The activation of dormant ependymal cells following spinal cord injury
Stem Cell Research & Therapy volume 14, Article number: 175 (2023)
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.
Traumatic spinal cord injury (SCI) induces pathological processes that lead to severe and irreversible deficits, thus affecting a patient's physical, psychological, and social well-being and significantly impacting health-care systems worldwide. The functional restoration of damaged tissue after SCI represents a lofty yet fundamental goal in regenerative medicine.
Therapeutic strategies for SCI include the application of combinations of molecules/drugs, biomaterials, optogenetic approaches, three-dimensional bioprinting technology, and stem cell therapy to promote the regeneration of severed/damaged nerves . The success of these strategies will be influenced by cell activity and communication within the niche after SCI, especially with resident stem cells whose activation, recruitment, and/or modulation could promote recovery. Stem cell transplantation represents a promising strategy for tissue regeneration after SCI, as these cells can survive post-administration and migrate to injured zones [2, 3]; however, critical challenges remain. For instance, preparing therapeutically relevant numbers of stem cells for autologous transplantation requires timespans that extend beyond the optimal treatment time window, while severe immune reaction or graft rejection also remains a significant concern related to allogenic stem cell transplantation [4, 5].
As previously mentioned, certain studies have validated the hypothesis that exogenous activation of endogenous progenitors represents a promising therapeutic strategy [6,7,8]; however, translating therapeutic strategies for SCI repair using ependymal cells from animal models to clinical studies should be approached cautiously. In humans of a certain age, injury can activate and stimulate dormant ependymal stem cells ; however, the central canal is mainly absent in the adult human spinal cord [2, 10]. Thus, the central canal in adulthood becomes substituted by a heterogeneous accumulation of astrocytes, ependymocytes, and perivascular pseudo-rosettes [10, 11]. Additional human studies will be crucial for characterizing the ependymal cell subset, their role after injury, and whether this NPC population possesses proliferative, migratory, and differentiation potential to contribute to spinal cord repair or plays a role in inhibiting/resolving glial scar formation with no regenerative potential.
Ependymal cells from spinal cord in vivo
The existence of spinal cord-resident ependymal cells has incited interest in endogenous strategies as a therapeutic tool [12, 13]. Radial glial cells, specialized cells in the developing nervous system, serve as primary progenitor cells in the central nervous system (CNS) and differentiate into ependymal cells (the possible endogenous neural stem cells [NSCs] of the adult spinal cord ) and astrocytes during early postnatal periods in mammals . Interestingly, a subpopulation of radial glial cells possesses deuterostomes, which participate in the early development of cilia, thereby reinforcing radial glia as ependymal cell progenitors . The ependymal cells that line the spinal cord's central canal  possess characteristic cilia that move cerebrospinal fluid. A subependymal layer containing small numbers of ciliated astrocytes, oligodendrocyte progenitors, and neurons borders the ependymal cells. Ependymal cells also form the neurogenic niche of the adult brain's subventricular zone (SVZ) in combination with dividing glial fibrillary acidic protein (GFAP)-expressing cells (B1 astrocytes), which take on a pinwheel-like cytoarchitecture . Monociliated B1 astrocytes form the core of pinwheels with NSCs-like characteristics, which are surrounded by cells with complex basal bodies with long cilia (often biciliated) comparable to ependymal cells . The ependymal layer in spinal cord tissue possesses a less elaborate form than the adult SVZ [18, 19], taking on a "pearl necklace"-like appearance with bi- or multi-ciliated cells . The most common ependymal cell type line the central canal of the spinal cord and possess two long motile cilia; however, studies have also described bi-nucleated cells with four cilia and cells with one or three cilia associated with large basal bodies . Additional cell subpopulations within the spinal cord central canal, such as GFAP-expressing astrocytes with a single cilium, display similarities to the B1 astrocytes of the SVZ .
Spinal cord-resident ependymal cells are morphologically and molecularly heterogeneous and may reside in different cell subpopulations [10, 20, 21]. Widely accepted, but not exclusive ependymal cell markers include SRY-box transcription factor 3 (Sox3), Sox9, CD15, CD133/prominin1, vimentin, Musashi1, CD24 , and Forkhead Box J1 (FoxJ1)  (Fig. 1). While only dorsally positioned ependymal cells express nestin/GFAP, all ependymal cells express the Sox2 transcription factor [12, 20, 22]. Ki67 expression (in addition to Nestin and Sox2) specifically characterizes the ependymal cells observed in injured tissue . Given the heterogeneity within the NSCs pool in the spinal cord , several recent studies sought to identify ependymal cell subpopulations with NSCs-like properties. Some studies identified cells expressing Msh homeobox 1 (Msx1) in a quiescent state  and Troy, TNF receptor superfamily member 19 (TNFRSF19) in an activated state . Single-cell RNA sequencing (scRNA-seq) experiments have revealed that activated ependymal cells gain stem cell features after injury before differentiating to astrocyte- or oligodendrocyte-lineage cells . Additional studies demonstrated that DNGR-1 expression marks a population of ventricular progenitors committed to an ependymal cell subset endowed with damage-responsive NSCs potential in adulthood. DNGR-1-traced ependymal cells possess latent regenerative potential and mobilize in response to local injury .
