Neural Stem/progenitor Cells Markers Research Articles

Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells

Background• Neural stem/progenitor cells (NSPCs) are multipotent self-renewing cells that can be isolated from the brain or spinal cord. As they need to be isolated from neural tissues, it is difficult to study human NSPCs. To facilitate NSPC research, we attempted to isolate NSPCs from dogs, as dogs share the environment and having many similar diseases with humans. We collected and established primary cultures of ependymal and subependymal cells from the central canal of the cervical spinal cord of adult dogs. To isolate pure NSPCs, we employed the monolayer culture and selective medium culture methods. We further tested the ability of the NSPCs to form neurospheres (using the suspension culture method) and evaluated their differentiation potential.Results• The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested.Conclusion• It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.

Transcription factors promote neural regeneration after spinal cord injury.

Transcription factors promote neural regeneration after spinal cord injury.

Establishment of a Reproducible Ischemic Stroke Model in Nestin-GFP Mice with High Survival Rates.

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

Low folate induces abnormal neuronal maturation and DNA hypomethylation of neuronal differentiation-related genes in cultured mouse neural stem and progenitor cells

Folate deficiency in a fetus is well known to cause neurodevelopment defects and development disorders. A low level of folate is also thought to be a risk for depression in adults. We have previously shown that post-weaning low folate induces neuronal immaturity in the dentate gyrus in mice, which suggests that low folate causes neuropsychological disorders via inhibition of neuronal maturation. In this study, we examined the effects of low folate on expression and epigenetic modification of genes involved in neuronal differentiation and maturation in primary mouse neural stem/progenitor cells (NSPCs) in vitro. An increase in Nestin (NSPC marker)-positive cells was observed in cells differentiated in a low folate medium for 3 days. An increase in βIII-tubulin (Tuj1: immature neuron marker)-positive cells and a decrease in microtubule-associated protein 2 (MAP2: mature neuron marker)-positive cells were observed in cells differentiated in a low folate medium for 7 days. In these cells, mRNA levels for genes involved in neuronal differentiation and maturation were altered. Hypomethylation of DNA, but not of histone proteins, was also observed at some promoters of these neuronal genes. The level of S-adenosylmethionine (SAM), a methyl donor, was decreased in these cells. The abnormalities in neural maturation and changes in gene expression in culture under low folate conditions were partially normalized by addition of SAM (5 μM). Based on these results, decreased SAM may induce DNA hypomethylation at genes involved in neuronal differentiation and maturation under low folate conditions, and this hypomethylation may be associated with low folate-induced neuronal immaturity.

Nylon mesh-based 3D scaffolds for the adherent culture of neural stem/progenitor cells

We developed novel scaffolds for the adherent culture of neural stem/progenitor cells on the woven mesh. Nylon mesh (NM) is an inert material for cell adhesion. We prepared polyacrylic acid-grafted nylon mesh (PAA-NM) by graft polymerization method using gamma-irradiation. Matrigel was covalently immobilized to the carboxyl groups in PAA-NM by chemical conjugation using 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to prepare the Matrigel-immobilized PAA-grafted nylon mesh (M-PAA-NM). Cell adhesion property of mouse neural stem/progenitor cells (NSPCs) between the NM, PAA-NM, and M-PAA-NM was different from each other. The neurosphere-like clusters of NSPCs were weakly bound to NM and PAA-NM without spreading. The NSPCs were firmly adhered to, spread, and covered the surface of M-PAA-NM. We evaluated the state of differentiation by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immnocytochemistry. A neuronal marker β III tubulin, a glial marker glial fibrillary acidic protein (GFAP) and a mature glial marker S100β were expressed at a low level in the cultured cells while immature NSPCs marker Nestin and Sox2 were slightly lower without significant statistical difference. We concluded that the M-PAA-NM is a good substrate for adherent culture of NSPCs without triggering their cell differentiation, and also provides the maintenance of their growth with fewer passages in comparison with the conventional suspension culture of NSPCs in neurospheres.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells.

Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons.Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied.Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs.Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Quadrella incana (Capparaceae) Leaf Extract Enhances Proliferation and Maintenance of Neural Stem/Progenitor Cells through Upregulating Glycolytic Flux and Redox Potential.

