Ependymal cells, a dormant population of ciliated progenitors found within the central canal of the spinal cord, undergo significant alterations after spinal cord injury (SCI). Understanding the molecular events that induce ependymal cell activation after SCI represents the first step toward controlling the response of the endogenous regenerative machinery in damaged tissues. This response involves the activation of specific signaling pathways in the spinal cord that promotes self-renewal, proliferation, and differentiation. We review our current understanding of the signaling pathways and molecular events that mediate the SCI-induced activation of ependymal cells by focusing on the roles of some cell adhesion molecules, cellular membrane receptors, ion channels (and their crosstalk), and transcription factors. An orchestrated response regulating the expression of receptors and ion channels fine-tunes and coordinates the activation of ependymal cells after SCI or cell transplantation. Understanding the major players in the activation of ependymal cells may help us to understand whether these cells represent a critical source of cells contributing to cellular replacement and tissue regeneration after SCI. A more complete understanding of the role and function of individual signaling pathways in endogenous spinal cord progenitors may foster the development of novel targeted therapies to induce the regeneration of the injured spinal cord.

• Klíčová slova
• Activation, Ependymal cells, Regeneration, Spinal cord injury,
• MeSH
• ependym metabolismus   MeSH
• iontové kanály metabolismus   MeSH
• lidé MeSH
• mícha MeSH
• neuroglie metabolismus   MeSH
• poranění míchy * terapie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• iontové kanály MeSH

Spinal cord injury is a devastating medical condition with no effective treatment. One approach to SCI treatment may be provided by stem cells (SCs). Studies have mainly focused on the transplantation of exogenous SCs, but the induction of endogenous SCs has also been considered as an alternative. While the differentiation potential of neural stem cells in the brain neurogenic regions has been known for decades, there are ongoing debates regarding the multipotent differentiation potential of the ependymal cells of the central canal in the spinal cord (SCECs). Following spinal cord insult, SCECs start to proliferate and differentiate mostly into astrocytes and partly into oligodendrocytes, but not into neurons. However, there are several approaches concerning how to increase neurogenesis in the injured spinal cord, which are discussed in this review. The potential treatment approaches include drug administration, the reduction of neuroinflammation, neuromodulation with physical factors and in vivo reprogramming.

• Klíčová slova
• astrocytes, ependymal stem cells, growth factors, neurogenesis, neuroinflammation, physical factors, reprogramming, spinal canal, spinal cord injury, valproic acid,
• MeSH
• buněčná diferenciace MeSH
• lidé MeSH
• mícha MeSH
• nervové kmenové buňky * MeSH
• neurogeneze MeSH
• neurony MeSH
• poranění míchy * terapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/β-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell-derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell-derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates β-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma-positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl-treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI.

• Klíčová slova
• GSK3 inhibition, Spinal cord injury, axonal growth, neurogenesis, stem cells,
• MeSH
• kinasa 3 glykogensynthasy antagonisté a inhibitory   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• multipotentní kmenové buňky účinky léků   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neurogeneze účinky léků   MeSH
• poranění míchy farmakoterapie   enzymologie   MeSH
• transplantace kmenových buněk MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinasa 3 glykogensynthasy MeSH

Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury.

• Klíčová slova
• glia limitans formation from grafted neural precursors, human-specific mRNA sequencing, immunodeficient rat, neuraxial neural precursor migration, subpial stem cell injection,
• MeSH
• krysa rodu Rattus MeSH
• nervové kmenové buňky metabolismus   MeSH
• parenchymatická tkáň cytologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The neurogenic niche of the subventricular zone (SVZ) in adult brain tissue takes the form of a pinwheel-like cytoarchitectural structure, with mono-ciliated astrocytes displaying neural stem cell (NSC) characteristics present in the core surrounded by ciliated ependymal cells. For the first time, we have demonstrated the formation of similar pinwheel structures in spinal cord and SVZ tissue-derived neurospheres cultured in vitro. To investigate whether the organization and integrity of these pinwheel structures depends on the appropriate organization of ciliated astrocytes and ependymal cells, we modified neurosphere cell arrangements via the application of the methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dc) or the antiviral drug ganciclovir (GCV) in transgenic mice expressing herpes simplex virus thymidine kinase from the GFAP promoter (GFAP-TK). Treatment of neurospheres with 5-aza-dc increased FoxJ1 expression, a crucial factor for ciliogenesis, by reducing methylation of the FoxJ1 CpG island. 5-aza-dc also increased the expression of the astrocyte marker GFAP and caused aberrant accumulation of ciliated astrocytes. However, the ablation of dividing astrocytes within neurospheres by GCV treatment led to an increase in the accumulation of ciliated ependymal cells, as evidenced by the increased expression of the ependymal cell markers Vimentin or CD24. While 5-aza-dc and GCV treatment differentially affected cell arrangement, both compounds significantly diminished the number of pinwheel structures present in neurospheres. Thus, we suggest that the ratio of ciliated astrocytes to ependymal cells plays a crucial role in the correct formation of the pinwheel structures in spinal cord tissue-derived neurospheres in vitro.

• Klíčová slova
• astrocytes, cilia, ependymal cells, neurospheres, pinwheels,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.

• Klíčová slova
• Amyotrophic lateral sclerosis (ALS), Bioinformatic tools to study xenografts, Human embryonic stem cell (hESC), Neural stem cell (NSC), Spinal cord, Spinal traumatic injury,
• MeSH
• buněčné linie MeSH
• lidé MeSH
• multipotentní kmenové buňky cytologie   MeSH
• nervové kmenové buňky cytologie   MeSH
• průtoková cytometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Oligodendrocyte progenitor cells (OPCs) play a pivotal role in both health and disease within the central nervous system, with oligodendrocytes, arising from resident OPCs, being the main myelinating cell type. Disruption in OPC numbers can lead to various deleterious health defects. Numerous studies have described techniques for isolating OPCs to obtain a better understanding of this cell type and to open doors for potential treatments of injury and disease. However, the techniques used in the majority of these studies involve several steps and are time consuming, with current culture protocols using serum and embryonic or postnatal cortical tissue as a source of isolation. We present a primary culture method for the direct isolation of functional adult rat OPCs, identified by neuron-glial antigen 2 (NG2) and platelet derived growth factor receptor alpha (PDGFrα) expression, which can be obtained from the adult spinal cord. Our method uses a simple serum-free cocktail of 3 growth factors - FGF2, PDGFAA, and IGF-I, to expand adult rat OPCs in vitro to 96% purity. Cultured cells can be expanded for at least 10 passages with very little manipulation and without losing their phenotypic progenitor cell properties, as shown by immunocytochemistry and RT-PCR. Cultured adult rat OPCs also maintain their ability to differentiate into GalC positive cells when incubated with factors known to stimulate their differentiation. This new isolation method provides a new source of easily accessible adult stem cells and a powerful tool for their expansion in vitro for studies aimed at central nervous system repair.

• Klíčová slova
• Adult spinal cord, CNS, Differentiation, Progenitor cells, Spinal cord injury,
• MeSH
• antigeny metabolismus   MeSH
• destičkový růstový faktor metabolismus   MeSH
• dospělé kmenové buňky cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• krysa rodu Rattus MeSH
• mícha cytologie   metabolismus   MeSH
• oligodendroglie cytologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• proteoglykany metabolismus   MeSH
• separace buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• chondroitin sulfate proteoglycan 4 MeSH Prohlížeč
• destičkový růstový faktor MeSH
• fibroblastový růstový faktor 2 MeSH
• insulin-like growth factor-1, rat MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• platelet-derived growth factor A MeSH Prohlížeč
• proteoglykany MeSH