The stem cell potential of ependymal cells in the adult spinal cord remains controversial. Neural stem cell progeny exerts a neurotrophic effect required for survival of neurons adjacent to the lesion and is required for maintaining the integrity of the injured spinal cord . Advances in lineage tracing and single-cell sequencing technologies have supported reports of the low contribution of ependymal cells to activated cells after SCI [27, 28]. Therefore, exploring whether ependymal cell activation represents a feasible approach to spinal cord repair after injury must also decipher the molecular mechanisms involved in activation, which extends into the first few weeks post-SCI . This knowledge may contribute to the development of novel therapeutic strategies in regeneration after SCI and the enhanced function of the endogenous regenerative machinery, thereby constituting an exciting alternative or complementary approach to cell transplantation strategies. Exogenous stem cells transplanted into the spinal cord of athymic rats or rat models of motor neuron disease induce endogenous stem cell activity and initiate intrinsic repair mechanisms ; however, whether transplanted cells favor paracrine activity and neurotrophic support, with possible positive effects on the endogenous ependymal cell population after SCI, remain unexplored. Recent research has shown that human fetal brain-derived NSCs and NPCs, embryonic stem cell-derived NPCs, and spinal cord-derived NSCs (ependymal cells) possess similar gene expression profiles; however, some differences have been observed. For instance, ependymal cells over-expressed a greater proportion of genes related to nerve function , while ependymal cells displayed enhanced survival and a greater propensity to differentiate into neurons after transplantation in the SCI in rats . In vivo comparisons have also underlined the optimal therapeutic effects of ependymal cells, which included electrophysiological and hindlimb functional recovery . On the other hand, recent studies have raised doubt regarding the status of the spinal cord ependymal region as a neurogenic niche and its involvement in cell replacement after lesions in adult humans [11, 18].
The microenvironmental conditions of the spinal cord may influence the previously noted poor neurogenic capacity of ependymal cells . In fact, cloned and expanded adult spinal cord ependymal cells can generate neurons and glia after transplantation into the adult rat dentate gyrus when exposed to the appropriate microenvironment . Cell transplantation in the spinal cord injury site could induce enhanced microenvironmental conditions that support spinal cord regeneration ; however, the inhibitory microenvironment that develops after SCI often causes transplanted NSCs and endogenous ependymal cells to differentiate into glial cells rather than neurons . To avoid these adverse effects, certain studies have attempted to improve microenvironmental conditions after SCI to promote the differentiation of exogenous and endogenous NSCs into neurons [35,36,37]. Niche activation via pharmacological agents could suffice to create a protective environment for newborn neurons [8, 38] or NSCs transplanted into the spinal cord, which could induce functional improvement in mice after injury [8, 39]. Therefore, enhancing the neurogenic potential of an ependymal cell or modifying the microenvironment represents an attractive strategy in SCI-focused regenerative medicine.
Epigenetic events can influence the in vivo expression of certain genes in ependymal cells in spinal cord tissue after SCI. Histone acetylation and DNA methylation are epigenetic modifications responsible of regulating patterns of gene expression and are crucial under normal physiological conditions, during development and pathological conditions. An analysis of transcriptional responses of neuronal and ependymal populations after SCI showed that are shaped by histone deacetylase 3 (HDAC3) activity . Based on the expression of ependymal markers Sox2, Foxj1, and regulatory subunit Of Type II PKA R-subunit domain containing 1 (Riiad1), the authors showed 1838 differentially expressed genes between ependymal cells and neurons. HDAC3 affected transcription of 448 genes in ependymal cells, with the majority (78%) repressed by HDAC3, obeying the general principle of gene repression caused by histone deacetylation. Authors showed that SCI caused a marked ependymal expansion with a substantial contraction of neuronal populations. The affected genes were connected to ECM organization, voltage-gated ion channels, as well as Wnt, Hedgehog, and platelet-derived growth factor B signaling. These results indicate a reactive gene profile of ependymal cells after injury . Thus, defining the epigenetic mechanisms that regulate progenitor cells after SCI would help to design strategies to promote the contribution of ependymal cells in wound healing and tissue preservation after injury.
Ependymal cells from spinal cord in vitro
While we still lack a complete understanding of the organization of ependymal cells grown as neurospheres in vitro , our laboratory recently reported the formation of pinwheel structures in spinal cord- and SVZ tissue-derived neurospheres cultured in vitro . Neurospheres are widely used in vitro three-dimensional culture system composed of free-floating clusters of proliferating neural stem cells. We have shown that this organotypic-like culture resembles the neurogenic niche organization of the adult SVZ. The pinwheel’s core contains the apical endings of B1 cells and in its periphery is consisted of the ependymal cells . We showed the presence of pinwheels in neurospheres obtained from spinal cord . We observed the alignment of these cores with apparent equidistant position, in a well-organized manner that may contribute to form round neurospheres (Fig. 2) (unpublished data). Neurospheres may offer an opportunity to study neurogenic mechanisms under normal or pathological conditions, thereby opening new perspectives for therapeutic interventions. Heterogeneous in vitro neurosphere cultures of ependymal cells obtained from the SVZ, olfactory bulb, and spinal cord [43, 44] revealed the expression of markers for radial glia (e.g., brain lipid-binding protein [BLBP], radial glial cell marker 2 [RC2], and glutamate/aspartate transporter [GLAST]). In parallel, it has been observed the expression of oligodendrocytes/neurons markers (e.g., achaete-scute complex-like 1 [Mash1], oligodendrocyte transcription factor 2 [Olig2], NK2 homeobox 2 [Nkx2.2], neural/glial antigen 2 [NG2], neuron cell surface ganglioside epitope [A2B5], and platelet-derived growth factor receptor [PDGFR]) , (Fig. 1). Notably, primary neurospheres derived from ependymal cells and grown in vitro exhibit limited self-renewal capacity [20, 46, 47]. However, a significant increase in proliferation activity was observed in vitro of neurospheres derived from ependymal cells isolated from rats 1 week after SCI as compared with cultures obtained from non-injured control rats. This means that in vitro neurosphere forming potential of ependymal cells increases after SCI [2, 48]. These cells we named induced ependymal cells. Interestingly, telomerase activity was augmented in these cells as well as the expression levels of both Sox2 and Oct4, factors which are critical for pluripotency, and self-renewal when compared to ependymal cells derived from uninjured animals. These activated (or induced) ependymal cells have capacity to differentiate into astrocytes and oligodendrocytes under defined in vitro conditions, although they lack robust neurogenic potential [12, 13, 32, 48,49,50]. It is crucial to determine the variety of molecular signals involved in activation of ependymal cells after SCI suggests that these mechanisms might be exploited to repair spinal cord. On the other hand, certain epigenetic mechanisms regulate the expression of proteins with a relevant presence in ependymal cells when grown as neurospheres in vitro.
Epigenetic regulation was observed in the expression of the ependymal marker FoxJ1 that was silenced by methylation of a CpG island. The forced DNA demethylation by treatment with 5-azacytidine (5-aza-dc) rescues FoxJ1 mRNA expression in neurospheres obtained from spinal cord . This hint suggests that epigenetic mechanisms can regulate expression of FoxJ1 a crucial protein with a role in an ependymal niche organization .
Ependymal cells from injured spinal cord transplanted in vivo
We have already shown that isolation of induced ependymal cells from SCI rats and subsequent transplantation in contusion models showed long-distance migration from the transplant site to the lesion zone participating in the improvement of functional locomotor recovery [2, 52]. These studies suggest that the ependymal cells undergo phenotypic and genotypic changes after SCI such as increase in self-renewal properties, better response to differentiation signals, and improved regenerative capacity. The study of Ohori et al.  showed the possibility to manipulate the neuronal and glial differentiation of endogenous NPCs in vivo. They give the evidence that overexpression of the proneural transcription factors Neurogenin2 and Mash1 together with treatment with growth factors stimulate neurogenesis and oligodendrogenesis, when injected into the injured spinal cord. Mobilizing endogenous NPCs and forced differentiation by growth factor treatment and genetic manipulations may lead to the development of novel cell replacement therapy for SCI.
Expression of SCI non-exclusive markers in ependymal cells after SCI
The confined nature of NSCs activity to spinal cord-resident ependymal cells has provided interest in this cell population as a therapeutic tool [12, 13, 46]. As mentioned above, ependymal cells in the intact spinal cord display limited self-renewal capacity ; thus, ependymal cells represent dormant cells in the intact spinal cord that becomes activated by injury . While a general and rapid loss of neural cell types, including stem/progenitor cells, occurs 1 day after SCI , surviving endogenous ependymal cells become activated in response to distinct types of injury by triggering injury-specific molecular events . Studies have reported that resident ependymal cells positively contribute to spinal cord regeneration by neurotrophic support, impairing cyst formation, or restricting the extent of secondary injury processes [3, 46]. Ependymal cells may also contribute to glial scar formation after SCI  but with minimal/local contribution and depending on direct damage in the ependymal layer . Regardless of the injury type, an early increase in proliferation represents a typical cellular response in ependymal cells after injury [2, 54, 57, 58] followed by migration toward the injury site [13, 16, 58].
While a range of factors contributes to molecular changes in ependymal cells after SCI, we briefly discuss the most critical transcription factors, cell adhesion/extracellular matrix molecules, receptors, and ion channels that may represent future targets for the therapeutic modulation of ependymal cells in the following chapters (Table 1).
After SCI, ependymal cells display well-orchestrated alterations to the expression of transcription factors involved in significant events such as cell viability, division, differentiation, migration, and cilia formation [59,60,61]. Within 3 days after injury, ependymal cell progeny leaves the central canal region and migrates toward the injury site [13, 16, 58]. The process of migration toward the injury may associate with the loss of ependymal phenotype, as judged by the reduced expression of typical transcription factors associated with ependymal cells such as Sox2, Sox3, and FoxJ1 . Ependymal cells microdissected from the central canal region 72 h after SCI in mice display an increase in the expression of cilia-associated transcription factor regulatory factor X4 (Rfx4), but a decrease in other factors such as regulatory factor X1 (Rfx1), tumor protein P53 (Trp53), and FoxJ1 . In adult rats, the downregulated expression of the BAF45D transcription factor (BRG1-associated factor 45D) in ependymal cells following SCI  correlates to the inhibition of neuronal differentiation, which acts indirectly by reducing the pool of neural progenitor cells (NPCs). This study partially supports the role of BAF45D in SCI-related neuropathology . The basic leucine zipper activating transcription factor 3 (ATF3), a critical transcription factor in axon regeneration, becomes rapidly upregulated in injured neurons after peripheral injury . ATF3 overexpression may contribute to neurite outgrowth by orchestrating alterations to gene expression in injured neurons , thereby contributing to spinal cord regeneration . ATF3 expression in ependymal cells from injured rats overlaps with nestin, vimentin, and Sox2 expression  while migrating ependymal cells from injured rats express both Sox9 and ATF3 [13, 60, 65]. Immunohistological analysis of dissected spinal cords demonstrated the importance of ATF3 localization; in this study, ATF3 translocated from the cytoplasm of ependymal cells to the nucleus after activation and mobilization . Thus, ATF3 could represent a reliable marker of activated NPCs in the rat spinal cord .
Olig2 belongs to the b-HLH transcription factor family and plays relevant roles during CNS development and regulates remyelination in models of demyelination CNS disorders. Although Olig2 expression in ependymal cells increases following SCI, scar tissue formation at later time points fails to induce Olig2 expression, which is restricted to uninjured tissue bordering the scar . Although the role of Olig2 in SCI and any possible therapeutic impact remain elusive, evidence suggests that ependymal cells from injured animals may contribute to the formation of myelinating oligodendrocytes to improve functional recovery [60, 65]. Latent lineage potential resident in NSCs enabled SCI repair; the authors reported that expression of OLIG2 in ependymal cells leads to the activation of the latent oligodendrocyte-lineage program, which could support the recovery of axon conduction after injury .
Cell adhesion and extracellular matrix molecules
Ependyma cells express cell adhesion molecules (e.g., E-cadherin, β1-integrin, and neural cell adhesion molecule [NCAM]) and extracellular matrix proteins (e.g., fibronectin, laminins, thrombospondin 2 [THBS2], and chondroitin sulfate proteoglycans [CSPGs]) from different subfamilies; however, their functions after SCI remain incompletely understood .
Cell adhesion molecules help maintain ependymal cell architecture, shape, survival, proliferation, and differentiation in the stem cell niche and support migration [67, 68]. While studies of E-cadherin have failed to demonstrate any differences in expression between control and SCI-affected dogs at the lesion epicenter or proximal sites , differences exist in the subcellular distribution of E-cadherin after SCI. While E-cadherin exclusively localizes to the apical section of ependymal cells in uninjured dogs, SCI induces the re-localization of E-cadherin to the cytosol and circumferential membrane . Taking into account that overexpression of E-cadherin facilitates motor function recovery following SCI by reducing the release of inflammatory cytokines in case of transplanted NSCs , future investigation is needed to determine whether E-cadherin could be efficient target in ependymal cells for regenerative purposes. β1-integrin also becomes robustly upregulated in ependymal cells following SCI, probably to induce ependymal cell migration to the injury site . β1-integrin expression by ependymal cells may play a critical role in astrocytic differentiation of ependymal cells in vivo following SCI by helping to maintain the stem/progenitor cell state. A study by North et al. demonstrated that ablation of β1-integrin expression in ependymal cells decreased the levels of stem cell markers in progeny but increased GFAP expression and astrocytic differentiation . Thus, the β1-integrin signaling system constitutes a potential therapeutic target to modify astrogliosis and limit the detrimental effects of glial scar formation after SCI . Moreno-Manzano et al.  reported increased NCAM expression in in vitro cultures of induced ependymal cells from SCI rats compared to ependymal cells from uninjured rats; however, how NCAM influences ependymal cell behavior after SCI remains unknown. In complete transaction spinal cord animal model, the expression level of NCAM is markedly elevated at 1 day and 3 days post-injury and strongly expressed in in motor neurons (3 days post-transection) and in dorsal sensory and corticospinal fiber tracts (8 days post-transection) . Bearing in mind, the role of NCAM in mediating cell migration, survival, neurite growth and synaptic plasticity , and its possible correlation with functional recovery of the spinal cord  suggesting a role for this protein in pathological development after SCI . By generating a molecular resource through RNA profiling of ependymal cells before and after injury, Chevreau et al.  observed the upregulated expression of the adhesive glycoprotein THBS2, besides the other signaling pathways. THBS2 has a role in mediating cell-to-cell and cell-to-matrix interactions and might contribute to the termination of post-trauma angiogenesis but there is little information concerning whether this molecular change could have relation to ependymal cell activity after injury.
Receptors and ion channels
The expression of specific receptors in the neurogenic niches of adult rodents and humans, including the central canal of the spinal cord, regulates stem cell responses after SCI.
Analysis of ependymal cells obtained by tissue laser microdissection after SCI also established an increase in oncostatin M (OSM) receptor (OSMR) ; furthermore, the same study reported OSM (inflammatory cytokine)-induced robust OSMR expression in spinal cord-derived neurospheres. This study shows that the OSM/OSMR pathway may regulate ependymal cell proliferation and differentiation, particularly the astrocytic fate of ependymal cells after SCI. Studies have indicated that stromal cell-derived factor 1 (SDF-1) plays a vital role in the chemotaxis of stem cells through an interaction with chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). Tysseling et al. observed CXCR4 in the ependymal cells surrounding the central canal; however, the CXCR4 expression pattern in the spinal cord altered 5 weeks after SCI with reduced CXCR4 in the ependymal layer . The reduced expression of this receptor by ependymal cells after injury suggests that CXCR4 does not control the migration of ependymal cell progeny toward the injury site ; instead, the SDF-1/CXCR4 axis may promote recovery after SCI by mediating the migration and attraction of bone marrow-derived mesenchymal stem cells .
Response to CNS injury also involves purinergic (P2) receptors ; however, the detailed contribution of P2 receptors and G protein-coupled receptors (P2Y) and ligand-gated ion channels (P2X) in ependymal cells under in vitro and in vivo conditions remains elusive. The metabotropic P2Y receptors respond to signaling molecules or agonists such as adenine and uridine nucleotides (ATP, ADP, UTP, and UDP) and nucleotide sugars (UDP-glucose). G protein-coupled receptor 17 (GPR17), a P2Y-like receptor responding to both uracil nucleotides (e.g., UDP-glucose) and cysteinyl-leukotrienes, is normally expressed by a subset of neurons, oligodendrocytes, and ependymal cells lining the central canal but not astrocytes . A study by Boccazzi et al. determined that P2Y-like GPR17 receptor could modulate the multipotency of oligodendrocyte precursor cells in vitro . GPR17 may function as a damage “sensor,” becoming activated by nucleotides and cysteinyl leukotrienes released in the lesioned area; however, GPR17 could also participate in post-injury responses. Ceruti et al. proposed an interesting dual and spatiotemporal-dependent role for GPR17 after SCI . They discovered that GPR17-mediated neuronal and oligodendrocyte death within the lesion early after injury; however, the injection of a specific GPR17 antisense oligonucleotide into the spinal cord impaired cell death and significantly ameliorated SCI-induced tissue damage and motor deficits. At later phases after injury, GPR17 may participate in beneficial remodeling and repair activated by danger signals through microglia/macrophages recruitment from distal parenchymal areas and move toward the lesioned zone. The induction of the astrocytes cell marker GFAP in GPR17-expressing ependymal cells suggested the initiation of repair mechanisms . Overall, these findings provide evidence for the designation of GPR17 as a target for therapeutic manipulation to promote remyelination and functional repair in SCI.
Ependymal cells grown as neurospheres in vitro respond to changes in ATP, ADP, and other nucleotides by activating the ionotropic P2X4 and P2X7 and metabotropic P2Y1 and P2Y4 purinergic receptors . Activation of ependymal cells by SCI downregulates the expression of the P2Y1 receptor and upregulates the expression of the P2Y4 receptor . The increased expression of the P2Y4 receptor in ependymal cells after injury could facilitate the expansion of mitotic neural precursors in vitro, while a decrease in P2Y1 could favor neuronal/glial differentiation . Ependymal cells from the intact rat spinal cord do not express functional P2Y2 receptors, as demonstrated by the poor calcium (Ca2+) response to the agonist Ap4A ; however, P2Y2 receptor expression becomes altered in rats after SCI . This study analyzed the spatiotemporal expression of P2Y2 receptors in the spinal cord after SCI and demonstrated a significant increase in P2Y2 mRNA between 2- and 28-day post-injury. Ionotropic P2X receptors, membrane ion channels permeable to sodium (Na+), potassium (K+), and Ca2+ open within milliseconds of ATP binding [81,82,83]. The effects of purinergic agonists on ependymal cells in the neonatal rat spinal cord suggest that P2X7 ion channel receptors and downstream cellular events (e.g., Ca2+ waves) represent possible targets to manipulate the response of the ependymal cell niche to ATP released after SCI [84, 85]. Highly selective pharmacological inhibition of P2X7R in rats by the administration of adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP)  or Brilliant Blue G (an analog of a commonly used food additive with low toxicity) [87, 88] reduces tissue damage and improves motor performance after SCI . The previous reports suggested an early and persistent increase in P2X4 and P2X7  receptor expression around the injury site after severe spinal cord contusion in rats; however, the transplantation of ependymal cells from injured animals in the rat SCI model reversed the increase in P2X4 and P2X7 expression . Related reports have noted the need for further preclinical investigations before evaluating the inhibition of the P2X7 receptor as a treatment for contusive SCI in clinical trials .
Connexin ion channels
Connexins (Cx) comprise a large family of transmembrane proteins that function in gap junction intercellular communication. Besides docking with connexins in neighboring cells, these ion channels form "hemichannels" or "connexons" that exist independently within an individual cell . Recently, the role of these ion channels in crucial stem cell-related processes, including self-renewal and differentiation, has become increasingly prominent [92, 93]. Importantly, connexins also play relevant roles in spinal cord physiology and functional recovery after SCI [94, 95].
Cx43 is the most widely studied connexin, contributing to the secondary expansion of traumatic SCI and playing a vital role in neuropathic pain . Administration of a Cx43 mimetic peptide after SCI in rats prompted a reduction in tissue damage and improved functional recovery, which might relate to the inhibited pathological opening of Cx43 hemichannels . Ependymal cells obtained from injured rats and cultured in vitro exhibited the downregulated expression of Cx37, Cx40, Cx43, and Cx50 compared to ependymal cells from uninjured rats . Interestingly, the plasma membrane represents the most frequent cell location for connexins; however, the location of Cx50 within the nucleus of ependymal cells  and astrocytes  suggests a potential role for this ion channel beyond cell-to-cell communication. A recent study reported that ependymal cell coupling increased after injury, paralleled by the upregulated expression of Cx26 ; however, Cx26 blockade reduced the injury-induced proliferation of ependymal cells. The authors suggest the altered expression of connexins as an early feature of ependymal cells after SCI, which may represent a target to improve the contribution of the central canal stem cell niche to repair .
Purinergic receptors and connexins are closely related families of cell membrane proteins that interact to coordinate molecular events in specific CNS cells to sense injury and activate suitable responses. Under pathological conditions such as SCI, glial activation depends on the communication between neurons and astrocytes mediated by connexin, pannexin, and purinergic receptors . Suadicani et al. discovered that the acute downregulation of Cx43 in mouse spinal cord astrocytes caused the decreased expression of the P2Y1 receptor and increased expression of the P2Y4 receptor [101, 102]. Other studies have reinforced the idea of a relationship between connexins and P2 receptors in NSCs. For instance, the reduced expression of P2Y1 receptors in Cx43-null mice alters Ca2+ signaling and NPC migration . Transplantation of ependymal cells from injured rats in host animals reversed the increased expression of P2X4 and P2X7 receptors  and Cx50  in the grafted region around the SCI. The absence of Cx50 expression in grafted ependymal cells from injured rats suggested a minor regenerative role or detrimental contribution of this ion channel to stem cell engraftment ; however, whether connexins and purinergic receptors function together to contribute to a more permissive environment for axon growth and cell survival after transplantation of NSCs remains to be elucidated.
An orchestrated modulation of gene expression profiles (affecting transcription factors, cell adhesion molecules, receptors, and ion channels) occurs during the transition of ependymal cells from uninjured animals into activated ependymal cells after SCI. Understanding the regulation of expression of these genes in ependymal cells in vitro and in vivo may provide new insight into parallel in vivo processes occurring after injury. Many studies in animal models have shown that injury-activated ependymal cells could contribute to the regenerative process ; however, the view of the spinal cord ependymal region as a neurogenic niche in adult humans remains under doubt due to the results from studies suggesting the lack of involvement of these cells in cell replacement processes after injury [11, 27, 103]. The various studies performed in different species and models and the requirement of detailed tracking studies to determine the origin and fate of cells before and after SCI may partially explain this controversy. This review summarized the main factors involved, including transcription factors and receptors, whose expression becomes significantly modulated in ependymal cells after SCI. Of note, the modulation of cell responses, such as ependymal cells after SCI, could avoid the exacerbated responses that contribute to secondary injury (e.g., inflammation and reactive oxidative damage) and undesired effects (such as pain). Pharmacological intervention to control the activation of the resident stem cells in the spinal cord represents a significant challenge to developing safe and efficient SCI repair/regeneration strategies.
Availability of data and materials
Spinal cord injury
Central nervous system
Neural stem cells
Single-cell RNA sequencing
- CpG Island:
Cytosine and guanine in a repeated sequence
Glial fibrillary acidic protein
SRY-box transcription factor 3
SRY-box transcription factor 9
Forkhead box J1
Msh homeobox 1
TNF receptor superfamily member 19
Marker of proliferation Ki-67
Histone deacetylase 3
Wingless-related integration site
Regulatory subunit of type II PKA R-subunit domain containing 1
Brain lipid-binding protein
Radial glial cell marker 2
Achaete-scute family b-HLH transcription factor 1
Basic helix-loop-helix transcription factor
Oligodendrocyte transcription factor 2
NK2 homeobox 2
Neural/glial antigen 2
Neuron cell surface ganglioside epitope
Platelet-derived growth factor receptor
POU class 5 homeobox 1
Cilia-associated transcription factor regulatory factor X4
Cilia-associated transcription factor regulatory factor X1
Tumor protein P53
Transcription factor (BRG1-associated factor 45D)
Double PHD fingers 2
Activating transcription factor 3
Neural cell adhesion molecule
Chondroitin sulfate proteoglycan
Stromal cell-derived factor 1
Chemokine receptor C-X-C motif chemokine receptor 4
- P2 receptors (P2Y, P2X):
G protein-coupled receptor 17
Ahuja CS, et al. Traumatic spinal cord injury. Nat Rev Dis Primers. 2017;3:17018.
Moreno-Manzano V, et al. Activated spinal cord ependymal stem cells rescue neurological function. Stem Cells. 2009;27(3):733–43.
Sabelstrom H, et al. Resident neural stem cells restrict tissue damage and neuronal loss after spinal cord injury in mice. Science. 2013;342(6158):637–40.
Veceric-Haler Z, et al. Autologous mesenchymal stem cells for treatment of chronic active antibody-mediated kidney graft rejection: report of the phase I/II clinical trial case series. Transpl Int. 2022;35:10772.
Bertaina A, Roncarolo MG. Graft engineering and adoptive immunotherapy: new approaches to promote immune tolerance after hematopoietic stem cell transplantation. Front Immunol. 2019;10:1342.
Jimenez Hamann MC, Tator CH, Shoichet MS. Injectable intrathecal delivery system for localized administration of EGF and FGF-2 to the injured rat spinal cord. Exp Neurol. 2005;194(1):106–19.
Xu B, et al. Transplantation of neural stem progenitor cells from different sources for severe spinal cord injury repair in rat. Bioact Mater. 2023;23:300–13.
Chu W, et al. Valproic acid arrests proliferation but promotes neuronal differentiation of adult spinal NSPCs from SCI rats. Neurochem Res. 2015;40(7):1472–86.
Cawsey T, et al. Nestin-positive ependymal cells are increased in the human spinal cord after traumatic central nervous system injury. J Neurotrauma. 2015;32(18):1393–402.
Garcia-Ovejero D, et al. The ependymal region of the adult human spinal cord differs from other species and shows ependymoma-like features. Brain. 2015;138(Pt 6):1583–97.
Paniagua-Torija B, et al. Cells in the adult human spinal cord ependymal region do not proliferate after injury. J Pathol. 2018;246(4):415–21.
Barnabe-Heider F, et al. Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell. 2010;7(4):470–82.
Meletis K, et al. Spinal cord injury reveals multilineage differentiation of ependymal cells. PLoS Biol. 2008;6(7):e182.
Jacquet BV, et al. FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain. Development. 2009;136(23):4021–31.
Spassky N, et al. Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis. J Neurosci. 2005;25(1):10–8.
Johansson CB, et al. Identification of a neural stem cell in the adult mammalian central nervous system. Cell. 1999;96(1):25–34.
Mirzadeh Z, et al. Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell. 2008;3(3):265–78.
Alfaro-Cervello C, et al. Biciliated ependymal cell proliferation contributes to spinal cord growth. J Comp Neurol. 2012;520(15):3528–52.
Hamilton LK, et al. Cellular organization of the central canal ependymal zone, a niche of latent neural stem cells in the adult mammalian spinal cord. Neuroscience. 2009;164(3):1044–56.
Sabourin JC, et al. A mesenchymal-like ZEB1(+) niche harbors dorsal radial glial fibrillary acidic protein-positive stem cells in the spinal cord. Stem Cells. 2009;27(11):2722–33.
Sabelstrom H, Stenudd M, Frisen J. Neural stem cells in the adult spinal cord. Exp Neurol. 2014;260:44–9.
Lee HJ, et al. SOX2 expression is upregulated in adult spinal cord after contusion injury in both oligodendrocyte lineage and ependymal cells. J Neurosci Res. 2013;91(2):196–210.
Namiki J, Tator CH. Cell proliferation and nestin expression in the ependyma of the adult rat spinal cord after injury. J Neuropathol Exp Neurol. 1999;58(5):489–98.
Ghazale H, et al. RNA profiling of the human and mouse spinal cord stem cell niches reveals an embryonic-like regionalization with MSX1(+) roof-plate-derived cells. Stem Cell Reports. 2019;12(5):1159–77.
Stenudd M, et al. Identification of a discrete subpopulation of spinal cord ependymal cells with neural stem cell properties. Cell Rep. 2022;38(9): 110440.
Frederico B, et al. DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential. Dev Cell. 2022;57(16):1957-1975 e9.
Shah PT, et al. Single-cell transcriptomics and fate mapping of ependymal cells reveals an absence of neural stem cell function. Cell. 2018;173(4):1045-1057 e9.
Xue X, et al. Lineage tracing reveals the origin of Nestin-positive cells are heterogeneous and rarely from ependymal cells after spinal cord injury. Sci China Life Sci. 2022;65(4):757–69.
McDonough A, Martinez-Cerdeno V. Endogenous proliferation after spinal cord injury in animal models. Stem Cells Int. 2012;2012: 387513.
Xu L, Mahairaki V, Koliatsos VE. Host induction by transplanted neural stem cells in the spinal cord: further evidence for an adult spinal cord neurogenic niche. Regen Med. 2012;7(6):785–97.
Okano H, Sawamoto K. Neural stem cells: involvement in adult neurogenesis and CNS repair. Philos Trans R Soc Lond B Biol Sci. 2008;363(1500):2111–22.
Shihabuddin LS, et al. Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci. 2000;20(23):8727–35.
Courtine G, Sofroniew MV. Spinal cord repair: advances in biology and technology. Nat Med. 2019;25(6):898–908.
Xue W, et al. Direct neuronal differentiation of neural stem cells for spinal cord injury repair. Stem Cells. 2021;39(8):1025–32.