Neural stem/progenitor cells (NSPCs) are self-renewing, multipotent cells located in the embryonic and adult central nervous system (CNS). Extensive preclinical and clinical studies have shed light on the potential of stem cell replacement therapy for various neurodegenerative diseases. The key prerequisite for the success of these clinical applications is the procurement of a sufficient number of high-quality NSPCs. In this study, we explored the biological activity of Quadrella incana leaf in NSPC homeostasis. We showed that the leaf extract of Quadrella incana upregulated NSPC marker and proliferative potential. On the other hand, Quadrella incana leaf suppressed spontaneous unintended NSPC differentiation. Mechanistically, Quadrella incana leaf contributed to the maintenance of NSPCs by upregulating glycolytic flux and redox potential.

A novel mouse model for the study of endogenous neural stem and progenitor cells after traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of death and disability in the US. Neural stem/progenitor cells (NSPCs) persist in the adult brain and represent a potential cell source for tissue regeneration and wound healing after injury. The Notch signaling pathway is critical for embryonic development and adult brain injury response. However, the specific role of Notch signaling in the injured brain is not well characterized. Our previous study has established a Notch1CR2-GFP reporter mouse line in which the Notch1CR2 enhancer directs GFP expression in NSPCs and their progeny. In this study, we performed closed head injury (CHI) in the Notch1CR2-GFP mice to study the response of injury-activated NSPCs. We show that CHI induces neuroinflammation, cell death, and the expression of typical TBI markers (e.g., ApoE, Il1b, and Tau), validating the animal model. In addition, CHI induces cell proliferation in GFP+ cells expressing NSPC markers, e.g., Notch1 and Nestin. A significant higher percentage of GFP+ astrocytes and GABAergic neurons was observed in the injured brain, with no significant change in oligodendrocyte lineage between the CHI and sham animal groups. Since injury is known to activate astrogliosis, our results suggest that injury-induced GFP+ NSPCs preferentially differentiate into GABAergic neurons. Our study establishes that Notch1CR2-GFP transgenic mouse is a useful tool for the study of NSPC behavior in vivo after TBI. Unveiling the potential of NSPCs response to TBI (e.g., proliferation and differentiation) will identify new therapeutic strategy for the treatment of brain trauma.

Histone Methylations Define Neural Stem/Progenitor Cell Subtypes in the Mouse Subventricular Zone

Neural stem/progenitor cells (NSPCs) persist in the mammalian brain throughout life and can be activated in response to the physiological and pathophysiological stimuli. Epigenetic reprogramming of NPSC represents a novel strategy for enhancing the intrinsic potential of the brain to regenerate after brain injury. Therefore, defining the epigenetic features of NSPCs is important for developing epigenetic therapies for targeted reprogramming of NSPCs to rescue neurologic function after injury. In this study, we aimed at defining different subtypes of NSPCs by individual histone methylations. We found the three histone marks, histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 trimethylation (H3K27me3), and histone H3 lysine 36 trimethylation (H3K36me3), to nicely and dynamically portray individual cell types during neurodevelopment. First, we found all three marks co-stained with NSPC marker SOX2 in mouse subventricular zone. Then, CD133, Id1, Mash1, and DCX immunostaining were used to define NSPC subtypes. Type E/B, B/C, and C/A cells showed high levels of H3K27me3, H3K36me3, and H3K4me3, respectively. Our results reveal defined histone methylations of NSPC subtypes supporting that epigenetic regulation is critical for neurogenesis and for maintaining NSPCs.

Potential of Adult Endogenous Neural Stem/Progenitor Cells in the Spinal Cord to Contribute to Remyelination in Experimental Autoimmune Encephalomyelitis.

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.

Malformations of Human Neocortex in Development - Their Progenitor Cell Basis and Experimental Model Systems.

Malformations of the human neocortex in development constitute a heterogeneous group of complex disorders, resulting in pathologies such as intellectual disability and abnormal neurological/psychiatric conditions such as epilepsy or autism. Advances in genomic sequencing and genetic techniques have allowed major breakthroughs in the field, revealing the molecular basis of several of these malformations. Here, we focus on those malformations of the human neocortex, notably microcephaly, and macrocephaly, where an underlying basis has been established at the level of the neural stem/progenitor cells (NPCs) from which neurons are directly or indirectly derived. Particular emphasis is placed on NPC cell biology and NPC markers. A second focus of this review is on experimental model systems used to dissect the underlying mechanisms of malformations of the human neocortex in development at the cellular and molecular level. The most commonly used model system have been genetically modified mice. However, although basic features of neocortical development are conserved across the various mammalian species, some important differences between mouse and human exist. These pertain to the abundance of specific NPC types and/or their proliferative capacity, as exemplified in the case of basal radial glia. These differences limit the ability of mouse models to fully recapitulate the phenotypes of malformations of the human neocortex. For this reason, additional experimental model systems, notably the ferret, non-human primates and cerebral organoids, have recently emerged as alternatives and shown to be of increasing relevance. It is therefore important to consider the benefits and limitations of each of these model systems for studying malformations of the human neocortex in development.

