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Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult Mammalian Brain
Abstract
Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.
... NSCs are primitive precursor cells with the ability to differentiate in the CNS. NSCs exist in the developing CNS and persist in certain areas of the brain in newborns and adults, appearing in the form of neuroepithelial cells, radial glial (RG) cells, subventricular zone (SVZ) astrocytes (type B cells), and the subgranular zone (SGZ) radial astrocytes, functioning as stem cells (Doetsch et al., 1999;Garcia et al., 2004). The symmetrical cell division process of neuroepithelial cells and RG cells, known as interkinetic nuclear migration, can maintain stem cell activity, whereas limited potential progenitor cells, such as intermediate progenitor cells (IPCs), no longer undergo interkinetic nuclear migration; therefore, interkinetic nuclear migration is a characteristic of NSCs in the CNS (Del Bene et al., 2008;Elkabetz et al., 2008). ...
... In the SVZ region of the lateral ventricle in adults, type B cells extend their basal processes towards blood vessels and their apical processes to contact the cerebrospinal fluid in the lateral ventricle. Type B cells undergo division to generate neural transiently amplifying progenitor cells (type C cells), which repeatedly divide to produce neuroblasts (type A cells) (Doetsch et al., 1999;Imayoshi et al., 2008). The newly generated neuroblasts migrate tangentially along chains to the olfactory bulb and differentiate into different subtypes of interneurons (Doetsch et al., 1999;Lim and Alvarez-Buylla, 2014). ...
... Type B cells undergo division to generate neural transiently amplifying progenitor cells (type C cells), which repeatedly divide to produce neuroblasts (type A cells) (Doetsch et al., 1999;Imayoshi et al., 2008). The newly generated neuroblasts migrate tangentially along chains to the olfactory bulb and differentiate into different subtypes of interneurons (Doetsch et al., 1999;Lim and Alvarez-Buylla, 2014). Another region in the adult CNS that generates new neurons is the SGZ of the hippocampal dentate gyrus. ...
Ischemic stroke is a major cause of mortality and disability worldwide, with limited treatment options available in clinical practice. The emergence of stem cell therapy has provided new hope to the field of stroke treatment via the restoration of brain neuron function. Exogenous neural stem cells are beneficial not only in cell replacement but also through the bystander effect. Neural stem cells regulate multiple physiological responses, including nerve repair, endogenous regeneration, immune function, and blood-brain barrier permeability, through the secretion of bioactive substances, including extracellular vesicles/exosomes. However, due to the complex microenvironment of ischemic cerebrovascular events and the low survival rate of neural stem cells following transplantation, limitations in the treatment effect remain unresolved. In this paper, we provide a detailed summary of the potential mechanisms of neural stem cell therapy for the treatment of ischemic stroke, review current neural stem cell therapeutic strategies and clinical trial results, and summarize the latest advancements in neural stem cell engineering to improve the survival rate of neural stem cells. We hope that this review could help provide insight into the therapeutic potential of neural stem cells and guide future scientific endeavors on neural stem cells.
... Although the repair capacity of the mature brain is limited, neural stem/progenitor cells (NSPCs) with multilineage differentiation potential are present in some brain regions throughout life [11]. Previous studies of mice expressing green fluorescent protein (GFP) under the control of the promoter for the NSPC marker nestin have shown that these cells are widely distributed during early brain development [12] but are restricted to specific neurogenic zones such as the subventricular zone (SVZ) and subgranular zone (SGZ) in the adult brain [12]. ...
... During the early embryonic stage, neurogenesis is detectable throughout the brain, including in the cortex. Mignone and colleagues (11) reported that GFP expression driven by the nestin promoter was first detectable on embryonic day 7 (E7), observed mainly in the neural plate at E8 and then distributed throughout the neuroepithelium by E10, but was largely restricted to specific regions such as ventricular zones by E12 and finally detectable only in conventional neurogenic regions such as the SVZ and SGZ by adulthood. Thus, NSPC activities, including NSPC-dependent neurogenesis, likely also decrease gradually with brain development outside regions such as the SVZ and SGZ [13,31]. ...
The neonatal brain is substantially more resistant to various forms of injury than the mature brain. For instance, the prognosis following ischemic stroke is generally poor in the elderly but favorable in neonates. Identifying the cellular and molecular mechanisms underlying reparative activities in the neonatal brain after ischemic injury may provide feasible targets for therapeutic interventions in adults. To this end, we compared the reparative activities in postnatal day 13 and adult (8–12-week-old) mouse brain following middle cerebral artery occlusion. Immunohistochemistry revealed considerably greater generation of ischemia-induced neural stem/progenitor cells (iNSPCs) expressing nestin or Sox2 in ischemic areas of the neonatal brain. The iNSPCs isolated from the neonatal brain also demonstrated greater proliferative activity than those isolated from adult mice. In addition, genes associated with neuronal differentiation were enriched in iNSPCs isolated from the neonatal brain according to microarray and gene ontogeny analyses. Immunohistochemistry further revealed considerably greater production of newborn doublecortin+ neurons at the sites of ischemic injury in the neonatal brain compared to the adult brain. These findings suggest that greater iNSPC generation and neurogenic differentiation capacities contribute to the superior regeneration of the neonatal brain following ischemia. Together, our findings may help identify therapeutic targets for enhancing the reparative potential of the adult brain following stroke.
... Homeostatic NSC-driven neurogenesis, particularly from the SVZ is mediated through an intermediary stage of DIx2-positive transit-amplifying C-type progenitor cells. EGF has previously been reported to convert these transit-amplifying C-type cells back to more NSC-like characteristics (Doetsch et al 1999, Doetsch et al 2002. We did not observe any DIx2 positive cells in either the SVZ or SGZ-derived uninjured control cultures at 20 DIV (S6). ...
... We did not observe any DIx2 positive cells in either the SVZ or SGZ-derived uninjured control cultures at 20 DIV (S6). This potentially indicates either that NSC proliferation and neurogenesis may originate from other cell types, i.e., not requiring an intermediary stage or proliferation through other progeny cell types, or that conversion of transit-amplifying C-type cells back towards neural lineage multipotent NSCs takes place in the presence of EGF, as previously reported by Doetsch and colleagues (Doetsch et al 1999, Doetsch et al 2002. If the latter, then our findings support previous observations and indicate that this EGF-driven conversion of transit-amplifying C-type cells may provide a starting point for pharmacological intervention following TBI. ...
The subventricular zone (SVZ) of the lateral ventricle and subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus represents neurogenic niches within the brain housing distinct populations of neural stem cells (NSCs), known for their exclusive capacity to sustain neurogenesis in the adult mammalian CNS. These niches respond to traumatic brain injury (TBI) by becoming activated, leading to NSC proliferation, a small number of which subsequently migrate towards the injury site, and differentiate predominantly into astrocytes. Although the capacity of activated NSCs to differentiate into neurons appears to be limited, it is intrinsically interesting to determine whether these cells may represent a potential source of new neurons that may replenish and replace damaged and lost neuronal tissue.
To address this question, it is necessary to understand the intrinsic behavior of NSCs derived from the activated SVZ and SGZ neurogenic niches after TBI, in terms of cell maturation, and differentiation capacity.
In this study, we induced a focal TBI lesion specifically targeting the SVZ or SGZ neurogenic niche in adult rats, harvested NSCs three days post-lesioning, and subsequently expanded them in culture. We found that the isolated NSCs displayed distinct proliferation, differentiation, and spontaneous organization in vitro , dependent on the activated niche of origin. Furthermore, these behaviors differed from NSCs derived from the SVZ or SGZ niche of uninjured control animals.
... From the adult subgranular zone of the dentate gyrus, the proliferation of non-radial and radial progenitors is responsible for the generation of intermediate progenitors from which new neuroblasts are produced. The new neuroblasts move to the inner granule zone where they differentiate into dentate granule cells, integrating themselves into the local circuitry (Lois et al., 1996;Doetsch et al., 1997Doetsch et al., , 1999. Their integration begins with the tonic stimulation produced by the GABA released in the extracellular space by local interneurons. ...
... Astrocytes are the most large, abundant, and widespread glial cells characterized by a long list of relevant functions. For instance, astrocytes have been reported to be able to self-renew similarly to stem cells to produce more immature progenitors and neuroblasts (Doetsch et al., 1999;Goldman, 2003). Starting from this observation, scientists tried to take control of this process assessing both in vitro and in vivo the possibility of reprogramming astrocytes into neurons via activated transcription factors and drugs, as a novel regenerative approach in the context of neurodegenerative disorders such as HD. ...
Huntington’s disease is a neurodegenerative disease caused by the expansion mutation of a cytosine-adenine-guanine triplet in the exon 1 of the HTT gene which is responsible for the production of the huntingtin (Htt) protein. In physiological conditions, Htt is involved in many cellular processes such as cell signaling, transcriptional regulation, energy metabolism regulation, DNA maintenance, axonal trafficking, and antiapoptotic activity. When the genetic alteration is present, the production of a mutant version of Htt (mHtt) occurs, which is characterized by a plethora of pathogenic activities that, finally, lead to cell death. Among all the cells in which mHtt exerts its dangerous activity, the GABAergic Medium Spiny Neurons seem to be the most affected by the mHtt-induced excitotoxicity both in the cortex and in the striatum. However, as the neurodegeneration proceeds ahead the neuronal loss grows also in other brain areas such as the cerebellum, hypothalamus, thalamus, subthalamic nucleus, globus pallidus, and substantia nigra, determining the variety of symptoms that characterize Huntington’s disease. From a clinical point of view, Huntington’s disease is characterized by a wide spectrum of symptoms spanning from motor impairment to cognitive disorders and dementia. Huntington’s disease shows a prevalence of around 3.92 cases every 100,000 worldwide and an incidence of 0.48 new cases every 100,000/year. To date, there is no available cure for Huntington’s disease. Several treatments have been developed so far, aiming to reduce the severity of one or more symptoms to slow down the inexorable decline caused by the disease. In this context, the search for reliable strategies to target the different aspects of Huntington’s disease become of the utmost interest. In recent years, a variety of studies demonstrated the detrimental role of neuronal loss in Huntington’s disease condition highlighting how the replacement of lost cells would be a reasonable strategy to overcome the neurodegeneration. In this view, numerous have been the attempts in several preclinical models of Huntington’s disease to evaluate the feasibility of invasive and non-invasive approaches. Thus, the aim of this review is to offer an overview of the most appealing approaches spanning from stem cell-based cell therapy to extracellular vesicles such as exosomes in light of promoting neurogenesis, discussing the results obtained so far, their limits and the future perspectives regarding the neural regeneration in the context of Huntington’s disease.
... Adult NSCs in the SVZ co-exist in a quiescent (qNSC) or an actively dividing (aNSC) state [36]. Upon activation, they give rise to TAPs, which in turn generate neuroblasts (NBs) that migrate to the OB and differentiate into interneurons [37]. ...
... In a comparable approach, we studied the influence of c-Cbl knock-out in NSCs on the formation of adult-born neurons in the OB in vivo using GLAST-CreERT2 mice allowing for the deletion of c-Cbl in radial glial cells and astrocytes. The former have been shown to represent NSCs in the SVZ (Type B cells) that exhibit characteristics of astrocytes, including the expression of GFAP and GLAST [37,42]. In conclusion, c-Cbl knock-out in TAPs or NSCs of the SVZ in vivo did not cause a significant effect on adult neurogenesis in vivo. ...
