DPC group had a broader cellular matrix and uniquely contained odontoblast-like cells co-expressing GFP. Vasculogenesis and neurogenesis were detected in both groups, with the former being more prominent in the REP + DPC group. Overall, the REP was achieved in mature mouse molars and DPC transplantation improved the outcomes by inducing the formation of odontoblast-like cells and greater vasculogenesis.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

Electrical stimulation is increasingly being used to modulate human cell behaviour for biotechnological research and therapeutics. Electrically conductive polymers (CPs) such as polypyrrole (PPy) are amenable to in vitro and in vivo cell stimulation, being easy to synthesise with different counter ions (dopants) to augment biocompatibility and cell-effects. Extending our earlier work, which showed that CP-mediated electrical stimulation promotes human neural stem cell differentiation, here we report using electroactive PPy containing the anionic dopant dodecylbenzenesulfonate (DBS) to modulate the fate determination of human induced pluripotent stem cells (iPSCs). Remarkably, the stimulation without conventional chemical inducers resulted in the iPSCs differentiating to cells of the three germ lineages-endoderm, ectoderm, and mesoderm. The unstimulated iPSC controls remained undifferentiated. Phenotypic characterisation further showed a robust induction to neuronal fate with electrical stimulation, again without customary chemical inducers. Our findings add to the growing body of evidence supporting the use of electrical stimulation to augment stem cell differentiation, more specifically, pluripotent stem cell differentiation, and especially neuronal induction. Moreover, we have shown the versatility of electroactive PPy as a cell-compatible platform for advanced stem cell research and translation, including identifying novel mechanisms of fate regulation, tissue development, electroceuticals, and regenerative medicine.

Thymosin β4 (Tβ4) is a small, 44-amino acid polypeptide. It has been implicated in multiple processes, including cell movement, angiogenesis, and stemness. Previously, we reported that melanoma cell lines differ in Tβ4 levels. Studies on stable clones with silenced TMSB4X expression showed that Tβ4 impacted adhesion and epithelial-mesenchymal transition progression. Here, we show that the cells with silenced TMSB4X expression exhibited altered actin cytoskeleton's organization and subcellular relocalization of two intermediate filament proteins: Nestin and Vimentin. The rearrangement of the cell cytoskeleton resulted in changes in the cells' topology, height, and stiffness defined by Young's modulus. Simultaneously, only for some A375 clones with a lowered Tβ4 level, we observed a decreased ability to initiate colony formation in soft agar, tumor formation in vivo, and alterations in Nanog's expression level transcription factor regulating stemness. Thus, we show for the first time that in A375 cells, biomechanical properties are not directly coupled to stemness features, and this cell line is phenotypically heterogeneous.

Spinal cord injury (SCI) is a devastating disease, which leads to paralysis and is associated to substantially high costs for the individual and society. At present, no effective therapies are available. Here, the use of mechanically-activated lipoaspirate adipose tissue (MALS) in a murine experimental model of SCI is presented. Our results show that, following acute intraspinal MALS transplantation, there is an engraftment at injury site with the acute powerful inhibition of the posttraumatic inflammatory response, followed by a significant progressive improvement in recovery of function. This is accompanied by spinal cord tissue preservation at the lesion site with the promotion of endogenous neurogenesis as indicated by the significant increase of Nestin-positive cells in perilesional areas. Cells originated from MALS infiltrate profoundly the recipient cord, while the extra-dural fat transplant is gradually impoverished in stromal cells. Altogether, these novel results suggest the potential of MALS application in the promotion of recovery in SCI.

We prepared cellulose nanofibrils-based (CNF), alginate-based and single-walled carbon nanotubes (SWCNT)-based inks for freeform reversible embedding hydrogel (FRESH) 3D bioprinting of conductive scaffolds. The 3D printability of conductive inks was evaluated in terms of their rheological properties. The differentiation of human neuroblastoma cells (SH-SY5Y cell line) was visualized by the confocal microscopy and the scanning electron microscopy techniques. The expression of TUBB3 and Nestin genes was monitored by the RT-qPCR technique. We have demonstrated that the conductive guidelines promote the cell differentiation, regardless of using differentiation factors. It was also shown that the electrical conductivity of the 3D printed scaffolds could be tuned by calcium-induced crosslinking of alginate, and this plays a significant role on neural cell differentiation. Our work provides a protocol for the generation of a realistic in vitro 3D neural model and allows for a better understanding of the pathological mechanisms of neurodegenerative diseases.

Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors' growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering.