The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation.

Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.

BACKGROUND: Mesenchymal stem cell-neural progenitors (MSC-NPs) are a bone marrow mesenchymal stem cell (MSC)-derived ex vivo manipulated cell product with therapeutic potential in multiple sclerosis (MS). The objective of this study was to determine efficacy of intrathecal (IT) MSC-NP treatment in patients with progressive MS.
METHODS: The study is a phase II randomized, double-blind, placebo-controlled clinical trial with a compassionate crossover design conducted at a single site. Subjects were stratified according to baseline Expanded Disability Status Scale (EDSS) (3.0-6.5) and disease subtype (secondary or primary progressive MS) and randomized into either treatment or placebo group to receive six IT injections of autologous MSC-NPs or saline every two months. The primary outcome was EDSS Plus, defined by improvement in EDSS, timed 25-foot walk (T25FW) or nine-hole peg test. Secondary outcomes included the individual components of EDSS Plus, the six-minute walk test (6MWT), urodynamics testing, and brain atrophy measurement.
RESULTS: Subjects were randomized into MSC-NP (n = 27) or saline (n = 27) groups. There was no difference in EDSS Plus improvement between the MSC-NP (33%) and saline (37%) groups. Exploratory subgroup analysis demonstrated that in subjects who require assistance for ambulation (EDSS 6.0-6.5) there was a significantly higher percentage of improvement in T25FW and 6MWT in the MSC-NP group (3.7% ± 23.1% and - 9.2% ± 18.2%) compared to the saline group (-54.4% ± 70.5% and - 32.1% ± 30.0%), (p = 0.030 and p = 0.036, respectively). IT-MSC-NP treatment was also associated with improved bladder function and reduced rate of grey matter atrophy on brain MRI. Biomarker analysis demonstrated increased MMP9 and decreased CCL2 levels in the cerebrospinal fluid following treatment.
CONCLUSIONS: Results from exploratory outcomes suggest that IT-MSC-NP treatment may be associated with a therapeutic response in a subgroup of MS patients.
BACKGROUND: ClinicalTrials.gov NCT03355365, registered November 14, 2017, https://clinicaltrials.gov/study/NCT03355365?term=NCT03355365&rank=1 .

Even though electromagnetic fields have been reported to assist endogenous neurogenesis, little is known about the exact mechanisms of their action. In this pilot study, we investigated the effects of pulsating extremely low-frequency electromagnetic fields on neural stem cell differentiation towards specific phenotypes, such as neurons and astrocytes. Neural stem cells isolated from the telencephalic wall of B6(Cg)-Tyrc-2J/J mouse embryos (E14.5) were randomly divided into three experimental groups and three controls. Electromagnetic field application setup included a solenoid placed within an incubator. Each of the experimental groups was exposed to 50Hz ELF-EMFs of varied strengths for 1 h. The expression of each marker (NES, GFAP, β-3 tubulin) was then assessed by immunocytochemistry. The application of high-strength ELF-EMF significantly increased and low-strength ELF-EMF decreased the expression of GFAP. A similar pattern was observed for β-3 tubulin, with high-strength ELF-EMFs significantly increasing the immunoreactivity of β-3 tubulin and medium- and low-strength ELF-EMFs decreasing it. Changes in NES expression were observed for medium-strength ELF-EMFs, with a demonstrated significant upregulation. This suggests that, even though ELF-EMFs appear to inhibit or promote the differentiation of neural stem cells into neurons or astrocytes, this effect highly depends on the strength and frequency of the fields as well as the duration of their application. While numerous studies have demonstrated the capacity of EMFs to guide the differentiation of NSCs into neuron-like cells or β-3 tubulin+ neurons, this is the first study to suggest that ELF-EMFs may also steer NSC differentiation towards astrocyte-like phenotypes.

UNASSIGNED: Cell therapy using neural progenitor cells (NPCs) is a promising approach for ischemic stroke treatment according to the results of multiple preclinical studies in animal stroke models. In the vast majority of conducted animal studies, the therapeutic efficacy of NPCs was estimated after intracerebral transplantation, while the information of the effectiveness of systemic administration is limited. Nowadays, several clinical trials aimed to estimate the safety and efficacy of NPCs transplantation in stroke patients were also conducted. In these studies, NPCs were transplanted intracerebrally in the subacute/chronic phase of stroke. The results of clinical trials confirmed the safety of the approach, however, the degree of functional improvement (the primary efficacy endpoint) was not sufficient in the majority of the studies. Therefore, more studies are needed in order to investigate the optimal transplantation parameters, especially the timing of cell transplantation after the stroke onset. This study aimed to evaluate the therapeutic effects of intra-arterial (IA) and intravenous (IV) administration of NPCs derived from induced pluripotent stem cells (iNPCs) in the acute phase of experimental stroke in rats. Induced pluripotent stem cells were chosen as the source of NPCs as this technology is perspective, has no ethical concerns and provides the access to personalized medicine.
UNASSIGNED: Human iNPCs were transplanted IA or IV into male Wistar rats 24 h after the middle cerebral artery occlusion stroke modeling. Therapeutic efficacy was monitored for 14 days and evaluated in comparison with the cell transplantation-free control group. Additionally, cell distribution in the brain was assessed.
UNASSIGNED: The obtained results show that both routes of systemic transplantation (IV and IA) significantly reduced the mortality and improved the neurological deficit of experimental animals compared to the control group. At the same time, according to the MRI data, only IA administration led to faster and prominent reduction of the stroke volume. After IA administration, iNPCs transiently trapped in the brain and were not detected on day 7 after the transplantation. In case of IV injection, transplanted cells were not visualized in the brain. The obtained data demonstrated that the systemic transplantation of human iNPCs in the acute phase of ischemic stroke can be a promising therapeutic strategy.

We report the analysis of 1 year of data from the first cohort of 15 patients enrolled in an open-label, first-in-human, dose-escalation phase I study (ClinicalTrials.gov: NCT03282760, EudraCT2015-004855-37) to determine the feasibility, safety, and tolerability of the transplantation of allogeneic human neural stem/progenitor cells (hNSCs) for the treatment of secondary progressive multiple sclerosis. Participants were treated with hNSCs delivered via intracerebroventricular injection in combination with an immunosuppressive regimen. No treatment-related deaths nor serious adverse events (AEs) were observed. All participants displayed stability of clinical and laboratory outcomes, as well as lesion load and brain activity (MRI), compared with the study entry. Longitudinal metabolomics and lipidomics of biological fluids identified time- and dose-dependent responses with increased levels of acyl-carnitines and fatty acids in the cerebrospinal fluid (CSF). The absence of AEs and the stability of functional and structural outcomes are reassuring and represent a milestone for the safe translation of stem cells into regenerative medicines.

BACKGROUND: Electrical stimulation is a noninvasive treatment method that has gained popularity in the treatment of spinal cord injury (SCI). Activation of spinal cord-derived neural stem/progenitor cell (SC-NSPC) proliferation and differentiation in the injured spinal cord may elicit considerable neural regenerative effects.
OBJECTIVE: This study aimed to explore the effect of electrical stimulation on the neurogenesis of SC-NSPCs.
METHODS: This study analyzed the effects of electrical stimulation on neurogenesis in rodent SC-NSPCs in vitro and in vivo and evaluated functional recovery and neural circuitry improvements with electrical stimulation using a rodent SCI model.
METHODS: Rats (20 rats/group) were assigned to sham (Group 1), SCI only (Group 2), SCI + electrode implant without stimulation (Group 3), and SCI + electrode with stimulation (Group 4) groups to count total SC-NSPCs and differentiated neurons and to evaluate morphological changes in differentiated neurons. Furthermore, the Basso, Beattie, and Bresnahan scores were analyzed, and the motor- and somatosensory-evoked potentials in all rats were monitored.
RESULTS: Biphasic electrical currents enhanced SC-NSPC proliferation differentiation and caused qualitative morphological changes in differentiated neurons in vitro. Electrical stimulation promoted SC-NSPC proliferation and neuronal differentiation and improved functional outcomes and neural circuitry in SCI models. Increased Wnt3, Wnt7, and β-catenin protein levels were also observed after electrical stimulation.
CONCLUSIONS: Our study proved the beneficial effects of electrical stimulation on SCI. The Wnt/β-catenin pathway activation may be associated with this relationship between electrical stimulation and neuronal regeneration after SCI.
CONCLUSIONS: The study confirmed the benefits of electrical stimulation on SCI based on cellular, functional, electrophysiological, and histological evidence. Based on these findings, we expect electrical stimulation to make a positive and significant difference in SCI treatment strategies.