The localization, expression, and physiological role of regulatory proteins in the neurogenic niches of the brain is fundamental to our understanding of adult neurogenesis. This study explores the expression and role of the E3-ubiquitin ligase, c-Cbl, in neurogenesis within the subventricular zone (SVZ) of mice. In vitro neurosphere assays and in vivo analyses were performed in specific c-Cbl knock-out lines to unravel c-Cbl’s role in receptor tyrosine kinase signaling, including the epidermal growth factor receptor (EGFR) pathway. Our findings suggest that c-Cbl is significantly expressed within EGFR-expressing cells, playing a pivotal role in neural stem cell proliferation and differentiation. However, c-Cbl’s function extends beyond EGFR signaling, as its loss upon knock-out stimulated progenitor cell proliferation in neurosphere cultures. Yet, this effect was not detected in hippocampal progenitor cells, reflecting the lack of the EGFR in the hippocampus. In vivo, c-Cbl exerted only a minor proneurogenic influence with no measurable impact on the formation of adult-born neurons. In conclusion, c-Cbl regulates neural stem cells in the subventricular zone via the EGFR pathway but, likely, its loss is compensated by other signaling modules in vivo.
... [7][8][9][10] Neural stem cells (NSCs) in the SVZ have genetically distinct characteristics from other localizations and may influence the development and progression of GBM. 11,12 Multiple lesions, including multifocal and multicentric GBM, are found in 0.5%-35% of all GBMs [13][14][15][16][17] and are associated with a worse prognosis than solitary lesions. 14 Several major genetic variants, including PTEN loss, TERT mutation, 18 and EGFR mutation, 19 are found in multiple lesions in GBMs. ...
... 40 NSCs in the SVZ have a genetically distinct feature from other localizations that may influence the development and progression of GBM. 11,12 NSCs have the ability to differentiate into neurons and glial cells and are abundant in fetal tissues. Owing to their high proliferative potential, NSCs have been implicated in the development of GBM. ...
Background
Glioblastoma (GBM) is a malignant brain tumor, with radiological and genetic heterogeneity. We examined the association between radiological characteristics and driver gene alterations.
Methods
We analyzed the driver genes of 124 patients with IDH-wildtype GBM with contrast enhancement using magnetic resonance imaging. We used a next-generation sequencing panel to identify mutations in driver genes and matched them with radiological information. Contrast-enhancing lesion localization of GBMs was classified into four groups based on their relationship with the subventricular zone (SVZ) and cortex (Ctx).
Results
The cohort included 69 men (55.6%) and 55 women (44.4%) with a mean age of 66.4 ± 13.3 years. EGFR and PDGFRA alterations were detected in 28.2% and 22.6% of the patients, respectively. Contrast-enhancing lesion touching both the SVZ and Ctx was excluded because it was difficult to determine whether it originated from the SVZ or Ctx. Contrast-enhancing lesions touching the SVZ but not the Ctx had significantly worse overall survival than non-SVZ lesions (441 days vs. 897 days, p=0.002). GBM touching only the Ctx had a better prognosis (901 days vs. 473 days, p<0.001) than non-Ctx lesions and was associated with EGFR alteration (39.4% vs. 13.2%, p=0.015). Multiple contrast lesions were predominant in PDGFRA alteration and RB1-wildtype (p=0.036 and p=0.031, respectively).
Conclusions
EGFR alteration was associated with cortical lesions. And PDGFRA alteration correlated with multiple lesions. Our results suggest that clarifying the association between driver genes and tumor localization may be useful in clinical practice, including prognosis prediction.
... In embryonic/fetal and early postnatal development, almost all of the cells in the developing neural tube are ascribed to NSCs [2]. Subsequently, the NSCs in the neural tube give rise to the neurons and glial cells (i.e., astrocytes and oligodendrocytes) in mammalian central nervous system (CNS) [3][4][5][6]. During this process, the polarization pattern generation in NSCs and the regulation of the cell-division cycle are the noticeably important determinants respectively [1,7]. ...
... Moreover, the fact that qNSCs express both Dio2 and Mct8 mRNA, and considering their location adjacent to the lateral ventricles, could suggest they take up T 4 delivered by TTR via the cerebrospinal fluid, where T 3 can also be found, although in lower concentrations than T 4 (Vancamp et al., 2019;Fame et al., 2024). As both SVZ-qNSCs and -astrocytes function as reservoirs for generating new neural progenitors (Doetsch et al., 1999;Ben et al., 2017), our data point out that their expression of all the regulators to modulate the intracellular T 3 availability is reminiscent of the role of radial glia during development (López-Espíndola et al., 2019). ...
... Adult neural stem cells are multipotent cells found within the subventricular zone (SVZ) of the olfactory bulb and the hippocampal dentate gyrus of the adult brain that can differentiate into neurons, glia, astroglia, and oligodendrocytes [57][58][59][60][61]. The advantages of adult neural stem cells are in the lack of ethical concerns associated with their use, their ability to be cultured, their potential for autologous transplantation, and their predetermined tendency to differentiate into cells of the neural lineage [62][63][64][65]. ...
Glioblastoma multiforme (GBM), the most common and lethal brain cancer, prognosis remains bleak with a median survival of about 15 months despite maximal surgical resection, radiotherapy, and temozolomide treatment. The difficulty associated with safely and effectively delivering therapeutics across the blood brain barrier (BBB) is a major challenge towards GBM treatment. Ongoing research and clinical trials, including attempts to deliver therapeutics within stem cells present possible solutions. The relationships between brain cancer pathology, stem cell properties, therapeutic advantages and disadvan- tages of various stem cell types, drug delivery methods, cancer stem cells, gene therapy, anti-cancer vaccines, chimeric antigen receptor therapies, and combination therapies are discussed.
... The hypothesis of a different responsiveness to NO in the SVZ and DG is supported by the diversity of cellular ⁄ molecular mechanisms by which neural stem cells produce intermediate precursors and then immature neurons in the two regions (Doetsch et al., 1999;Kempermann et al., 2004;Seri et al., 2004), and also by the modulators known to affect specifically one of the two neurogenic niches (Shingo et al., 2003;Mechawar et al., 2004;Abrous et al., 2005). Although, in both cases, stem cells are represented by astrocytes, the SVZ generates a highly proliferative cell population, the transit amplifying cells or type C cells that, later on, differentiate into migrating neuroblasts (Doetsch et al., 1997), whereas in the DG, only a few mitosis occur in the intermediate cell population, the D cells (Kempermann et al., 2004;Seri et al., 2004). ...
... Therefore, the observed results in this preliminary experiment are not solely a reflection of the differentiation potential of NSCs as a whole, but rather the combined output of various distinct subpopulations. Moreover, the microenvironment or "niche" in which NSCs reside can further contribute to their heterogeneity [34,35]. Differences in niche characteristics such as the availability of growth factors, extracellular matrix composition, and cell-cell interactions can influence NSC behavior and differentiation [36]. ...
Even though electromagnetic fields have been reported to assist endogenous neurogenesis, little is known about the exact mechanisms of their action. In this pilot study, we investigated the effects of pulsating extremely low-frequency electromagnetic fields on neural stem cell differentiation towards specific phenotypes, such as neurons and astrocytes. Neural stem cells isolated from the telencephalic wall of B6(Cg)-Tyrc-2J/J mouse embryos (E14.5) were randomly divided into three experimental groups and three controls. Electromagnetic field application setup included a solenoid placed within an incubator. Each of the experimental groups was exposed to 50Hz ELF-EMFs of varied strengths for 1 h. The expression of each marker (NES, GFAP, β-3 tubulin) was then assessed by immunocytochemistry. The application of high-strength ELF-EMF significantly increased and low-strength ELF-EMF decreased the expression of GFAP. A similar pattern was observed for β-3 tubulin, with high-strength ELF-EMFs significantly increasing the immunoreactivity of β-3 tubulin and medium- and low-strength ELF-EMFs decreasing it. Changes in NES expression were observed for medium-strength ELF-EMFs, with a demonstrated significant upregulation. This suggests that, even though ELF-EMFs appear to inhibit or promote the differentiation of neural stem cells into neurons or astrocytes, this effect highly depends on the strength and frequency of the fields as well as the duration of their application. While numerous studies have demonstrated the capacity of EMFs to guide the differentiation of NSCs into neuron-like cells or β-3 tubulin+ neurons, this is the first study to suggest that ELF-EMFs may also steer NSC differentiation towards astrocyte-like phenotypes.
... In vitro evidence has indicated the presence of stem cells [1,2]. These cells are thought to be located in or adjacent to the ependymal layer of the central canal in the spinal cord [3][4][5][6][7]. In the normal adult spinal cord of the rat, Horner et al. (2000) [8] reported the presence of turnover of glial progenitors and mature glial cells, particularly in the white matter. ...
The existence of proliferating cells in the intact spinal cord, their distribution and phenotype, are well studied in rodents. A limited number of studies also address the proliferation after spinal cord injury, in non-human primates. However, a detailed description of the quantity, distribution and phenotype of proliferating cells at different anatomical levels of the intact adult non-human primate spinal cord is lacking at present. In the present study, we analyzed normal spinal cord tissues from adult macaque monkeys (Macaca fuscata), infused with Bromo-2′-deoxyuridine (BrdU), and euthanized at 2h, 2 weeks, 5 weeks and 10 weeks after BrdU. We found a significantly higher density of BrdU + cells in the gray matter of cervical segments as compared to thoracic or lumbar segments, and a significantly higher density of proliferating cells in the posterior as compared to the anterior horn of the gray matter. BrdU + cells exhibited phenotype of microglia or endothelial cells (∼50%) or astroglial and oligodendroglial cells (∼40%), including glial progenitor phenotypes marked by the transcription factors Sox9 and Sox10. BrdU + cells also co-expressed other transcription factors known for their involvement in embryonic development, including Emx2, Sox1, Sox2, Ngn1, Olig1, Olig2, Olig3. In the central canal, BrdU + cells were located along the dorso-ventral axis and co-labeled for the markers Vimentin and Nestin. These results reveal the extent of cellular plasticity in the spinal cord of non-human primates under normal conditions.
... Neural stem cells (NSCs) in the adult central nervous system (CNS) are capable of selfrenewal and generation of mature neurons, oligodendrocytes and astrocytes. NSCs are actively dividing in the subventricular zone (SVZ) [1] and the subgranular zone (SGZ) [2] in the brain and are found in a quiescent state in the spinal cord [3]. These NSC niches are under strict regulatory cues ensuring a balance between cell proliferation and quiescence, thus providing sufficient numbers of cells for differentiation while preserving the NSC pool. ...
Adult neural stem cells (NSC) are a potential source for the regeneration of damaged tissue during neuropathological conditions, but much remains unexplored. In an attempt to study the influence of neuroinflammation on NSCs, we generated a transgenic reporter rat strain that expresses the Discosoma sp . red (DsRed) fluorophore in NSCs and subjected it to traumatic brain injury (TBI). Transcriptomic analysis of NSCs isolated from TBI revealed an enrichment of stress response genes that pertained to endoplasmic reticulum (ER) stress and integrated stress response (ISR). Downstream analysis on NSC cultures pinpointed IL-1α as a trigger of ISR in these cells. At concentration levels similar to the ones measured post-TBI in rats, IL-1α induced the translation of activating transcription factor 4 (ATF4), an ISR master regulator. Further, ATF4 was necessary for the IL-1α -dependent induction of a senescent profile in NSCs, which included a metabolic shift towards glycolysis, induction of senescence-associated secretory phenotype, SASP, and cell cycle arrest. In summary, the ISR/ATF4 pathway seems to play a major role in NSC function during neuroinflammation and provides a therapeutic tool for protecting the NSC pool during these conditions.
... 4,5 Adult SVZ NSCs have been reported to retain astroglial characteristics. 16,19 C7 also expressed extracellular matrix-related genes PTN, BCAN, NCAN, and SPARCL1, 20 and gap/tight junction genes CLDN1/12, COX43/GJA1, and TJP1 (Table S3). 3,21,22 C4 expressed genes associated with embryonic development and neuronal programs such as ASCL1, SOX2, CD24, SOX4, and SOX11 (Figures 2C-2E; Table S3), [23][24][25] and neurogenic oRG transcription factors HES6, NHLH1, and CBFA2T2 (Table S3). ...