Xu B, et al. A dual functional scaffold tethered with EGFR antibody promotes neural stem cell retention and neuronal differentiation for spinal cord injury repair. Adv Healthc Mater. 2017;6(9)
Fan C, et al. Cetuximab and Taxol co-modified collagen scaffolds show combination effects for the repair of acute spinal cord injury. Biomater Sci. 2018;6(7):1723–34.
Li X, et al. Promotion of neuronal differentiation of neural progenitor cells by using EGFR antibody functionalized collagen scaffolds for spinal cord injury repair. Biomaterials. 2013;34(21):5107–16.
Rodriguez-Jimenez FJ, et al. Activation of neurogenesis in multipotent stem cells cultured in vitro and in the spinal cord tissue after severe injury by inhibition of glycogen synthase Kinase-3. Neurotherapeutics. 2021;18(1):515–33.
Abematsu M, et al. Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a mouse model of spinal cord injury. J Clin Invest. 2010;120(9):3255–66.
Wahane S, et al. Diversified transcriptional responses of myeloid and glial cells in spinal cord injury shaped by HDAC3 activity. Sci Adv. 2021;7(9):eabd8811.
Lee JH, et al. NeuroCore formation during differentiation of neurospheres of mouse embryonic neural stem cells. Stem Cell Res. 2020;43: 101691.
Rodriguez-Jimenez FJ, et al. Organized neurogenic-niche-like pinwheel structures discovered in spinal cord tissue-derived neurospheres. Front Cell Dev Biol. 2019;7:334.
Liu Z, Martin LJ. Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human. J Comp Neurol. 2003;459(4):368–91.
Reynolds BA, Rietze RL. Neural stem cells and neurospheres–re-evaluating the relationship. Nat Methods. 2005;2(5):333–6.
Dromard C, et al. NG2 and Olig2 expression provides evidence for phenotypic deregulation of cultured central nervous system and peripheral nervous system neural precursor cells. Stem Cells. 2007;25(2):340–53.
Stenudd M, Sabelstrom H, Frisen J. Role of endogenous neural stem cells in spinal cord injury and repair. JAMA Neurol. 2015;72(2):235–7.
Fiorelli R, et al. The adult spinal cord harbors a population of GFAP-positive progenitors with limited self-renewal potential. Glia. 2013;61(12):2100–13.
Weiss S, et al. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci. 1996;16(23):7599–609.
Shihabuddin LS, et al. Induction of mature neuronal properties in immortalized neuronal precursor cells following grafting into the neonatal CNS. J Neurocytol. 1996;25(2):101–11.
Carlen M, et al. Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke. Nat Neurosci. 2009;12(3):259–67.
Paez-Gonzalez P, et al. Ank3-dependent SVZ niche assembly is required for the continued production of new neurons. Neuron. 2011;71(1):61–75.
Gomez-Villafuertes R. Contribution of purinergic receptors to spinal cord injury repair: stem cell-based neuroregeneration. Neural Regen Res. 2016;11(3):418–9.
Ohori Y, et al. Growth factor treatment and genetic manipulation stimulate neurogenesis and oligodendrogenesis by endogenous neural progenitors in the injured adult spinal cord. J Neurosci. 2006;26(46):11948–60.
Horky LL, et al. Fate of endogenous stem/progenitor cells following spinal cord injury. J Comp Neurol. 2006;498(4):525–38.
Barnabe-Heider F, Frisen J. Stem cells for spinal cord repair. Cell Stem Cell. 2008;3(1):16–24.
Ren Y, et al. Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury. Sci Rep. 2017;7:41122.
Yamamoto S, et al. Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord. J Neurosci. 2001;21(24):9814–23.
Mothe AJ, Tator CH. Advances in stem cell therapy for spinal cord injury. J Clin Invest. 2012;122(11):3824–34.
Wang Z, et al. BAF45D downregulation in spinal cord ependymal cells following spinal cord injury in adult rats and its potential role in the development of neuronal lesions. Front Neurosci. 2019;13:1151.
Mladinic M, et al. ATF3 is a novel nuclear marker for migrating ependymal stem cells in the rat spinal cord. Stem Cell Res. 2014;12(3):815–27.
Chevreau R, et al. RNA profiling of mouse ependymal cells after spinal cord injury identifies the oncostatin pathway as a potential key regulator of spinal cord stem cell fate. Cells. 2021;10(12):3332.
Tsujino H, et al. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury. Mol Cell Neurosci. 2000;15(2):170–82.
Seijffers R, Allchorne AJ, Woolf CJ. The transcription factor ATF-3 promotes neurite outgrowth. Mol Cell Neurosci. 2006;32(1–2):143–54.
Wang LF, et al. Activating transcription factor 3 promotes spinal cord regeneration of adult zebrafish. Biochem Biophys Res Commun. 2017;488(3):522–7.
Panayiotou E, Malas S. Adult spinal cord ependymal layer: a promising pool of quiescent stem cells to treat spinal cord injury. Front Physiol. 2013;4:340.
Llorens-Bobadilla E, et al. A latent lineage potential in resident neural stem cells enables spinal cord repair. Science. 2020;370(6512):eabb8795.
Morante-Redolat JM, Porlan E. Neural stem cell regulation by adhesion molecules within the subependymal niche. Front Cell Dev Biol. 2019;7:102.
Doetsch F. A niche for adult neural stem cells. Curr Opin Genet Dev. 2003;13(5):543–50.
Moore SA, Oglesbee MJ. Spinal cord ependymal responses to naturally occurring traumatic spinal cord injury in dogs. Vet Pathol. 2015;52(6):1108–17.
Chen D, et al. E-cadherin regulates biological behaviors of neural stem cells and promotes motor function recovery following spinal cord injury. Exp Ther Med. 2019;17(3):2061–70.
North HA, et al. beta1-Integrin alters ependymal stem cell BMP receptor localization and attenuates astrogliosis after spinal cord injury. J Neurosci. 2015;35(9):3725–33.