Adult hippocampal neurogenesis (AHN) gives rise to new neurons throughout life. This phenomenon takes place in more than 120 mammalian species, including humans, yet its occurrence in the latter was questioned after one study proposed the putative absence of neurogenesis markers in the adult human hippocampus. In this regard, we showed that prolonged fixation impedes the visualization of Doublecortin+ immature neurons in this structure, whereas other authors have suggested that a dilated post-mortem delay (PMD) underlies these discrepancies. Nevertheless, the individual and/or additive contribution of fixation and the PMD to the detection (or lack thereof) of other AHN markers has not been studied to date. To address this pivotal question, we used a tightly controlled experimental design in mice, which allowed the dissection of the relative contribution of the aforementioned factors to the visualization of markers of individual AHN stages. Fixation time emerged as the most prominent factor globally impeding the study of this process in mice. Moreover, the visualization of other particularly sensitive epitopes was further prevented by prolonged PMD. These results are crucial to disambiguate current controversies related to the occurrence of AHN not only in humans but also in other mammalian species.

Lead (Pb) exposure causes immeasurable damage to multiple human systems, particularly the central nervous system (CNS). In this study, human induced pluripotent stem cells (hiPSCs) were differentiated into neural progenitor cells (NPCs) to investigate the neurotoxic effects of Pb. The hiPSCs were treated with 0, 0.5, 1.0, 2.5, 5.0 and 10.0 μmol/L Pb for 7 days, whereas embryoid bodies (EBs) and NPCs were treated with 0, 0.1, 0.5, and 1.0 μmol/L Pb for 7 days. Pb exposure disrupted the cell cycle and caused apoptosis in hiPSCs, EBs, and NPCs. Besides, Pb inhibited the differentiation of NPCs and EBs. Whole exome sequencing revealed 2509, 2413, and 1984 single nucleotide variants (SNVs) caused by Pb in hiPSCs, EBs, and NPCs, respectively. The common mutation sites in the exon region were mostly nonsynonymous mutations. We identified 18, 19, and 18 common deleterious mutations in hiPSCs, EBs, and NPCs, respectively. Additionally, Online Mendelian Inheritance in Man database analysis revealed 30, 20, and 13 genes related to CNS disorders in hiPSCs, EBs, and NPCs, respectively. Our findings suggest that this in vitro model may supplement animal models and be applied to the study of neurodevelopmental toxicity in the future.

Oligodendrocytes are the myelinating cells of the central nervous system that facilitate efficient signal transduction. The loss of these cells and the associated myelin sheath can lead to profound functional deficits. Moreover, oligodendrocytes also play key roles in mediating glial-neuronal interactions, which further speaks to their importance in health and disease. Neural progenitor cells (NPCs) are a promising source of cells for the treatment of oligodendrocyte-related neurological diseases due to their ability to differentiate into a variety of cell types, including oligodendrocytes. However, the efficiency of oligodendrocyte differentiation is often low. In this study, we induced the expression of the Olig2 transcription factor in tripotent NPCs using a doxycycline-inducible promoter, such that the extent of oligodendrocyte differentiation could be carefully regulated. We characterized the differentiation profile and the transcriptome of these inducible oligodendrogenic NPCs (ioNPCs) using a combination of qRT-PCR, immunocytochemistry and RNA sequencing with gene ontology (GO) and gene set enrichment analysis (GSEA). Our results show that the ioNPCs differentiated into a significantly greater proportion of oligodendrocytes than the NPCs. The induction of Olig2 expression was also associated with the upregulation of genes involved in oligodendrocyte development and function, as well as the downregulation of genes involved in other cell lineages. The GO and GSEA analyses further corroborated the oligodendrocyte specification of the ioNPCs.