The existence of neural stem cells (NSCs) in adult human brain neurogenic regions remains unresolved. To address this, we created a cell atlas of the adult human subventricular zone (SVZ) derived from fresh neurosurgical samples using single-cell transcriptomics. We discovered 2 adult radial glia (RG)-like populations, aRG1 and aRG2. aRG1 shared features with fetal early RG (eRG) and aRG2 were transcriptomically similar to fetal outer RG (oRG). We also captured early neuronal and oligodendrocytic NSC states. We found that the biological programs driven by their transcriptomes support their roles as early lineage NSCs. Finally, we show that these NSCs have the potential to transition between states and along lineage trajectories. These data reveal that multipotent NSCs reside in the adult human SVZ.
... Increasing evidence indicates that the cells of origin of GBM are likely neural stem cells in the subventricular zone (SVZ) of the adult human brain. The SVZ is a layer between the lateral ventricle, corpus callosum, and striatum, which has the largest number of neural stem cells in the brain [5][6][7]. These cells can contain many of the driver mutations that give rise to GBM, share molecular features with GBM cells, and display migratory patterns from the SVZ to the tumor. ...
Rapid neuronal inhibition in the brain is mediated by γ-aminobutyric acid (GABA) activation of GABAA receptors. The GABRA5 gene, which encodes the α5 subunit of the GABAA receptor, has been implicated in an aggressive subgroup of medulloblastoma (MB), a type of pediatric brain tumor. However, the possible role of GABAA receptor subunits in glioma remains poorly understood. Here, we examined the expression of genes encoding GABAA receptor subunits in different types of glioma, and its possible association with patient prognosis assessed by overall survival (OS). Data were obtained from the French and The Cancer Genome Atlas Brain Lower Grade Glioma (TCGA-LGG) datasets and analyzed for expression of GABAA receptor subunit genes. OS was calculated using the Kaplan–Meier estimate. We found that genes GABRA2, GABRA3, GABRB3, GABRG1, and GABRG2 showed a significant association with OS, with higher gene expression indicating better prognosis. In patients with GBM, high expression of GABRA2 was associated with shorter OS, whereas, in contrast, higher levels of GABRB3 were associated with better prognosis indicated by longer OS. In patients with lower grade gliomas, GABRA3, GABRB3, GABRG1, and GABRG2, were associated with longer OS. High GABRB3 expression was related to longer survival when low grade glioma types were analyzed separately. Our results suggest an overall association between higher expression of most genes encoding GABAA receptor subunits and better prognosis in different types of glioma. Our findings support the possibility that down-regulation of GABAA receptors in glioma contributes to promoting tumor progression by reducing negative inhibition. These findings might contribute to further evaluation of GABAA receptors as a therapeutic target in glioma.
... Relative to the control group, these cells exhibited reduced GLT-1 expression and significant increases in GFAP and GLAST expression. Such changes have also been observed in radial glia and astrocytes [30], supporting the notion that a considerable proportion of radial glial cells are present in GLDC27-FiPS4F-1 iAs [31,32]. Elevated GFAP expression has also been linked to reactive phenotypes associated with pathological conditions [26,32,33]. ...
The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine–glycine–one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.
... The additional small pictures show the magnified images of the dorsolateral corner of SVZ. The cells in the SVZ are classified into three major types [1], namely radial glia-like cells (GFAP-positive cells) [10], transient amplifying cells (MASH1-positive cells) [11], and neuroblast cells (doublecortin (Dcx) [12][13][14][15][16]. Different antibodies (anti-GFAP, anti-MASH1, and anti-Dcx) were used as co-labels with HDAC8 to detect the types of NSCs/NPCs (Figure 1b). These findings suggest the involvement of HDAC8 in neurogenesis in the adult brain. ...
In the adult mammalian brain, neurons are produced from neural stem cells (NSCs) residing in two niches—the subventricular zone (SVZ), which forms the lining of the lateral ventricles, and the subgranular zone in the hippocampus. Epigenetic mechanisms contribute to maintaining distinct cell fates by suppressing gene expression that is required for deciding alternate cell fates. Several histone deacetylase (HDAC) inhibitors can affect adult neurogenesis in vivo. However, data regarding the role of specific HDACs in cell fate decisions remain limited. Herein, we demonstrate that HDAC8 participates in the regulation of the proliferation and differentiation of NSCs/neural progenitor cells (NPCs) in the adult mouse SVZ. Specific knockout of Hdac8 in NSCs/NPCs inhibited proliferation and neural differentiation. Treatment with the selective HDAC8 inhibitor PCI-34051 reduced the neurosphere size in cultures from the SVZ of adult mice. Further transcriptional datasets revealed that HDAC8 inhibition in adult SVZ cells disturbs biological processes, transcription factor networks, and key regulatory pathways. HDAC8 inhibition in adult SVZ neurospheres upregulated the cytokine-mediated signaling and downregulated the cell cycle pathway. In conclusion, HDAC8 participates in the regulation of in vivo proliferation and differentiation of NSCs/NPCs in the adult SVZ, which provides insights into the underlying molecular mechanisms.
... In the canonical neurogenic niches, newborn neurons are generated by astrocyte-like GFAP-positive NSCs during both embryonic [18] and postnatal stages [3,[5][6][7]19]. In the hypothalamus, a diencephalic region identified as neurogenic, tanycytes, cells derived from embryonic radial glial cells, represent a subpopulation of NSPCs [20]. ...
... In this region, newborn cells will move toward the olfactory bulb and act as interneurons. 4,5 Hippocampus is a highly plastic region of the mammalian brain, performing a vital function in episodic and locational memory. 6 The newborn cells in the hippocampus DG communicate with others by integrating into neural circuits 7,8 and then increases the DG synaptic plasticity, which is essential in memory management. ...
Background
Glucocorticoids (GCs) are steroidal hormones produced by the adrenal cortex. A physiological‐level GCs have a crucial function in maintaining many cognitive processes, like cognition, memory, and mood, however, both insufficient and excessive GCs impair these functions. Although this phenomenon could be explained by the U‐shape of GC effects, the underlying mechanisms are still not clear. Therefore, understanding the underlying mechanisms of GCs may provide insight into the treatments for cognitive and mood‐related disorders.
Methods
Consecutive administration of corticosterone (CORT, 10 mg/kg, i.g.) proceeded for 28 days to mimic excessive GCs condition. Adrenalectomy (ADX) surgery was performed to ablate endogenous GCs in mice. Microinjection of 1 μL of Ad‐mTERT‐GFP virus into mouse hippocampus dentate gyrus (DG) and behavioral alterations in mice were observed 4 weeks later.
Results
Different concentrations of GCs were shown to affect the cell growth and development of neural stem cells (NSCs) in a U‐shaped manner. The physiological level of GCs (0.01 μM) promoted NSC proliferation in vitro, while the stress level of GCs (10 μM) inhibited it. The glucocorticoid synthesis blocker metyrapone (100 mg/kg, i.p.) and ADX surgery both decreased the quantity and morphological development of doublecortin (DCX)‐positive immature cells in the DG. The physiological level of GCs activated mineralocorticoid receptor and then promoted the production of telomerase reverse transcriptase (TERT); in contrast, the stress level of GCs activated glucocorticoid receptor and then reduced the expression of TERT. Overexpression of TERT by AD‐mTERT‐GFP reversed both chronic stresses‐ and ADX‐induced deficiency of TERT and the proliferation and development of NSCs, chronic stresses‐associated depressive symptoms, and ADX‐associated learning and memory impairment.
Conclusion
The bidirectional regulation of TERT by different GCs concentrations is a key mechanism mediating the U‐shape of GC effects in modulation of hippocampal NSCs and associated brain function. Replenishment of TERT could be a common treatment strategy for GC dysfunction‐associated diseases.
... Self-renewing, multipotent, and GFAP-expressing neural stem cells (NSCs) transform into neural progenitor cells (NPCs) that generate neurons and glial cells (18,19) . Because of the developmental potential and plasticity of NSCs, they are ideal candidates for glioma cells of origin because multiple oncogenic mutations are required for gliomagenesis, and the self-renewal and proliferative properties of NSCs may result in the endogenous accumulation of somatic mutations (20,21) . ...
... Aging of stem cells is a natural and inevitable biological process characterized by degenerative changes that are influenced by cell-intrinsic pathways and extrinsic factors within stem cell niches 1 . In the rodent brain, most neural stem and progenitor cells (NSPCs) with the ability to self-renew and differentiate into neurons persist in the subventricular zone (SVZ) 2,3 . Within the SVZ, neural stem cells (NSCs) are identified as glial-like type B1 cells that can be activated to divide at a comparatively slower rate. ...
The decline in stem cell function during aging may affect the regenerative capacity of mammalian organisms; however, the gene regulatory mechanism underlying this decline remains unclear. Here we show that the aging of neural stem and progenitor cells (NSPCs) in the male mouse brain is characterized by a decrease in the generation efficacy of proliferative NSPCs rather than the changes in lineage specificity of NSPCs. We reveal that the downregulation of age-dependent genes in NSPCs drives cell aging by decreasing the population of actively proliferating NSPCs while increasing the expression of quiescence markers. We found that epigenetic deregulation of the MLL complex at promoters leads to transcriptional inactivation of age-dependent genes, highlighting the importance of the dynamic interaction between histone modifiers and gene regulatory elements in regulating transcriptional program of aging cells. Our study sheds light on the key intrinsic mechanisms driving stem cell aging through epigenetic regulators and identifies potential rejuvenation targets that could restore the function of aging stem cells.
... This occurrence of neurogenesis in the adult rodent brain was further evidenced in the 1990s with the fast development of new technologies, including 5-bromo-2′deoxyuridine labeling and antibody-based biomarker detection approaches (Ming and Song, 2005;Jurkowski et al., 2020). In the adult rodent brain, subventricular zone astrocytes were discovered to be NSCs around the year 2000 (Doetsch et al., 1999); in subsequent animal studies, Lindvall and Kokaia (2015) found the feasibility of stimulating endogenous neurogenesis in the subventricular zone to promote neural repair in the striatum and cortex. ...
Traumatic brain injury is a serious medical condition that can be attributed to falls, motor vehicle accidents, sports injuries and acts of violence, causing a series of neural injuries and neuropsychiatric symptoms. However, limited accessibility to the injury sites, complicated histological and anatomical structure, intricate cellular and extracellular milieu, lack of regenerative capacity in the native cells, vast variety of damage routes, and the insufficient time available for treatment have restricted the widespread application of several therapeutic methods in cases of central nervous system injury. Tissue engineering and regenerative medicine have emerged as innovative approaches in the field of nerve regeneration. By combining biomaterials, stem cells, and growth factors, these approaches have provided a platform for developing effective treatments for neural injuries, which can offer the potential to restore neural function, improve patient outcomes, and reduce the need for drugs and invasive surgical procedures. Biomaterials have shown advantages in promoting neural development, inhibiting glial scar formation, and providing a suitable biomimetic neural microenvironment, which makes their application promising in the field of neural regeneration. For instance, bioactive scaffolds loaded with stem cells can provide a biocompatible and biodegradable milieu. Furthermore, stem cells-derived exosomes combine the advantages of stem cells, avoid the risk of immune rejection, cooperate with biomaterials to enhance their biological functions, and exert stable functions, thereby inducing angiogenesis and neural regeneration in patients with traumatic brain injury and promoting the recovery of brain function. Unfortunately, biomaterials have shown positive effects in the laboratory, but when similar materials are used in clinical studies of human central nervous system regeneration, their efficacy is unsatisfactory. Here, we review the characteristics and properties of various bioactive materials, followed by the introduction of applications based on biochemistry and cell molecules, and discuss the emerging role of biomaterials in promoting neural regeneration. Further, we summarize the adaptive biomaterials infused with exosomes produced from stem cells and stem cells themselves for the treatment of traumatic brain injury. Finally, we present the main limitations of biomaterials for the treatment of traumatic brain injury and offer insights into their future potential.
... Likewise, there were no differences in the cell types transduced by these viruses, primarily consisting of glial fibrillary acidic protein (GFAP)-negative/immature neuron marker doublecortin (DCX)-negative or GFAPnegative/DCX-positive cells ( Figures S4C and S4D), corresponding to TAP and their resulting neuroblast progeny. A minority of cells were GFAP + , indicating that a small number of self-renewing NSCs (Doetsch et al., 1999), or proliferating parenchymal astrocytes (Garcia et al., 2004) (see histogram in Figure S4D) had been transduced. Neither microglial (CD45 + ) nor oligodendroglial (NG2 + ) cells were identified in brains injected with either control or Neu-rog2-encoding viruses. ...
Although adult subependymal zone (SEZ) neural stem cells mostly generate GABAergic interneurons, a small progenitor population expresses the proneural gene Neurog2 and produces glutamatergic neurons. Here, we determined whether Neurog2 could respecify SEZ neural stem cells and their progeny toward a glutamatergic fate. Retrovirus-mediated expression of Neurog2 induced the glutamatergic lineage markers TBR2 and TBR1 in cultured SEZ progenitors, which differentiated into functional glutamatergic neurons. Likewise, Neurog2-transduced SEZ progenitors acquired glutamatergic neuron hallmarks in vivo. Intriguingly, they failed to migrate toward the olfactory bulb and instead differentiated within the SEZ or the adjacent striatum, where they received connections from local neurons, as indicated by rabies virus-mediated monosynaptic tracing. In contrast, lentivirus-mediated expression of Neurog2 failed to reprogram early SEZ neurons, which maintained GABAergic identity and migrated to the olfactory bulb. Our data show that NEUROG2 can program SEZ progenitors toward a glutamatergic identity but fails to reprogram their neuronal progeny.
... However, strikingly, Dio2 was also detected at moderate levels in neurons, despite the prevailing notion that they almost uniquely express Dio3, and Dio2 is mainly expressed in astrocytes (40,41), suggesting neurons could activate T 4 themselves, to some extent. Mct8 was highly expressed in endothelial cells forming the blood-brain-barrier (BBB), aligning with previous observations in mice, primates and humans (16,42) (44,45), our data point out that their expression of all the regulators to modulate the intracellular T 3 availability is reminiscent of the role of radial glia during development (16). ...
... In the adult brain, some areas retain the capacity of generating new neurons throughout an individual's lifespan (Jurkowski et al., 2020). In particular, the two most studied neurogenic niches are the subgranular zone (SGZ) of the dentate gyrus of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle (Doetsch et al., 1999;Seri et al., 2001;Obernier and Alvarez-Buylla, 2019). In the dentate gyrus, glia-like stem cells, called type-1, express Glial Fibrillary Acidic Protein (GFAP), Sex Determining Region Y-Box 2 (Sox2), and nestin, and mature into progenitor cells, classified as type-2a, positive for nestin and Sox2 (nestin + /Sox2 + ), type-2b positive for nestin and doublecortin (nestin + /DCX + ) or type-3 (DCX + ; Filippov et al., 2003;Fukuda et al., 2003;Kronenberg et al., 2003;Steiner et al., 2006). ...
Throughout adulthood neural stem cells divide in neurogenic niches–the dentate gyrus of the hippocampus and the subventricular zone–producing progenitor cells and new neurons. Stem cells self-renew, thus preserving their pool. Furthermore, the number of stem/progenitor cells in the neurogenic niches decreases with age. We have previously demonstrated that the cyclin-dependent kinase inhibitor p16Ink4a maintains, in aged mice, the pool of dentate gyrus stem cells by preventing their activation after a neurogenic stimulus such as exercise (running). We showed that, although p16Ink4a ablation by itself does not activate stem/progenitor cells, exercise strongly induced stem cell proliferation in p16Ink4a knockout dentate gyrus, but not in wild-type. As p16Ink4a regulates stem cell self-renewal during aging, we sought to profile the dentate gyrus transcriptome from p16Ink4a wild-type and knockout aged mice, either sedentary or running for 12 days. By pairwise comparisons of differentially expressed genes and by correlative analyses through the DESeq2 software, we identified genes regulated by p16Ink4a deletion, either without stimulus (running) added, or following running. The p16Ink4a knockout basic gene signature, i.e., in sedentary mice, involves upregulation of apoptotic, neuroinflammation- and synaptic activity-associated genes, suggesting a reactive cellular state. Conversely, another set of 106 genes we identified, whose differential expression specifically reflects the pattern of proliferative response of p16 knockout stem cells to running, are involved in processes that regulate stem cell activation, such as synaptic function, neurotransmitter metabolism, stem cell proliferation control, and reactive oxygen species level regulation. Moreover, we analyzed the regulation of these stem cell-specific genes after a second running stimulus. Surprisingly, the second running neither activated stem cell proliferation in the p16Ink4a knockout dentate gyrus nor changed the expression of these genes, confirming that they are correlated to the stem cell reactivity to stimulus, a process where they may play a role regulating stem cell activation.
... LV neural stem cells (LV NSCs) could be quiescent or actively dividing (2,3). Upon activation, quiescent LV NSCs can divide asymmetrically for self-renewal or differentiate into transient amplifying intermediate progenitors (TAPs) (2,4,5). These progenitors undergo symmetrical division and differentiation, producing young migrating doublecortin-positive (DCX + ) neuroblasts (6,7). ...
Neurogenesis and proliferation of neural stem cells (NSCs) in the subventricular zone (SVZ) are controlled by both intrinsic molecular pathways and extrinsic signaling cues, including neural circuits. One such circuit, the ACC-subep-ChAT+ circuit, has been identified as a regulator of ventral SVZ neurogenesis by modulating the proliferation of LV NSCs. However, the specific neural signals that promote the proliferation activity of LV NSCs have remained largely unknown. In this study, we uncover a molecular mechanism underlying the cellular activation and proliferation of quiescent NSCs (qNSCs) in the lateral ventricle SVZ (LV-SVZ) mediated by the cortical circuit. Our findings demonstrate that postnatal and adult LV qNSCs are triggered by the cortical circuit through ChAT+ neuron stimulation, consequently resulting in the activation of muscarinic 3 receptors (M3) expressed on LV qNSCs. This, in turn, triggers inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activation, causing intracellular calcium release. Subsequently, the proliferation of LV qNSCs occurs through the downstream regulation of the calcium/calmodulin dependent protein kinase II delta (CAMK2D) and the MAPK10 signaling pathway. These findings shed light on the molecular regulatory mechanisms that govern LV qNSCs and emphasize the significant role of the cortical circuit in promoting their proliferative activation within the ventral LV-SVZ.
... Given that primary cilia play a vital role in the neurogenic process [42], one could postulate that both Abracl mRNA and protein should be expressed in the VZ, where ciliated progenitors reside; our results show that neither Abracl mRNA nor Abracl were expressed in the VZ. As distinct molecular mechanisms regulate cell cycle progression and differentiation within VZ and SVZ (for instance the progenitors in the VZ use cyclin D1 while those in the SVZ cyclin D2; [43][44][45][46][47]), it is possible that the primary cilia structure is not identical in these zones. In addition, primary cilia play a role in regulating the migratory rhythm and the directionality of the cells in both radial and tangential migration, as well as in the reorientation from tangential to radial migration during the laminar allocation of the interneurons [48]. ...
Abracl (ABRA C-terminal-like protein) is a small, non-typical winged-helix protein that shares similarity with the C-terminal domain of the protein ABRA (Actin-Binding Rho-Activating protein). The role of Abracl in the cell remains elusive, although in cancer cells, it has been implicated in proliferation, migration and actin dynamics. Our previous study showed that Abracl mRNA was expressed in the dividing cells of the subpallial subventricular zone (SVZ), in the developing cortical plate (CP), and in the diencephalic SVZ; however, the molecular identities of the Abracl-expressing cell populations were not defined in that work. In this study, we use double immunofluorescence to characterize the expression of Abracl on sections of embryonic murine (E11.5-E18.5) and feline (E30/31-E33/34) telencephalon; to this end, we use a battery of well-known molecular markers of cycling (Ki67, Ascl1, Dlx2) or post-mitotic (Tubb3, Gad65/67, Lhx6 and Tbr1) cells. Our experiments show that Abracl protein has, compared to the mRNA, a broader expression domain, including, apart from proliferating cells of the subpallial and diencephalic SVZ, post-mitotic cells occupying the subpallial and pallial mantle (including the CP), as well as subpallial-derived migrating interneurons. Interestingly, in late embryonic developmental stages, Abracl was also transiently detected in major telencephalic fiber tracts.
Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the “canonical” megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.
It has long been asserted that failure to recover from central nervous system diseases is due to the system’s intricate structure and the regenerative incapacity of adult neurons. Yet over recent decades, numerous studies have established that endogenous neurogenesis occurs in the adult central nervous system, including humans’. This has challenged the long-held scientific consensus that the number of adult neurons remains constant, and that new central nervous system neurons cannot be created or renewed. Herein, we present a comprehensive overview of the alterations and regulatory mechanisms of endogenous neurogenesis following central nervous system injury, and describe novel treatment strategies that target endogenous neurogenesis and newborn neurons in the treatment of central nervous system injury. Central nervous system injury frequently results in alterations of endogenous neurogenesis, encompassing the activation, proliferation, ectopic migration, differentiation, and functional integration of endogenous neural stem cells. Because of the unfavorable local microenvironment, most activated neural stem cells differentiate into glial cells rather than neurons. Consequently, the injury-induced endogenous neurogenesis response is inadequate for repairing impaired neural function. Scientists have attempted to enhance endogenous neurogenesis using various strategies, including using neurotrophic factors, bioactive materials, and cell reprogramming techniques. Used alone or in combination, these therapeutic strategies can promote targeted migration of neural stem cells to an injured area, ensure their survival and differentiation into mature functional neurons, and facilitate their integration into the neural circuit. Thus can integration replenish lost neurons after central nervous system injury, by improving the local microenvironment. By regulating each phase of endogenous neurogenesis, endogenous neural stem cells can be harnessed to promote effective regeneration of newborn neurons. This offers a novel approach for treating central nervous system injury.
Cancers commonly reprogram translation and metabolism, but little is known about how these two features coordinate in cancer stem cells. Here we show that glioblastoma stem cells (GSCs) display elevated protein translation. To dissect underlying mechanisms, we performed a CRISPR screen and identified YRDC as the top essential transfer RNA (tRNA) modification enzyme in GSCs. YRDC catalyzes the formation of N⁶-threonylcarbamoyladenosine (t⁶A) on ANN-decoding tRNA species (A denotes adenosine, and N denotes any nucleotide). Targeting YRDC reduced t⁶A formation, suppressed global translation and inhibited tumor growth both in vitro and in vivo. Threonine is an essential substrate of YRDC. Threonine accumulated in GSCs, which facilitated t⁶A formation through YRDC and shifted the proteome to support mitosis-related genes with ANN codon bias. Dietary threonine restriction (TR) reduced tumor t⁶A formation, slowed xenograft growth and augmented anti-tumor efficacy of chemotherapy and anti-mitotic therapy, providing a molecular basis for a dietary intervention in cancer treatment.
Over the last decades, thyroid hormones (THs) signaling has been established as a key signaling cue for the proper maintenance of brain functions in adult mammals, including humans. One of the most fascinating roles of THs in the mature mammalian brain is their ability to regulate adult neurogliogenic processes. In this respect, THs control the generation of new neuronal and glial progenitors from neural stem cells (NSCs) as well as their final differentiation and maturation programs. In this review, we summarize current knowledge on the cellular organization of adult rodent neurogliogenic niches encompassing well-established niches in the subventricular zone (SVZ) lining the lateral ventricles, the hippocampal subgranular zone (SGZ), and the hypothalamus, but also less characterized niches in the striatum and the cerebral cortex. We then discuss critical questions regarding how THs availability is regulated in the respective niches in rodents and larger mammals as well as how modulating THs availability in those niches interferes with lineage decision and progression at the molecular, cellular, and functional levels. Based on those alterations, we explore the novel therapeutic avenues aiming at harnessing THs regulatory influences on neurogliogenic output to stimulate repair processes by influencing the generation of either new neurons (i.e. Alzheimer’s, Parkinson’s diseases), oligodendrocytes (multiple sclerosis) or both (stroke). Finally, we point out future challenges, which will shape research in this exciting field in the upcoming years.