Neural Stem/Progenitor Cells of the Spinal Cord are Activated in Response to Tail Loss in the Leopard Gecko

As for many lizards, the leopard gecko (Eublepharis macularius) is able to self‐detach its tail as an anti‐predation strategy and subsequently regenerate a replacement. The replacement tail includes a regenerated spinal cord. We hypothesize that endogenous neural stem progenitor cells (NSPCs) are present in the original spinal cord and that these cells are activated during regeneration. As many stem cells are slow cycling, we used a Bromodeoxyuridine (BrdU) pulse‐chase experiment to identify label‐retaining populations in the original spinal cord. We also conducted a spatiotemporal characterization of spinal cord cells before and during regeneration using a panel of common NSPC markers (including Sox2, Sox9, Vimentin, βIII‐Tubulin and Musashi‐1, and the proliferation marker PCNA). BrdU retaining cells were still present after a 20‐week chase. Prior to injury, expression of NSPC markers is limited, but co‐localization of BrdU and Sox2 in ependymal cells was observed. Following tail loss, ependymal cells in the original stump of the tail become highly proliferative and express our panel of NSPC markers. Outgrowth of the new tail is matched by proliferating, NSPC marker‐positive ependymal cells. These cells appear to contribute to regenerating spinal cord. We conclude that ependymal cells are a likely source of endogenous NSPCs in the gecko and that they are activated following tail loss.NSERC Grant: 400358

Postnatal age governs the extent of differentiation of hippocampal CA1 and CA3 subfield neural stem/progenitor cells into neurons and oligodendrocytes

While neural stem/progenitor cells (NSCs) in the dentate gyrus of the hippocampus have been extensively characterized, the behavior of NSCs in the CA1 and CA3 subfields of the hippocampus is mostly unclear. Therefore, we compared the in vitro behavior of NSCs expanded from the micro-dissected CA1 and CA3 subfields of postnatal day (PND) 4 and 12 Fischer 344 rats. A small fraction (∼1%) of dissociated cells from CA1 and CA3 subfields of both PND 4 and 12 hippocampi formed neurospheres in the presence of EGF and FGF-2. A vast majority of neurosphere cells expressed NSC markers such as nestin, Sox-2 and Musashi-1. Differentiation assays revealed the ability of these NSCs to give rise to neurons, astrocytes, and oligodendrocytes. Interestingly, the overall neuronal differentiation of NSCs from both subfields decreased with age (23–28% at PND4 to 5–10% at PND12) but the extent of oligodendrocyte differentiation from NSCs increased with age (24–32% at PND 4 to 45–55% at PND 12). Differentiation of NSCs into astrocytes was however unchanged (40–48%). Furthermore, NSCs from both subfields gave rise to GABA-ergic neurons including subclasses expressing markers such as calbindin, calretinin, neuropeptide Y and parvalbumin. However, the fraction of neurons that expressed GABA decreased between PND4 (59–67%) and PND 12 (25–38%). Additional analyses revealed the presence of proliferating NSC-like cells (i.e. cells expressing Ki-67 and Sox-2) in different strata of hippocampal CA1 and CA3 subfields of both PND4 and PND 12 animals. Thus, multipotent NSCs persist in both CA1 and CA3 subfields of the hippocampus in the postnatal period. Such NSCs also retain their ability to give rise to both GABA-ergic and non-GABA-ergic neurons. However, their overall neurogenic potential declines considerably in the early postnatal period.

Cell of Origin Determines Tumor Phenotype in an Oncogenic Ras/p53 Knockout Transgenic Model of High-Grade Glioma

