Mannitol inhibits the proliferation of neural stem cell by a p38 mitogen-activated protein kinase-dependent signaling pathway / 中华创伤杂志(英文版)

PURPOSE@#Mannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure. However, long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes. Given that neural stem cell (NSC) is a subpopulation of main regenerative cells in the central nervous system after injury, the effect of mannitol on NSC is still elusive. The present study aims to elucidate the role of mannitol in NSC proliferation.@*METHODS@#C57 mice were derived from the animal house of Zunyi Medical University. A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation. Initially, mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining. In order to investigate the impact of mannitol on NSC proliferation, both cell counting kit-8 assays and neurospheres formation assays were conducted. The in vitro effects of mannitol were examined at various doses and time points. In order to elucidate the role of Aquaporin 4 (AQP4) in the suppressive effect of mannitol on NSC proliferation, various assays including reverse transcription polymerase chain reaction, western blotting, and immunocytochemistry were conducted on control and mannitol-treated groups. Additionally, the phosphorylated p38 (p-p38) was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation. Finally, to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent (MAPK) signaling pathway in the observed inhibition of NSC proliferation by mannitol, SB203580 was employed. All data were analyzed using SPSS 20.0 software (SPSS, Inc., Chicago, IL). The statistical analysis among multiple comparisons was performed using one-way analysis of variance (ANOVA), followed by Turkey's post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test. Comparisons between 2 groups were determined using Student's t-test, if the data exhibited a normal distribution using a Shapiro-Wilk normality test. Meanwhile, data were shown as median and interquartile range and analyzed using the Mann-Whitney U test, if the data failed the normality test. A p < 0.05 was considered as significant difference.@*RESULTS@#Primary NSC were isolated from the mice, and the characteristics were identified using immunostaining analysis. Thereafter, the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8, neurospheres formation, and immunostaining of Nestin and Ki67 assays. During the process of mannitol suppressing NSC proliferation, the expression of AQP4 mRNA and protein was downregulated, while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction, immunostaining, and western blotting assays. Subsequently, the administration of SB203580, one of the p38 MAPK signaling pathway inhibitors, partially abrogated this inhibitory effect resulting from mannitol, supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol.@*CONCLUSIONS@#Mannitol inhibits NSC proliferation through downregulating AQP4, while upregulating the expression of p-p38 MAPK.

The Oncogenesis of Glial Cells in Diffuse Gliomas and Clinical Opportunities / 神经科学通报·英文版

Glioma is the most common and lethal intrinsic primary tumor of the brain. Its controversial origins may contribute to its heterogeneity, creating challenges and difficulties in the development of therapies. Among the components constituting tumors, glioma stem cells are highly plastic subpopulations that are thought to be the site of tumor initiation. Neural stem cells/progenitor cells and oligodendrocyte progenitor cells are possible lineage groups populating the bulk of the tumor, in which gene mutations related to cell-cycle or metabolic enzymes dramatically affect this transformation. Novel approaches have revealed the tumor-promoting properties of distinct tumor cell states, glial, neural, and immune cell populations in the tumor microenvironment. Communication between tumor cells and other normal cells manipulate tumor progression and influence sensitivity to therapy. Here, we discuss the heterogeneity and relevant functions of tumor cell state, microglia, monocyte-derived macrophages, and neurons in glioma, highlighting their bilateral effects on tumors. Finally, we describe potential therapeutic approaches and targets beyond standard treatments.

A Spacetime Odyssey of Neural Progenitors to Generate Neuronal Diversity / 神经科学通报·英文版

To understand how the nervous system develops from a small pool of progenitors during early embryonic development, it is fundamentally important to identify the diversity of neuronal subtypes, decode the origin of neuronal diversity, and uncover the principles governing neuronal specification across different regions. Recent single-cell analyses have systematically identified neuronal diversity at unprecedented scale and speed, leaving the deconstruction of spatiotemporal mechanisms for generating neuronal diversity an imperative and paramount challenge. In this review, we highlight three distinct strategies deployed by neural progenitors to produce diverse neuronal subtypes, including predetermined, stochastic, and cascade diversifying models, and elaborate how these strategies are implemented in distinct regions such as the neocortex, spinal cord, retina, and hypothalamus. Importantly, the identity of neural progenitors is defined by their spatial position and temporal patterning factors, and each type of progenitor cell gives rise to distinguishable cohorts of neuronal subtypes. Microenvironmental cues, spontaneous activity, and connectional pattern further reshape and diversify the fate of unspecialized neurons in particular regions. The illumination of how neuronal diversity is generated will pave the way for producing specific brain organoids to model human disease and desired neuronal subtypes for cell therapy, as well as understanding the organization of functional neural circuits and the evolution of the nervous system.