Chemogenetic modulation, via the use of designer receptors exclusively activated by designer drugs (DREADDs), is an established in vivo approach that allows cell-type-specific and spatiotemporally defined modulation of the brain. It combines both a genetic and a pharmacological aspect and can be used in multiple cell types such as neurons and glia. Several DREADDs have been developed, of which the muscarinic G-protein-coupled receptor DREADDs are the most frequently used. We will focus on viral vectors as delivery approach of the receptors and will describe the method for intracranial delivery of viral vectors in detail. Several aspects need to be considered regarding the choice of the DREADD agonist upon its administration. Next, we will describe the protocol that has been used for the visualization of DREADDs ex vivo. Through the measurement of local field potentials in hippocampal slices, it has been successfully demonstrated that astrocytic Gq- and Gi-DREADD modulation induces long-term synaptic potentiation. Finally, we will elaborate on other approaches to study chemogenetic modulation of astrocytes at glutamatergic synapses.
Disease-relevant in vivo tumor models are essential tools for both discovery and translational research. Here, we describe a highly genetically tractable technique for generating immunocompetent somatic glioblastoma (GBM) mouse models using piggyBac transposition and CRISPR-Cas9-mediated gene editing in wild-type mice. We describe steps to deliver plasmids into subventricular zone endogenous neural stem cells by injection and electroporation, leading to the development of adult tumors that closely recapitulate the histopathological, molecular, and cellular features of human GBM.
For complete details on the use and execution of this protocol, please refer to Garcia-Diaz et al.¹
Mouse postnatal neural stem cells (pNSCs) can be expanded in vitro in the presence of epidermal growth factor and fibroblast growth factor 2 and upon removal of these factors cease proliferation and generate neurons, astrocytes, and oligodendrocytes. The genetic requirements for self-renewal and lineage-commitment of pNSCs are incompletely understood. In this study, we show that the transcription factors NFIA and NFIB, previously shown individually, to be essential for the normal commitment of pNSCs to the astrocytic lineage in vivo, are jointly required for normal self-renewal of pNSCs in vitro and in vivo. Using conditional knockout alleles of Nfia and Nfib, we show that the simultaneous loss of these two genes under self-renewal conditions in vitro reduces the expression of the proliferation markers PCNA and Ki67, eliminates clonogenicity of the cells, reduces the number of cells in S phase, and induces aberrant differentiation primarily into the neuroblast lineage. This phenotype requires the loss of both genes and is not seen upon loss of Nfia or Nfib alone, nor with combined loss of Nfia and Nfix or Nfib and Nfix. These data demonstrate a unique combined requirement for both Nfia and Nfib for pNSC self-renewal.
This chapter reviews functional characteristics which define the ventricular/subarachnoid space and the cellular and molecular contents of the cerebrospinal fluid. Systems underlying the elaboration of CSF and its movement of fluid in the parenchyma extracellular space are reviewed in detail in chapter “Cerebrospinal and Interstitial Fluids: Production, Outflow, and Circulation.” Factors governing the dynamics of rostrocaudal molecule distribution in the CSF are reviewed in chapters “Emerging Insights into the Interstitial Distribution of Neuraxial Therapeutics via the Cerebrospinal Fluid Compartment” and “CSF Flow Dynamics in Relation to Intrathecal Drug Transport.”
Nuclear pore complexes (NPCs) are highly dynamic macromolecular protein structures that facilitate molecular exchange across the nuclear envelope. Aberrant NPC functioning has been implicated in neurodegeneration. The translocated promoter region (Tpr) is a critical scaffolding nucleoporin (Nup) of the nuclear basket, facing the interior of the NPC. However, the role of Tpr in adult neural stem/precursor cells (NSPCs) in Alzheimer's disease (AD) is unknown. Using super-resolution (SR) and electron microscopy, we defined the different subcellular localizations of Tpr and phospho-Tpr (P-Tpr) in NSPCs in vitro and in vivo. Elevated Tpr expression and reduced P-Tpr nuclear localization accompany NSPC differentiation along the neurogenic lineage. In 5xFAD mice, an animal model of AD, increased Tpr expression in DCX+ hippocampal neuroblasts precedes increased neurogenesis at an early stage, before the onset of amyloid-β plaque formation. Whereas nuclear basket Tpr interacts with chromatin modifiers and NSPC-related transcription factors, P-Tpr interacts and co-localizes with cyclin-dependent kinase 1 (Cdk1) at the nuclear chromatin of NSPCs. In hippocampal NSPCs in a mouse model of AD, aberrant Tpr expression was correlated with altered NPC morphology and counts, and Tpr was aberrantly expressed in postmortem human brain samples from patients with AD. Thus, we propose that altered levels and subcellular localiza-tion of Tpr in CNS disease affect Tpr functionality, which in turn regulates the architecture and number of NSPC NPCs, possibly leading to aberrant neurogenesis.
Neural stem/progenitor cells live in an intricate cellular environment, the neurogenic niche, which supports their function and enables neurogenesis. The niche is made of a diversity of cell types, including neurons, glia and the vasculature, which are able to signal to and are structurally organised around neural stem/progenitor cells. While the focus has been on how individual cell types signal to and influence the behaviour of neural stem/progenitor cells, very little is actually known on how the niche is assembled during development from multiple cellular origins, and on the role of the resulting topology on these cells. This review proposes to draw a state-of-the art picture of this emerging field of research, with the aim to expose our knowledge on niche architecture and formation from different animal models (mouse, zebrafish and fruit fly). We will span its multiple aspects, from the existence and importance of local, adhesive interactions to the potential emergence of larger-scale topological properties through the careful assembly of diverse cellular and acellular components.
Alzheimer's disease (AD) affects various brain cell types, including astrocytes, which are the most abundant cell types in the central nervous system (CNS). Astrocytes not only provide homeostatic support to neurons but also actively regulate synaptic signaling and functions and become reactive in response to CNS insults through diverse signaling pathways including the JAK/STAT, NF-κB, and GPCR-elicited pathways. The advent of new technology for transcriptomic profiling at the single-cell level has led to increasing recognition of the highly versatile nature of reactive astrocytes and the context-dependent specificity of astrocyte reactivity. In AD, reactive astrocytes have long been observed in senile plaques and have recently been suggested to play a role in AD pathogenesis and progression. However, the precise contributions of reactive astrocytes to AD remain elusive, and targeting this complex cell population for AD treatment poses significant challenges. In this review, we summarize the current understanding of astrocyte reactivity and its role in AD, with a particular focus on the signaling pathways that promote astrocyte reactivity and the heterogeneity of reactive astrocytes. Furthermore, we explore potential implications for the development of therapeutics for AD. Our objective is to shed light on the complex involvement of astrocytes in AD and offer insights into potential therapeutic targets and strategies for treating and managing this devastating neurodegenerative disorder.
The loss or failure of an organ/tissue stands as one of the healthcare system's most prevalent, devastating, and costly challenges. Strategies for neural tissue repair and regeneration have received significant attention due to their particularly strong impact on patients' well‐being. Many research efforts are dedicated not only to control the disease symptoms but also to find solutions to repair the damaged tissues. Neural tissue engineering (TE) plays a key role in addressing this problem and significant efforts are being carried out to develop strategies for neural repair treatment. In the last years, active materials allowing to tune cell–materials interaction are being increasingly used, representing a recent paradigm in TE applications. Among the most important stimuli influencing cell behavior are the electrical and mechanical ones. In this way, materials with the ability to provide this kind of stimuli to the neural cells seem to be appropriate to support neural TE. In this scope, this review summarizes the different biomaterials types used for neural TE, highlighting the relevance of using active biomaterials and electrical stimulation. Furthermore, this review provides not only a compilation of the most relevant studies and results but also strategies for novel and more biomimetic approaches for neural TE.
Stroke enhances proliferation of neural precursor cells within the subventricular zone (SVZ) and induces ectopic migration of newborn cells towards the site of injury. Here, we characterize the identity of cells arising from the SVZ after stroke and uncover a mechanism through which they facilitate neural repair and functional recovery. With genetic lineage tracing, we show that SVZ-derived cells that migrate towards cortical photothrombotic stroke in mice are predominantly undifferentiated precursors. We find that ablation of neural precursor cells or conditional knockout of VEGF impairs neuronal and vascular reparative responses and worsens recovery. Replacement of VEGF is sufficient to induce neural repair and recovery. We also provide evidence that CXCL12 from peri-infarct vasculature signals to CXCR4-expressing cells arising from the SVZ to direct their ectopic migration. These results support a model in which vasculature surrounding the site of injury attracts cells from the SVZ, and these cells subsequently provide trophic support that drives neural repair and recovery.
The subventricular zone (SVZ) is a neurogenic niche that contributes to homeostasis and repair after brain injury. However, the effects of mild traumatic brain injury (mTBI) on the divergence of the regulatory DNA landscape within the SVZ and its link to functional alterations remain unexplored. In this study, we meticulously mapped the transcriptome atlas of murine SVZ and its responses to mTBI at the single-cell level. We observed cell-specific gene expression changes following mTBI and unveiled diverse cell-to-cell interaction networks that influence a wide array of cellular processes. Moreover, we reported novel neurogenesis lineage trajectories and related key transcription factors, which we subsequently validated through loss-of-function experiments. Specifically, we validated the role of Tcf7l1 , a cell cycle gene regulator, in promoting neural stem cell differentiation towards the neuronal lineage after mTBI, providing a potential target for regenerative medicine. Overall, our study profiles an SVZ transcriptome reference map, which underlies the differential cellular behavior in response to mTBI. The identified key genes and pathways that may ameliorate brain damage or facilitate neural repair serve as valuable resources for drug discovery in the context of mTBI.
Many of the mechanisms underlying neural development are basically similar in vertebrates and invertebrates. Among vertebrates, popular species for experimental studies are zebrafish, the South African clawed toad, the chick embryo and mice. In mice, many spontaneously occurring mutations affecting the cerebral cortex and the cerebellum have been described. Their molecular analysis, combined with transgenic technology to achieve ectopic gene expression and targeted gene ablation, has made the mouse the mammal of choice for molecular genetic studies of early development.In this chapter, mechanisms of development will be discussed with emphasis on neural induction (► Sect. 2.2), cell lineage studies and fate mapping (► Sect. 2.3), pattern formation of the forebrain and the hindbrain (► Sect. 2.4), specification of cell fate from the spinal cord to the telencephalon (► Sect. 2.5), neurogenesis, gliogenesis and migration, of the cerebral cortex in particular (► Sect. 2.6), axon outgrowth and guidance, focussing on the corpus callosum, the pyramidal tract, thalamocortical projections and the formation of topographic maps (► Sect. 2.7) and programmed cell death (► Sect. 2.8).KeywordsMechanisms of developmentGene expressionNeural inductionCell lineageFate mappingSpecification of cell fateNeurogenesisGliogenesisAxon outgrowthFormation topographic mapsProgrammed cell death
Brain injuries due to trauma or stroke are major causes of adult death and disability. Unfortunately, few interventions are effective for post-injury repair of brain tissue. After a long debate on whether endogenous neurogenesis actually happens in the adult human brain, there is now substantial evidence to support its occurrence. Although neurogenesis is usually significantly stimulated by injury, the reparative potential of endogenous differentiation from neural stem/progenitor cells is usually insufficient. Alternatively, exogenous stem cell transplantation has shown promising results in animal models, but limitations such as poor long-term survival and inefficient neuronal differentiation make it still challenging for clinical use. Recently, a high focus was placed on glia-to-neuron conversion under single-factor regulation. Despite some inspiring results, the validity of this strategy is still controversial. In this review, we summarize historical findings and recent advances on neurogenesis strategies for neurorepair after brain injury. We also discuss their advantages and drawbacks, as to provide a comprehensive account of their potentials for further studies.