Tzeng SF, et al. Expression of neural cell adhesion molecule in spinal cords following a complete transection. Life Sci. 2001;68(9):1005–12.
Ronn LC, Hartz BP, Bock E. The neural cell adhesion molecule (NCAM) in development and plasticity of the nervous system. Exp Gerontol. 1998;33(7–8):853–64.
Tysseling VM, et al. SDF1 in the dorsal corticospinal tract promotes CXCR4+ cell migration after spinal cord injury. J Neuroinflammation. 2011;8:16.
Wang GD, et al. The SDF-1/CXCR4 axis promotes recovery after spinal cord injury by mediating bone marrow-derived from mesenchymal stem cells. Oncotarget. 2017;8(7):11629–40.
Franke H, Krugel U, Illes P. P2 receptors and neuronal injury. Pflugers Arch. 2006;452(5):622–44.
Ceruti S, et al. The P2Y-like receptor GPR17 as a sensor of damage and a new potential target in spinal cord injury. Brain. 2009;132(Pt 8):2206–18.
Boccazzi M, et al. A new role for the P2Y-like GPR17 receptor in the modulation of multipotency of oligodendrocyte precursor cells in vitro. Purinergic Signal. 2016;12(4):661–72.
Gomez-Villafuertes R, et al. Purinergic receptors in spinal cord-derived ependymal stem/progenitor cells and their potential role in cell-based therapy for spinal cord injury. Cell Transpl. 2015;24(8):1493–509.
Rodriguez-Zayas AE, Torrado AI, Miranda JD. P2Y2 receptor expression is altered in rats after spinal cord injury. Int J Dev Neurosci. 2010;28(6):413–21.
North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002;82(4):1013–67.
Scemes E, Duval N, Meda P. Reduced expression of P2Y1 receptors in connexin43-null mice alters calcium signaling and migration of neural progenitor cells. J Neurosci. 2003;23(36):11444–52.
Scemes E, et al. Connexin and pannexin mediated cell-cell communication. Neuron Glia Biol. 2007;3(3):199–208.
Miras-Portugal MT, et al. Nucleotides in neuroregeneration and neuroprotection. Neuropharmacology. 2016;104:243–54.
Marichal N, et al. Purinergic signalling in a latent stem cell niche of the rat spinal cord. Purinergic Signal. 2016;12(2):331–41.
Wang X, et al. P2X7 receptor inhibition improves recovery after spinal cord injury. Nat Med. 2004;10(8):821–7.
Remy M, et al. An in vivo evaluation of Brilliant Blue G in animals and humans. Br J Ophthalmol. 2008;92(8):1142–7.
Jiang LH, et al. Brilliant blue G selectively blocks ATP-gated rat P2X(7) receptors. Mol Pharmacol. 2000;58(1):82–8.
Peng W, et al. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. Proc Natl Acad Sci USA. 2009;106(30):12489–93.
Marcillo A, et al. A reassessment of P2X7 receptor inhibition as a neuroprotective strategy in rat models of contusion injury. Exp Neurol. 2012;233(2):687–92.
Hofer A, Dermietzel R. Visualization and functional blocking of gap junction hemichannels (connexons) with antibodies against external loop domains in astrocytes. Glia. 1998;24(1):141–54.
Rodriguez-Jimenez FJ, et al. Connexin 50 expression in ependymal stem progenitor cells after spinal cord injury activation. Int J Mol Sci. 2015;16(11):26608–18.
Ke Q, et al. Connexin 43 is involved in the generation of human-induced pluripotent stem cells. Hum Mol Genet. 2013;22(11):2221–33.
Lee IH, et al. Glial and neuronal connexin expression patterns in the rat spinal cord during development and following injury. J Comp Neurol. 2005;489(1):1–10.
Tonkin RS, et al. Gap junction proteins and their role in spinal cord injury. Front Mol Neurosci. 2014;7:102.
Huang C, et al. Critical role of connexin 43 in secondary expansion of traumatic spinal cord injury. J Neurosci. 2012;32(10):3333–8.
Mao Y, et al. Systemic administration of connexin43 mimetic peptide improves functional recovery after traumatic spinal cord injury in adult rats. J Neurotrauma. 2017;34(3):707–19.
Rodriguez-Jimenez FJ, et al. Connexin 50 modulates Sox2 expression in spinal-cord-derived ependymal stem/progenitor cells. Cell Tissue Res. 2016;365(2):295–307.
Fabbiani G, et al. Connexin signaling is involved in the reactivation of a latent stem cell niche after spinal cord injury. J Neurosci. 2020;40(11):2246–58.
Wang A, Xu C. The role of connexin43 in neuropathic pain induced by spinal cord injury. Acta Biochim Biophys Sin (Shanghai). 2019;51(6):555–61.
Suadicani SO, Brosnan CF, Scemes E. P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci. 2006;26(5):1378–85.
Suadicani SO, et al. Acute downregulation of Cx43 alters P2Y receptor expression levels in mouse spinal cord astrocytes. Glia. 2003;42(2):160–71.
Muthusamy N, et al. Foxj1 expressing ependymal cells do not contribute new cells to sites of injury or stroke in the mouse forebrain. Sci Rep. 2018;8(1):1766.
We acknowledge Servier Medical Art for providing elements within Figure 1. We thank Dr. Stuart P. Atkinson for article reviewing and English language editing.
This work was supported by funds for research from the Institute of Health Carlos III PI18/00286 and PI21/00157 (SE) FEDER (European Regional Development Fund) and the project "Centre of Reconstructive Neuroscience," registration number CZ.02.1.01/0.0./0.0/15_003/0000419.
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Rodriguez-Jimenez, F.J., Jendelova, P. & Erceg, S. The activation of dormant ependymal cells following spinal cord injury. Stem Cell Res Ther 14, 175 (2023). https://doi.org/10.1186/s13287-023-03395-4
- Spinal cord injury
- Ependymal cells