Temporal and spatial stability of the EM/PM molecular subtypes in adult diffuse glioma / 医学前沿

Detailed characterizations of genomic alterations have not identified subtype-specific vulnerabilities in adult gliomas. Mapping gliomas into developmental programs may uncover new vulnerabilities that are not strictly related to genomic alterations. After identifying conserved gene modules co-expressed with EGFR or PDGFRA (EM or PM), we recently proposed an EM/PM classification scheme for adult gliomas in a histological subtype- and grade-independent manner. By using cohorts of bulk samples, paired primary and recurrent samples, multi-region samples from the same glioma, single-cell RNA-seq samples, and clinical samples, we here demonstrate the temporal and spatial stability of the EM and PM subtypes. The EM and PM subtypes, which progress in a subtype-specific mode, are robustly maintained in paired longitudinal samples. Elevated activities of cell proliferation, genomic instability and microenvironment, rather than subtype switching, mark recurrent gliomas. Within individual gliomas, the EM/PM subtype was preserved across regions and single cells. Malignant cells in the EM and PM gliomas were correlated to neural stem cell and oligodendrocyte progenitor cell compartment, respectively. Thus, while genetic makeup may change during progression and/or within different tumor areas, adult gliomas evolve within a neurodevelopmental framework of the EM and PM molecular subtypes. The dysregulated developmental pathways embedded in these molecular subtypes may contain subtype-specific vulnerabilities.

GID complex regulates the differentiation of neural stem cells by destabilizing TET2 / 医学前沿

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.

Effect of electroacupuncture on proliferation of hippocampal neural stem cells in young mice with Alzheimer's disease / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) on the proliferation of endogenous neural stem cells in the hippocampus of young mice with Alzheimer's disease (AD), so as to explore its mechanisms underlying improvement of AD.@*METHODS@#Forty 1.5-month-old APP/PS1 transgenic male mice were randomly divided into an EA group and a model group, 20 mice in each group, and other 20 C57BL/6J male mice of the same age were used as the normal control group. EA (intermittment wave 10 Hz, 2 mA) was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Shenshu" (BL 23) for 20 min, once a day, 6 days a week for 16 weeks. H.E. staining was used to assess histopathological changes of neurons of the hippocampal dentate gyrus. Immunohistochemical stain was used to detect the expression of 5-bromodeoxyuridine (BrdU)-positive in the hippocampus, and immunofluorescence double-labeled technique was used to detect the number of proliferated positive neurons of hippocampal neural stem cells. The expression levels of brain derived neurotrophic factor (BDNF) and Nestin mRNA and protein were detected by using real-time PCR and Western blot, separately.@*RESULTS@#The immunoactivity of BrdU, and the expression levels of BDNF and Nestin mRNA and protein in the hippocampus in the model group were significantly lower than in the normal control group (P<0.01, P<0.05), and considerably higher in the EA group than in the model group (P<0.01, P<0.05). The number of BrdU/NeuN dual labeled neurons was slightly increased in the model group than in the normal control group (P>0.05), and evidently increased in the EA group relevant to the model group (P<0.05), suggesting a proliferation of hippocampal neural stem cells. After modeling, the neurons of hippocampal dentate gyrus were arranged loosely and irregularly and their structure was fuzzy, with an appearance of different degrees of nuclear pyknosis, whereas in the EA group, the neuronal contour was clear and the nuclear structure was relatively distinct.@*CONCLUSION@#EA can activate the proliferation of neural stem cells in the hippocampus in AD mice, which may contribute to its function in improving the neuronal structure by upregulating the expression of BDNF.

Neonatal Exposure to Propofol Interferes with the Proliferation and Differentiation of Hippocampal Neural Stem Cells and the Neurocognitive Function of Rats in Adulthood via the Akt/p27 Signaling Pathway / 生物医学与环境科学（英文）

Objective@#Neonatal exposure to propofol has been reported to cause neurotoxicity and neurocognitive decline in adulthood; however, the underlying mechanism has not been established.@*Methods@#SD rats were exposed to propofol on postnatal day 7 (PND-7). Double-immunofluorescence staining was used to assess neurogenesis in the hippocampal dentate gyrus (DG). The expression of p-Akt and p27 were measured by western blotting. The Morris water maze, novel object recognition test, and object location test were used to evaluate neurocognitive function 2-month-old rats.@*Results@#Phosphorylation of Akt was inhibited, while p27 expression was enhanced after neonatal exposure to propofol. Propofol also inhibited proliferation of neural stem cells (NSCs) and decreased differentiation to neurons and astroglia. Moreover, the neurocognitive function in 2-month-old rats was weakened. Of significance, intra-hippocampal injection of the Akt activator, SC79, attenuated the inhibition of p-AKT and increase of p27 expression. SC79 also rescued the propofol-induced inhibition of NSC proliferation and differentiation. The propofol-induced neurocognition deficit was also partially reversed by SC79.@*Conclusion@#Taken together, these results suggest that neurogenesis is hindered by neonatal propofol exposure. Specifically, neonatal propofol exposure was shown to suppress the proliferation and differentiation of NSCs by inhibiting Akt/p27 signaling pathway.