The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogeneous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent on their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here, we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to the impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.
Background:
Electroacupuncture (EA) is given to assist in the treatment of MS, which is an effective therapeutic method. However, the therapy mechanism of EA related to stem cells in the treatment of MS is not yet known. In this study, we used a classic animal model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) to evaluate the therapeutic effect of EA at Zusanli (ST36) acupoint in EAE and shed light on its potential roles in the effects of stem cells in vivo.
Methods:
The EAE animal models were established. From the first day after immunization, EAE model mice received EA at ST36 acupoint, named the EA group. The weight and clinical score of the three groups were recorded for 28 days. The demyelination, inflammatory cell infiltration, and markers of neural stem cells (NSCs), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs) were compared.
Results:
We showed that EAE mice treated with EA at ST36 acupoint, were suppressed in demyelination and inflammatory cell infiltration, and thus decreased clinical score and weight loss and mitigated the development of EAE when compared with the EAE group. Moreover, our data revealed that the proportions of NSCs, HSCs, and MSCs increased in the EA group compared with the EAE group.
Conclusions:
Our study suggested that EA at ST36 acupoint was an effective nonpharmacological therapeutic protocol that not only reduced the CNS demyelination and inflammatory cell infiltration in EAE disease but also increased the proportions of various stem cells. Further study is necessary to better understand how EA at the ST36 acupoint affects EAE.
Important features of adult neuronal number, location, and type are a consequence of early embryonic events that occur before neurons have differentiated. We have measured cell number during embryogenesis of the rat CNS. Markers that are expressed in the proliferating neuronal precursor are required to study the mechanisms controlling their proliferation and differentiation. By applying immunohistochemistry, fluorescence-activated cell sorting, and 3H-thymidine auto-radiography to dissociated rat CNS cells, we show that the monoclonal antibody Rat 401 recognizes a cell population with proliferative, temporal, and quantitative features expected of neuronal precursors.
The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.
We have isolated a tripotential glial precursor cell population from spinal cords of E13.5 rats. In vitro, these A2B5+E-NCAM- glial-restricted precursor (GRP) cells can undergo extensive self-renewal, and can differentiate into oligodendrocytes and two distinct astrocyte populations, but do not differentiate into neurons. The differentiation potential of GRP cells is retained through at least three cycles of expansion and recloning. Unlike oligodendrocyte-type 2 astrocyte progenitor cells, freshly isolated GRP cells do not respond to platelet-derived growth factor as a mitogen or survival factor, nor do GRP cells differentiate into oligodendrocytes--or even survive--when plated in mitogen-free chemically defined medium. Exposure to fetal calf serum induces GRP cells to differentiate into A2B5- fibroblast-like astrocytes, whereas growth in the presence of basic fibroblast growth factor and ciliary neurotrophic factor induces the generation of A2B5+ process-bearing astrocytes. The early appearance of GRP cells during spinal cord development suggests that they may represent the earliest GRP cell population.
Neurogenesis in the mammalian central nervous system is believed to end in the period just after birth; in the mouse striatum
no new neurons are produced after the first few days after birth. In this study, cells isolated from the striatum of the adult
mouse brain were induced to proliferate in vitro by epidermal growth factor. The proliferating cells initially expressed nestin,
an intermediate filament found in neuroepithelial stem cells, and subsequently developed the morphology and antigenic properties
of neurons and astrocytes. Newly generated cells with neuronal morphology were immunoreactive for gamma-aminobutyric acid
and substance P, two neurotransmitters of the adult striatum in vivo. Thus, cells of the adult mouse striatum have the capacity
to divide and differentiate into neurons and astrocytes.
Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can
proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of
these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the
survivors.
Important features of adult neuronal number, location, and type are a consequence of early embryonic events that occur before neurons have differentiated. We have measured cell number during embryogenesis of the rat CNS. Markers that are expressed in the proliferating neuronal precursor are required to study the mechanisms controlling their proliferation and differentiation. By applying immunohistochemistry, fluorescence-activated cell sorting, and 3H-thymidine auto-radiography to dissociated rat CNS cells, we show that the monoclonal antibody Rat 401 recognizes a cell population with proliferative, temporal, and quantitative features expected of neuronal precursors.
The nature of proliferative cells in the subgranular zone (SGZ) of the hippocampal region and the fate of their progeny was analyzed by 3H-thymidine (3H-TdR) autoradiography combined with immunocytochemistry at the light and electron microscopic levels in 18 rhesus monkeys ranging in age from late gestation to 17 years. Our analysis indicates that, during the last quarter of gestation and the first 3 postnatal months, the SGZ produces both glial and neuronal cells. These 2 major classes of cells originate from the 2 precursor lines and, following their mitotic division, migrate to the granular layer. During the juvenile period (4-6 months of age), neuronal production tapers off and most postmitotic cells remaining within the SGZ differentiate into glial elements. In postpubertal animals (3 years and older), the 3H-TdR-labeled cells in the dentate gyrus belong to several non-neuronal classes. The largest group was immunoreactive to the glial fibrillary acidic protein (GFAP) at both the light and electron microscopic levels, indicating their astrocytic nature. The remaining 3H-TdR-labeled, GFAP-negative cells had ultra-structural characteristics of either microglia, oligodendroglia, or their progenitory stem cells. Therefore, there is a continuing addition and/or turnover of the glial cells in the dentate gyrus of sexually mature monkeys, but, in contrast to the massive neurogenesis reported in adult rodents, the production of new neurons could not be detected after puberty. The significance of a stable population of neurons in the hippocampal formation of mature primates is discussed in relation to its possible function in memory.
The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.
The goal of this study was to examine the ability of basic fibroblast growth factor (FGF-2) to promote reactivity and/or proliferation of astrocytes in vivo following brain injury, and the possible mechanisms involved. A small bilateral lesion in the motor-sensory cortex was performed, and either FGF-2, FGF-2 plus heparan sulfate, heparan sulfate, or saline was applied unilaterally in a piece of Gelfoam within the wound cavity. Following lesions, there was an increase in FGF-2 and FGF receptor (FGFR) immunoreactivities in the area surrounding the lesion in all the treatment groups. Rats that received treatment with recombinant FGF-2 alone showed an increase in the density of astrocytes as compared to the control group. The same group of rats exhibited an increase in the density of cells displaying FGF-2 immunoreactivity and cells displaying FGFR-1 immunoreactivity and cells displaying FGFR-1 immunoreactivity, and also an induction of FGF-2 mRNA in the tissue surrounding the lesion. The group of rats that received FGF-2 combined with heparan sulfate showed a larger increase in the same cellular parameters. Our results suggest that the FGF-2/FGFR system is involved in the regulation of astrocytic reactivity and/or proliferation in the brain and its action is potentiated by heparan sulfate. The action of FGF-2 on CNS injury appears to be part of an autocrine cascade that involves induction of FGF-2 and its receptor, thereby enhancing the ability of astrocytes to respond to FGF-2.
During development of mammalian cerebral cortex, two classes of glial cells are thought to underlie the establishment of cell patterning. In the embryonic period, migration of young neurons is supported by a system of radial glial cells spanning the thickness of the cortical wall. In the neonatal period, neuronal function is assisted by the physiological support of a second class of astroglial cell, the astrocyte. Here, we show that expression of embryonic radial glial identity requires extrinsic soluble signals present in embryonic forebrain. Moreover, astrocytes reexpress features of radial glia in vitro in the presence of the embryonic cortical signals and in vivo after transplantation into embryonic neocortex. These findings suggest that the transformation of radial glia cells into astrocytes is regulated by availability of inducing signals rather than by changes in cell potential.
In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results focus attention on how information in external stimuli is translated into the number and types of differentiated cells in the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding of the origin and stability of cell type in the central nervous system.
To analyze cell lineage in the murine cerebral cortex, we infected progenitor cells with a recombinant retrovirus, then used the retroviral gene product to identify the descendants of infected cells. Cortices were infected on E12-E14 either in vivo or following dissociation and culture. In both cases, nearly all clones contained either neurons or glia, but not both. Thus, neuronal and glial lineages appear to diverge early in cortical development. To analyze the distribution of clonally related cells in vivo, clonal boundaries were reconstructed from serial sections. Perinatally (E18-PN)), clonally related cells were radially arrayed as they migrated to the cortical plate. Thus, clonal cohorts traverse a similar radial path. Following migration (PN7-PN23), neuronal clones generally remained radially arrayed, while glial clones were variable in orientation, suggesting that these two cell types accumulate in different ways. Neuronal clones sometimes spanned the full thickness of the cortex. Thus, a single progenitor can contribute neurons to several laminae.
To determine whether fibrillary astrocytes proliferate in response to brain injury, cells identified as fibrillary astrocytes using immunoperoxidase technique for glial fibrillary acidic protein (GFAP) were examined for uptake of radiolabeled thymidine by autoradiography. In injured mouse brain, autoradiographic label was present over nuclei of immunoreactive fibrillary astrocytes in the lesion site 1 hr following injection of radiolabeled thymidine. The data suggest that fibrillary astrocytes which are sufficiently differentiated to accumulate GFAP retain the capacity to proliferate in response to injury.
Neural stem cells are maintained in the subventricular zone (SVZ) of the adult mammalian brain. Here, we review the cellular organization of this germinal layer and propose lineage relationships of the three main cell types found in this area. The majority of cells in the adult SVZ are migrating neuroblasts (type A cells) that continue to proliferate. These cells form an extensive network of tangentially oriented pathways throughout the lateral wall of the lateral ventricle. Type A cells move long distances through this network at high speeds by means of chain migration. Cells in the SVZ network enter the rostral migratory stream (RMS) and migrate anteriorly into the olfactory bulb, where they differentiate into interneurons. The chains of type A cells are ensheathed by slowly proliferating astrocytes (type B cells), the second most common cell type in this germinal layer. The most actively proliferating cells in the SVZ, type C, form small clusters dispersed throughout the network. These foci of proliferating type C cells are in close proximity to chains of type A cells. We discuss possible lineage relationships among these cells and hypothesize which are the neural stem cells in the adult SVZ. In addition, we suggest that interactions between type A, B, and C cells may regulate proliferation and initial differentiation within this germinal layer. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 234–248, 1998
The properties and fate of the cells of the subependymal layer of the anterior lateral ventricle and its rostral extension into the olfactory bulb were examined. In one experiment, histological analysis was made of this structure in a large group of rats, ranging in age from newborn to adults. It was established that the ventricular subependymal layer and its rostral extension are present as proliferative and migratory matrices throughout the period studied, with relatively little reduction in size from birth to adulthood. In another, autoradiographic study, the proliferation and migration of cells of this system, and their destination and mode of differentiation, were studied in rats that were injected with thymidine-H3 at 30 days of age and killed at intervals ranging from 1 hour to 180 days. There was a declining gradient in cell proliferation in a caudorostral direction from a high level near the lateral ventricle to the absence of cell proliferation in the olfactory bulb. The labeled cells that were present in high proportion near the lateral ventricle in the rats killed 1–24 hours after injection had further multiplied and moved to the middle portion of the “rostral migratory stream” by the third day, and were located in the subependymal layer of the olfactory bulb by the sixth day after injection. By the twentieth day the labeled cells disappeared from the subependymal layer of the olfactory bulb and were distributed throughout the internal granular layer. The differentiated cells were tentatively identified as granular nerve cells and neuroglia cells. These results established that the major target structure of cell production in the subependymal layer of the lateral ventricle in young-adult rats is the olfactory bulb, with only moderate contribution made to the anterior neocortex and basal ganglia. It was postulated that the function of cell migration to the olfactory bulb is the renewal of its cell population.