Human high-grade gliomas (HGGs) are known for their histologic diversity. To address the role of cell of origin in glioma phenotype, transgenic mice were generated in which oncogenic Ras and p53 deletion were targeted to neural stem/progenitor cells (NSPCs) and mature astrocytes. The hGFAP-Cre/Kras/p53 mice develop multifocal HGGs that vary histopathologically and with respect to the expression of markers associated with NSPCs. One HGG pattern strongly expressed markers of NSPCs and arose near the subventricular zone. Additional nonoverlapping patterns that recapitulate human HGG variants were present simultaneously in the same brain. These neoplastic foci were more often cortical or leptomeningeal based, and the neoplastic cells lacked expression of NSPC markers. To determine whether cell of origin determines tumor phenotype, astrocytes and NSPCs were harvested from neonatal mutant pups. Onorthotopic transplantation, early-passage astrocytes and NSPCs formed tumors that differed in engraftment rates, latency to clinical signs, histopathology, and protein expression. Astrocyte-derivedtumors were more aggressive, had giant-cell histology, and glial fibrillary acidic protein expression. The NSPC-derived tumors retained NSPC markers and showed evidence of differentiation along astrocytic, oligodendroglial, and neuronal lineages. These results indicate that identical tumorigenic stimuli produce markedly different glioma phenotypes, depending on the differentiation status of the transformed cell.

Leptomeningeal-Derived Doublecortin-Expressing Cells in Poststroke Brain

Increasing evidence indicates that neural stem/progenitor cells (NSPCs) reside in many regions of the central nervous system (CNS), including the subventricular zone (SVZ) of the lateral ventricle, subgranular zone of the hippocampal dentate gyrus, cortex, striatum, and spinal cord. Using a murine model of cortical infarction, we recently demonstrated that the leptomeninges (pia mater), which cover the entire cortex, also exhibit NSPC activity in response to ischemia. Pial-ischemia-induced NSPCs expressed NSPC markers such as nestin, formed neurosphere-like cell clusters with self-renewal activity, and differentiated into neurons, astrocytes, and oligodendrocytes, although they were not identical to previously reported NSPCs, such as SVZ astrocytes, ependymal cells, oligodendrocyte precursor cells, and reactive astrocytes. In this study, we showed that leptomeningeal cells in the poststroke brain express the immature neuronal marker doublecortin as well as nestin. We also showed that these cells can migrate into the poststroke cortex. Thus, the leptomeninges may participate in CNS repair in response to brain injury.

Expression of the Ubiquitin Ligase HRD1 in Neural Stem/Progenitor Cells of the Adult Mouse Brain

Neural stem/progenitor cells (NSCs) reside in the subventricular zone (SVZ) and subgranular zone of the hippocampal dentate gyrus in adult mammals. The ubiquitin ligase HRD1 is associated with degradation of amyloid precursor protein and believed to be specifically expressed in neurons and not in astrocytes. We investigated expression of HRD1 using immunohistochemistry and found colocalization of HRD1 with the NSC marker protein nestin and glial fibrillary acidic protein in the NSCs of the SVZ (the SVZ astrocytes) but not in the hippocampus. In the hippocampal dentate gyrus, HRD1 is localized in the nucleus of nestin-positive cells.

Primary neural stem/progenitor cells expressing endostatin or cytochrome P450 for gene therapy of glioblastoma.

Despite recent technical improvements in surgical excision techniques and adjuvant radio- and chemotherapy, the clinical outcome of patients with grade IV astrocytoma (glioblastoma) remains very poor, with a median survival of less than 12 months. A promising approach to therapy employs gene-engineered neural stem/progenitor cells (NSCs) as a cellular therapeutic delivery system, to track glioblastoma cells and deliver anticancer molecules. However, most results on their tumor tropism have been derived by immortalized NSCs. We now report that primary murine gene-engineered NSCs displayed in vivo tropism for glioblastoma cells. Ten days after injection into the brain, many NSCs continued to express the transgene and the NSC marker, nestin. NSCs transduced with a retroviral vector co-expressing a secretable form of human endostatin and eGFP (NSC/endo-eGFP) released potentially antiangiogenetic concentrations of endostatin into the culture medium. Conditioned medium of NSC/endo-eGFP cells inhibited the growth of mouse pulmonary microvascular endothelial cells (PMVECs). A good correlation between endostatin levels and PMVEC growth inhibition was observed. In NSCs co-expressing cytochrome P450 2B6 (CYP2B6) and eGFP (NSC/CYP2B6-eGFP), the forced expression of CYP2B6 resulted in intracellular activation of CPA and subsequent cell death. In the presence of NSC/CYP2B6-eGFP, we observed CPA cytotoxicity to DsRed-expressing U87Mg glioma cells. In vivo treatment of intracranial GL-261 glioblastoma with NSC/endo-eGFP caused a 65% reduction in tumor size, compared to untreated control mice or mice treated with NSC/eGFP cells. Our data suggest that primary NSCs transduced with retroviral vectors expressing endostatin and/or CYP2B6 have a potential role in glioblastoma therapy.