Effects of Porphyromonas gingivalis injected through tail vein on the expressions of biomarkers in neural stem cells and neurons of wild-type rats hippocampus / 中华口腔医学杂志

Objectives: To study the effects of Porphyromonas gingivalis (Pg) injected through tail vein on the molecular expression levels of biomarkers of neural stem cells (NSC) and neurons in the hippocampus of wild-type adult rats, and the effects on hippocampal neurogenesis. Methods: Eighteen male Sprague-Dawley (SD) rats were randomly divided into 3 groups based on the table of random numbers (n=6 in each group). In low-intensity group and high-intensity group, rats were injected intravenously through tail vein with 200 μl Pg ATCC33277 [1.0×103 and 1.0×108 colony forming unit (CFU), respectively] 3 times per week for 8 weeks. In the sham group, 200 μl of phosphate buffer saline (PBS) was given instead. Behavioral tests: the navigation and the exploration tests using Morris water maze (MWM) were applied to evaluate learning and memory ability of rats. Immunohistochemistry was performed to detect cells positively expressing nestin, doublecortin (DCX) and neuronal nuclei (NeuN) in the subgranular zone (SGZ) of rats in each group. Western blotting was used to evaluate the expression levels of nestin, DCX and NeuN in rat hippocampus. Results: Learning and memory abilities: on day 5 of navigation test, the lagency time was 22.83 (16.00, 38.34) s in the high-intensity group, significantly longer than the sham group [5.59 (5.41, 6.17) s] (t=-11.17, P<0.001). There were no significant differences between the low-intensity group [9.85 (8.75, 21.01) s] and the sham group (t=-6.83, P=0.080). Results in the exploration test showed that, in the high-intensity group, the number of fime crossing over the previous platform area within 60 s was 1.50 (1.00, 2.00), significantly less than the sham group [4.00 (2.75, 4.00)] (t=9.75, P=0.003); no significant differences between the low-intensity group [2.50 (2.00, 3.00)] and the sham one (t=4.50, P=0.382). Immunohistochemistry showed that the nestin+ cell density in the low-intensity group [(35.36±4.32) cell/mm2] and high-intensity group [(26.51±5.89) cell/mm2] were significantly lower than the sham group [(59.58±14.15) cell/mm2] (t=24.21, P=0.018; t=33.07, P=0.005); as for the mean absorbance of DCX+ cells, the low-intensity group (0.007±0.002) and the high-intensity group (0.006±0.002) were significantly lower than the sham group (0.011±0.001) (t=0.004, P=0.018; t=0.006, P=0.005); compared with the sham group [(1.13±0.14)×103 cell/mm2], the density of NeuN+ neurons in the high-intensity group [(0.75±0.08)×103 cell/mm2] was significantly reduced (t=0.38, P=0.017), and was not significantly changed in the low-intensity group [(0.88±0.19)×103 cell/mm2] (t=0.25, P=0.075). Western blotting results showed that, compared with the sham group, the expression levels of nestin, DCX, and NeuN were significantly reduced in the high-intensity group (t=0.74, P<0.001; t=0.18, P=0.014; t=0.35, P=0.008), but were not statistically changed in the low-intensity group (t=0.18, P=0.108; t=0.08, P=0.172; t=0.19, P=0.077). Conclusions: Pg injected through tail vein may reduce learning and memory abilities of wild-type rats, and may reduce the number of nestin, DCX, and NeuN-positive cells, and the protein expression levels of the above molecules in the hippocampus.

20-Hydroxyecdysone Improves Neuronal Differentiation of Adult Hippocampal Neural Stem Cells in High Power Microwave Radiation-Exposed Rats / 生物医学与环境科学（英文）

Objective@#The hippocampus is thought to be a vulnerable target of microwave exposure. The aim of the present study was to investigate whether 20-hydroxyecdysone (20E) acted as a fate regulator of adult rat hippocampal neural stem cells (NSCs). Furthermore, we investigated if 20E attenuated high power microwave (HMP) radiation-induced learning and memory deficits.@*Methods@#Sixty male Sprague-Dawley rats were randomly divided into three groups: normal controls, radiation treated, and radiation+20E treated. Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system. The learning and memory abilities of the rats were assessed using the Morris water maze test. Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation. In addition, hematoxylin & eosin staining, western blotting, and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure.@*Results@#The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro. Furthermore, 20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure. Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein (FRZB) reduced the 20E-induced nuclear translocation of β-catenin, while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro.@*Conclusion@#These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs, where it played a role in attenuating HPM radiation-induced learning and memory deficits.