Ciliary development was studied in the cells of the neural canal of chick embryos incubated from 60 hours to 7 days.
It was found that centrioles move after the last mitosis to the cell periphery where one of them enters into contact (terminal contact) with the cell membrane; the other centriole remains close by, its axis aligned along the axis of the former.
The cell membrane was seen afterwards bulging at the contact point, and the content of the ciliary bud thus formed is only constituted at the beginning of a varied number of vesicles of about 140 Å diameter.
The ciliary bud becomes elongated shortly after filaments start becoming organized in the bud matrix.
Roughly coinciding with the initiation of filament organization the centrioles move inward and the cilium becomes deeply invaginated in the cell. At the end of ciliary growth the centriole moves again toward the surface and the cilium emerges in the neural canal lumen.
Radial glial cells (epithelial cells of Ramón y Cajal) impregnated by a modified del Rio Hortega rapid Golgi method were studied in the occipital lobes of 38 rhesus monkeys from embryonic day 48 (E48) to birth which occurs at E165 and in 27 postnatal animals to day 365 (P365). Some radial glial cells are already recognized at E48 by their bipolar shape and elongated radial fiber, which terminates with characteristic endfeet on the walls of blood vessels or at the pial surface. At slightly older ages-between E60 and E70-all cells spanning the cerebral wall develop lamellate expansions along their radial fiber and their endfeet become PAS positive. After E60, some radial glia detach from the ventricular surface and their somas become displaced outwards in the cerebral wall. After this age, radial glial cells are easily distinguished from migrating neurons by their larger oval nucleus located in the ventricular or subventricular zone, radial fiber extending outwards to the pial surface where it terminates in one or more endfeet, and the delicate lamellate expansions on both radial fiber and soma.
Displaced radial glial cells have more closely packed lamellate expansions and display a range of transitional shapes leading to either fibrous or protoplasmic astrocytes. Between E95 and E140, when neuron migration to the visual cortex tapers off, perikarya of displaced radial glial cells form a conspicuous band at the outer border of the subventricular zone. Numerous transitional forms are present in the cortical plate at this age. After birth, fewer radial glial fibers are present in occipital lobe and their length is difficult to determine in the convoluted lateral cerebral wall expanded up to 10–20 mm. However, at P7 and P20, many radial fibers still span the medial cerebral wall in the depth of the calcarine fissure where it remains less than 2 mm thick. Even here, no fibers spanning the cerebral wall were seen in 17 animals from P50 to P200 despite the presence of well-impregnated transitional forms situated near the lateral ventricle and myriad astrocytes dispersed throughout the hemisphere. By P365, end of the first year, the few short remaining radial fibers belong to ependymal cells or mature astrocytes while all immature transitional forms have disappeared.
To determine whether fibrillary astrocytes proliferate in response to brain injury, cells identified as fibrillary astrocytes using immunoperoxidase technique for glial fibrillary acidic protein (GFAP) were examined for uptake of radiolabeled thymidine by autoradiography. In injured mouse brain, autoradiographic label was present over nuclei of immunoreactive fibrillary astrocytes in the lesion site 1 hr following injection of radiolabeled thymidine. The data suggest that fibrillary astrocytes which are sufficiently differentiated to accumulate GFAP retain the capacity to proliferate in response to injury.
The cycle time of the proliferating glial cells outside the subependymal layer of the lateral ventricle as well as that of endothelial cells was studied autoradiographically in the brains of adult and untreated mice. To determine the mean cycle time two independent methods were used. A mean cycle time of about 20 hours was obtained for glial and endothelial cells from the decrease of the mean grain number/nucleus as a function of time after tritiated thymidine (3H-TdR) injection.
Another group of experiments utilized the “method of labeled S phases.” With this method the passage of labeled cells through successive S phases is observed. Passing through S phase following 3H-TdR injection the 3H-labeled cells are double labeled by an additional 14C-TdR injection. This method again resulted in a cycle time of 20 hours for glial and endothelial cells. From the present work and a former study (Korr et al., 11973) the following cell cycle parameters were derived: Cycle time 20 hours; S phase 9.4 hours; G2 less than three hours; (G2 + M) five hours; G1 five hours. The growth fraction of glial cells related to all glial cells is only 0.004.
Furthermore, the present experiments show that in the case of glial cells 17% of the daughter cells after mitosis become pyknotic and are eliminated from the glial cell population. Apart from this cell loss, after mitosis about onefourth of the daughter cells do not enter the next S phase. These cells leave the growth fraction and are replaced by a corresponding number of non-proliferating glial cells. There is a relatively extensive permanent exchange of cells between the growth fraction and non-growth fraction of glial cell.
In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.
The cerebral cortex of the mammalian brain has expanded rapidly during the course of evolution and acquired structurally distinguishable areas devoted to separate functions. In some brain regions, topographic restrictions to cell intermixing occur during embryonic development. As a means of examining experimentally whether such restrictions occur during formation of functional subdivisions in the rat neocortex, clonally related neocortical cells were marked by retroviral-mediated transfer of a histochemical marker gene. Clonal boundaries were determined by infection of the developing brain with a library of genetically distinct viruses and amplification of single viral genomes by the polymerase chain reaction. Many clonally related neurons in the cerebral cortex became widely dispersed across functional areas of the cortex. Specification of cortical areas therefore occurs after neurogenesis.
Neurogenesis in the adult avian brain is restricted to the telencephalon. New neurons originate in the ventricular zone (VZ) from cells that have not been identified. We mapped the position of [3H]thymidine-labeled cells in the walls of the ventricles of the adult canary brain. Labeled VZ cells were restricted to the telencephalon (lateral ventricles) and concentrated in "hot spots". The coincidence of these hot spots with regions rich in radial cells suggested that radial cells may be the cells undergoing mitosis. We used smears prepared from fragments of the VZ containing the hot spots to show directly that radial cells accumulate [3H]thymidine. In addition, grain counts at different survival times demonstrated that these cells divide. Hot spots of VZ cell division also coincided with sites of neuronal origin. We suggest that radial cell division may give rise to new neurons.
An antibody prepared against adult canary brain, 40E-C, stains ventricular zone cells that send long, unbranched processes into the forebrain parenchyma. We identify these cells as radial glia. The same antibody also stains a subset of brain astroglia and reacts with nonbrain material such as mesenchyme, Sertoli cells, and the Z-line of muscle. A weaker reaction is given by erythrocytes and some endothelial cells. 40E-C also reacts with the radial glia of the developing rat brain but fails to show any such glia in adult rodent brain. Western blot analysis shows that this antibody recognizes vimentin, a molecule shared by all 40E-C-positive cell types. We believe that the presence of radial glia in the adult avian forebrain and their apparent absence in mammals is related to neurogenesis in adulthood, which occurs in birds and much less or not at all in mammals. In addition, the presence of radial glia in adult birds may also relate to other, still-hypothetical, differences in the physiology of adult avian and mammalian brains.
Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.
The mechanism of transformation of the overtly similar cells of the neural plate into the numerous and diverse cell types of the mature vertebrate central nervous system (CNS) can better be understood by studying the clonal development of isolated CNS precursor cells. Here I describe a culture system in which blast cells (cells capable of division) isolated from embryonic day 13.5-14.5 rat forebrain can divide and differentiate into a variety of clonal types. Most clones contain only neurons or glia; 22% contain both neurons and non-neuronal cells. For the division of blast cells, live conditioning cells need to be present indicating that environmental signals influence proliferation. Heterogeneous clones develop in homogeneous culture conditions, so factors intrinsic to the blast cells are probably important in determining the number and type of clonal progeny.
Coronal sections of the cerebral wall from developing ferrets (newborn to adult) were double‐stained with antibodies to vimentin and glial fibrillary acidic protein (GFAP). At birth, the dominant glial population was radial glia and these cells labeled only for vimentin. A small population of immature astrocytes in the cortical plate was double labeled for GFAP and vimentin. In successive days, the number of vimentin‐positive radial glia gradually decreased and they disappeared entirely at about 21 days. During this same period, the double‐stained astrocytes increased in number and were distributed throughout the cortical plate and intermediate zone. After 6 weeks of age the astrocytes were mostly confined to the developing white matter. Around this time they gradually lost their vimentin staining, and in the adult no vimentin‐positive elements were seen except at the ependymal surface.
In newborn ferrets single radial glial cells were also visualized by applying the carbocyanine dye DiI onto the pial surface of fixed brains. While most radial glia extended from the ventricular zone to the pial surface, a substantial fraction of them had lost their contact to the ventricular zone. Their somata were displaced into the subventricular zone and lower portion of the intermdiate zone.
The possibility that radial glia transform into astrocytes was directly tested by injecting fluorescent dyes under the pial surface of newborn ferrets at a time when virtually no GFAP‐positive astrocytes are present. The tracer, which was taken up in the upper portion of the cortical plate, stained the radial glial cell somata in the ventricular zone in a similar way as the dye DiI did in the fixed brains. As the radial glial cells disappeared at successively longer survival times, the tracer was ultimately found within newly formed GFAP‐positive astrocytes. These results provide strong support for the hypothesis that radial glia cells are the immature form of astrocytes (Choi and Lapham: Brain Res. 148: 295–311, ′78; Schmechel and Rakic: Anat. Embryol. (Berl.) 156: 115–152, ′79), and also show that, at least in the ferret cortex, the transformation is accompanied by a change in the expression of intermediate filament protein.
Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.
Cellular topography within the highly polarized surface epithelia can be used to identify the location of the stem cells. In some instances, this can be quite precise and allows the characteristics of stem cells to be studied. Our current knowledge of the stem cell population in murine epidermis and small intestinal crypts is reviewed. In the epidermis, the stem cells would appear to make up about 10% of the basal layer and are distributed towards the centre of the basal layer component of the epidermal proliferative unit. These cells have a long cell cycle and are probably the same cells that retain both tritiated thymidine and radioactively labelled carcinogens for long periods of time. This label retention permits the labelling of the putative stem cell compartment. Over recent years, there has been an accumulation of information indicating various types of heterogeneity within the basal layer, much of which can be interpreted in relation to cellular hierarchies. In the small intestine, cell positions can be fairly precisely identified and the stem cell zone identified. Complex modelling of a wide range of cell kinetic experiments suggests that each crypt contains between 4 and 16 steady state functional stem cells. Radiobiological experiments suggest that up to 32 cells may be capable of clonal regeneration. The repopulation of the clonogenic cell compartment has been determined and the doubling time measured to be 19.7 h. Such studies should throw further light on the behaviour of stem cells and identify the timing of periods of increased and decreased cell proliferation (activation and suppression of controls).
We describe a cell-lineage marking system applicable to the vertebrate nervous system. The basis of the technique is gene transfer using the retroviral vector system. We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat promoter, with simultaneous expression of the Tn5 neo gene from the simian virus 40 early promoter. This expression has allowed us to detect individual infected cells histochemically. We applied this marking technique to the study of lineage relationships in the developing vertebrate nervous system, both in vivo and in culture. In the rat retina, we injected virus in vivo and histochemically identified clones of marked neural cells. In addition, we used this virus to infect cultures of rat cerebral cortex and have analyzed the clonal relationships of morphologically different neural cell types. The host range of the marking system extends to avian as well as mammalian species. Thus, this system should have broad applicability as a means of gene transfer and expression in the nervous system.
We have used a retroviral vector that codes for the bacterial enzyme beta-galactosidase to study cell lineage in the rat cerebral cortex. This vector has been used to label progenitor cells in the cerebral cortices of rat embryos during the period of neurogenesis. When these embryos are allowed to develop to adulthood, the clones of cells derived from the marked progenitor cells can be identified histochemically. In this way, we can ask what are the lineage relationships between different neural cell types. From these studies, we conclude that there are two distinct types of progenitor cells in the developing cortex. One generates only grey matter astrocytes, whereas the second gives rise to neurones - both pyramidal and nonpyramidal - and to another class of cells that we have tentatively identified as glial cells of the white matter. We have also been able to address the question of how neurones are dispersed in the cortex during histogenesis. It had been previously hypothesized that clonally related neurones migrated radially to form columns in the mature cortex. However, we find that clones of neurones do not form radial columns; rather, they tend to occupy the same or neighbouring cortical laminae and to be spread over several hundreds of micrometers of cortex in the horizontal dimension. This spread occurs in both mediolateral and rostrocaudal directions.
Brain astroglia in normal adult rats stained weakly or not at all with an antibody to epidermal growth factor receptor (EGFR). A dramatic change took place after injury. The astrocytes adjacent to an entorhinal ablation and in deafferented areas of the hippocampus showed prominent EGFR immunoreactivity. Cells that were EFGR-immunoreactive also stained intensely with an antibody to glial fibrillary acidic protein (GFAP). The localization and the time course of appearance of EGFR/GFAP immunoreactivity suggests that EGFR may be involved in the conversion of a normal into a reactive astrocyte.
To analyze cell lineage in the murine cerebral cortex, we infected progenitor cells with a recombinant retrovirus, then used the retroviral gene product to identify the descendants of infected cells. Cortices were infected on E12-E14 either in vivo or following dissociation and culture. In both cases, nearly all clones contained either neurons or glia, but not both. Thus, neuronal and glial lineages appear to diverge early in cortical development. To analyze the distribution of clonally related cells in vivo, clonal boundaries were reconstructed from serial sections. Perinatally (E18-PN0), clonally related cells were radially arrayed as they migrated to the cortical plate. Thus, clonal cohorts traverse a similar radial path. Following migration (PN7-PN23), neuronal clones generally remained radially arrayed, while glial clones were variable in orientation, suggesting that these two cell types accumulate in different ways. Neuronal clones sometimes spanned the full thickness of the cortex. Thus, a single progenitor can contribute neurons to several laminae.
The morphology and the development of the cells in the subependymal layer and of granule cells of the olfactory bulb were examined by Nissl and Golgi staining in postnatal rats. The subependymal layer around the anterior lateral ventricle extends into the center of the olfactory bulb. The mitotic indexes in the subependymal layer are high at the level of the anterior horn of the lateral ventricle and very low inside the olfactory bulb during the first 3 weeks after birth. Golgi-stained subependymal cells are classified into two main groups. One group consists of smoothly contoured bipolar cells with leading processes tipped by large growth cones and with trailing processes. They make up a majority of Golgi-stained subependymal cells during the first 3 weeks of age, and smaller numbers of them continue to exist at 37 and 60 days. They migrate with their growth cones oriented toward the olfactory bulb from the level of the anterior lateral ventricle into the granular layer of the olfactory bulb, where they differentiate into the definitive granule cells: their somata enlarge; the leading processes elongate, branch, sprout many gemmules, and become the peripheral processes; and the trailing processes become the basal dendrites. The other group contains relatively large cells with many cytoplasmic processes that are considered to belong to the glial cell line.
The glial fibrillary acidic (GFA) protein, a brain specific protein extracted from severely gliosed human tissue, is not species specific; cross-reaction occurs between anti-human GFA protein antibodies and brain extracts of rabbit, guinea pig, rat and dog. Using anti-GFA protein antiserum, astrocytes are selectively stained with the indirect immunofluorescence technique in both normal and pathological (gliosed) brain tissue.
Strong labeling of the cells in the subependymal layer was produced by stereotaxic injection of 5 μCi of ³ H‐thymidine into the left lateral ventricle of the brain of one and a quarter month old rats weighing about 100 gm. These animals were sacrificed by glutaraldehyde perfusion from two hours to 21 days later. Blocks of corpus callosum with adjacent subependymal and ependymal layers were excised from the injected and non‐injected sides, and embedded in Epon; 0.5 μ thick sections were radioautographed and stained with toluidine blue.
In the subependymal region, on both injected and non‐injected sides, there was an immediate uptake of label by many cells followed by an increase and later a decrease in the percent cells labeled. In the corpus callosum while at first the percent labeling of glial cells was rather low, it did increase slowly with time and, after seven days, exceeded that in the subependymal region. These results were interpreted as indicating that cells arising in the subependymal layer had migrated into the corpus callosum.
Up to four days after injection, most of the label in corpus callosum was present in immature‐looking cells resembling the cells of the subependymal layer and referred to as free subependymal cells. With time, the percent labeling decreased in these cells while increasing in some of the glial cells. A labeling peak was observed for light oligodendrocytes at four to seven days and for dark oligodendrocytes at 21 days, whereas labeling of medium shade oligodendrocytes occurred at intermediate times. The succession of labeling peaks indicated a sequence of development from free subependymal cells through light and medium shade to dark oligodendrocytes. Few astrocytes carried label at any time; those which did seemed to have arisen from the transformation of labeled free subependymal cells. Microglia were unlabeled at two hours, but their percent labeling was high at 4–14 days. While the labeling of other glial cells reflected their physiological behavior, the labeling of microglia was a consequence of the trauma produced by the injection 0f tracer into the ventricle.
In conclusion, cells coming from the subependymal layer appear to migrate into the corpus callosum where, in 100 gm rats, many of them transform into oligodendrocytes and a few into astrocytes.
The proliferation of glial cells outside the subependymal layer of the lateral ventricle as well as of endothelial cells was studied autoradiographically in the brain of the adult and untreated mouse. The double labeling method with 3H- and 14C-thymidine was applied in order to show experimentally the existence of a DNA synthesis phase (S phase) and to measure its duration. Adult mice received a first injection of 14C-thymidine, two or four hours later a second injection of 3H-thymidine and were sacrificed one hour after the last injection by perfusion fixation. Double layer autoradiographs were made from serial sections of the region from the corpus callosum/commissura anterior up to the corpus callosum/commissura fornicis ventralis in order to register purely 3H-, doubly 3H- and 14C-, and purely 14C-labeled nuclei. From the ratio of all 3H-labeled cells with and without 14C to the purely 3H-labeled cells a DNA synthesis phase of 9.4 ± 0.5 hours for glial cells and one of 11.0 ± 2.2 hours for endothelial cells was obtained. Based on the first appearance of labeled mitoses and labeled pairs of glial cells after injections of labeled thymidine the G2 phase was estimated to be < three hours and G2 + M about five hours. The duration of the measured S phase as well as the appearance of labeled mitoses about three hours after application of labeled thymidine are very similar to these cycle parameters in many other somatic cells in different kinds of animals. This has led to the conclusion that a well-defined DNA synthesis phase with doubling of the DNA content and a successive mitosis also exists in glial and endothelial cells of the adult mouse brain.
We have identified a cell type in 7-day-old rat optic nerve that differentiates into a fibrous astrocyte if cultured in the presence of fetal calf serum and into an oligodendrocyte if cultured in the absence of serum. In certain culture conditions some of these cells acquire a mixed phenotype, displaying properties of both astrocytes and oligodendrocytes. These observations suggest that fibrous astrocytes and oligodendrocytes develop from a common progenitor cell and provide a striking example of developmental plasticity and environmental influence in the differentiation of CNS glial cells.
The nervous system of adult mammals, unlike the rest of the organs in the body, has been considered unique in its apparent inability to replace neurons following injury. However, in certain regions of the brain, neurogenesis occurs postnatally and continues through adulthood. The nature, fate, and longevity of cells undergoing proliferation within the CNS are unknown. These cells are increasingly becoming the focus of intense scrutiny; this is a recent development that has led to considerable controversy over the appropriate terminology to describe neural cells as they pass through different stages of proliferation, migration, and differentiation. Continuing studies detailing the properties of mitotic populations in the adult CNS will provide a better understanding of the nature of these cells during their development and should lead to a more consistent nomenclature. Studies of neural precursors isolated from the embryonic brain have indicated that many subgroups of cells undergo mitosis and subsequent differentiation into neurons and glia in vitro. A number of substances, such as growth factors and substrate molecules, are essential for these processes and also for lineage restriction and fate determination of these cells. Recent studies have shown that cells with proliferative capabilities can also be isolated from the adult brain. The nature of these cells is unknown, but there is evidence that both multipotent cells (stem cells) and lineage-restricted cells (neuroblasts or glioblasts) are resident within the mature CNS and that they can be maintained and induced to divide and differentiate in response to many of the same factors that influence their embryonic counterparts. Presently, it is unclear how many potentially quiescent precursor cells exist in the adult brain or what combination of growth factors and substrate molecules is involved in the proliferation and differentiation of these cells. Some of these questions are currently being addressed by using immortalized neural precursors or growth factor-expanded populations of primary precursors to model precursor responsiveness to environmental manipulations. Because in vitro culture conditions are unlikely to provide all of the factors necessary for inducing the proliferation and differentiation of neural precursors, recent studies have explored the properties of well-characterized precursor populations after implantation back into specific regions of the developing or adult CNS. These studies have highlighted the importance of the microenvironment in precursor differentiation and further suggested that precursor plasticity is a characteristic that is probably common to neural precursors throughout the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)
Stem cells isolated from the CNS of both embryonic and adult mice undergo extensive proliferation in the presence of epidermal growth factor (EGF). Removal of EGF determines the differentiation of these cells into neurons and glia. We have recently demonstrated that basic fibroblast growth factor (bFGF) regulates the proliferation of EGF-generated progenitors of the embryonic mouse striatum. We report here that bFGF induces proliferation of some EGF-generated precursors of the adult mouse striatum which, in turn, differentiate in vitro into cells possessing neuron-like morphology and neuronal antigenic properties. These results demonstrate that EGF and bFGF can act sequentially to regulate the de novo generation of neurons from the adult mouse CNS in vitro and suggest the existence of a lineage relationship between EGF- and bFGF-responsive progenitor cells of the adult murine brain.
Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.
Neuroectoderm cells in the cortical ventricular zone generate many diverse cell types, maintain the ventricular zone during embryonic life and create another germinal layer, the subventricular zone, which persists into adulthood. In other vertebrate tissues, including skin, intestine, blood and neural crest, stem cells are important in maintaining a germinal population and generating differentiated progeny. By following the fates of single ventricular zone cells in culture, we show here that self-renewing, multipotential stem cells are present in the embryonic rat cerebral cortex. Forty per cent of these stem cells produced all three principal cell types of the central nervous system: neurons, astrocytes and oligodendrocytes. Stem cells constituted about 7% of cortical clones; in contrast, over 80% consisted of small numbers of neurons or glia. We suggest that multipotential stem cells may be the ancestors of other cortical progenitor cells that exhibit more limited proliferation and more restricted repertoires of progeny fates.
We have examined the region-specific expression of mRNAs for four members of rat FGF receptor family, FGFR-1, FGFR-2 FGFR-3, and FGFR-4, in rat brain by in situ hybridization. The FGFR-1, FGFR-2, and FGFR-3 mRNAs were expressed widely but differentially in the brain. However, the FGFR-4 mRNA was not expressed in the brain. The FGFR-1 mRNA was strongly expressed in several regions including the hippocampus, cerebellum, and pedunculopotine tegmental nucleus. The FGFR-2 mRNA expression was high in the choroid plexus, and moderate in the fiber-rich regions (the corpus callosum, external capsule, and internal capsule) and the olfactory bulb. The FGFR-3 mRNA was expressed diffusely in the brain. We have also examined the cellular localization of these mRNAs in the brain. Although the FGFR-1 mRNA was expressed preferentially in neurons, the FGFR-2 and FGFR-3 mRNAs were expressed preferentially in glial cells. The present findings that the FGFR-1, FGFR-2, and FGFR-3 mRNAs were expressed widely but with region- and cell-specificity in the brain indicate that these receptors have different roles in the brain.