Total Ginsenoside Extract from Panax ginseng Enhances Neural Stem Cell Proliferation and Neuronal Differentiation by Inactivating GSK-3β / 中国结合医学杂志

OBJECTIVE@#To study the effects of total ginsenosides (TG) extract from Panax ginseng on neural stem cell (NSC) proliferation and differentiation and their underlying mechanisms.@*METHODS@#The migration of NSCs after treatment with various concentrations of TG extract (50, 100, or 200 µ g/mL) were monitored. The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays. NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2 (MAP2). The GSK-3β/β-catenin pathway in TG-treated NSCs was examined by Western blot assay. The NSCs with constitutively active GSK-3β mutant were made by adenovirus-mediated gene transfection, then the proliferation and differentiation of NSCs mediated by TG were further verified.@*RESULTS@#TG treatment significantly enhanced NSC migration (P<0.01 or P<0.05) and increased the proliferation of NSCs (P<0.01 or P<0.05). TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression (P<0.01 or P<0.05). TG extract also significantly induced GSK-3β phosphorylation at Ser9, leading to GSK-3β inactivation and, consequently, the activation of the GSK-3β/β-catenin pathway (P<0.01 or P<0.05). In addition, constitutive activation of GSK-3β in NSCs by the transfection of GSK-3β S9A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation (P<0.01 or P<0.05).@*CONCLUSION@#TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3β.

Extrapolating neurogenesis of mesenchymal stem/stromal cells on electroactive and electroconductive scaffolds to dental and oral-derived stem cells / 国际口腔科学杂志·英文版

The high neurogenic potential of dental and oral-derived stem cells due to their embryonic neural crest origin, coupled with their ready accessibility and easy isolation from clinical waste, make these ideal cell sources for neuroregeneration therapy. Nevertheless, these cells also have high propensity to differentiate into the osteo-odontogenic lineage. One strategy to enhance neurogenesis of these cells may be to recapitulate the natural physiological electrical microenvironment of neural tissues via electroactive or electroconductive tissue engineering scaffolds. Nevertheless, to date, there had been hardly any such studies on these cells. Most relevant scientific information comes from neurogenesis of other mesenchymal stem/stromal cell lineages (particularly bone marrow and adipose tissue) cultured on electroactive and electroconductive scaffolds, which will therefore be the focus of this review. Although there are larger number of similar studies on neural cell lines (i.e. PC12), neural stem/progenitor cells, and pluripotent stem cells, the scientific data from such studies are much less relevant and less translatable to dental and oral-derived stem cells, which are of the mesenchymal lineage. Much extrapolation work is needed to validate that electroactive and electroconductive scaffolds can indeed promote neurogenesis of dental and oral-derived stem cells, which would thus facilitate clinical applications in neuroregeneration therapy.

Cannabidiol prevents depressive-like behaviors through the modulation of neural stem cell differentiation / 医学前沿

Chronic stress impairs radial neural stem cell (rNSC) differentiation and adult hippocampal neurogenesis (AHN), whereas promoting AHN can increase stress resilience against depression. Therefore, investigating the mechanism of neural differentiation and AHN is of great importance for developing antidepressant drugs. The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to be effective against depression. However, whether CBD can modulate rNSC differentiation and hippocampal neurogenesis is unknown. Here, by using the chronic restraint stress (CRS) mouse model, we showed that hippocampal rNSCs mostly differentiated into astrocytes under stress conditions. Moreover, transcriptome analysis revealed that the FoxO signaling pathway was involved in the regulation of this process. The administration of CBD rescued depressive-like symptoms in CRS mice and prevented rNSCs overactivation and differentiation into astrocyte, which was partly mediated by the modulation of the FoxO signaling pathway. These results revealed a previously unknown neural mechanism for neural differentiation and AHN in depression and provided mechanistic insights into the antidepressive effects of CBD.

Effect of advanced maternal age on development of hippocampal neural stem cells in offspring rats / 中国当代儿科杂志

OBJECTIVE@#To study the effect of advanced maternal age (AMA) on the development of hippocampal neural stem cells in offspring rats.@*METHODS@#Ten 3-month-old and ten 12-month-old female Sprague-Dawley rats were housed individually with 3-month-old male rats (1:1, n=20), whose offspring rats were assigned to a control group and an AMA group. A total of 40 rats were randomly selected from each group. Immunofluorescence assay and Western blot were used to localize and determine the levels of protein expression of Nestin and doublecortin (DCX) on day 7 as well as neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) on day 28 (n=8 for each marker). Immunofluorescence assay was also used to localize the hippocampal expression of polysialylated isoforms of neural cell adhesion molecule (PSA-NCAM) on day 14 (n=8 for each marker).@*RESULTS@#According to the Western blot results, the AMA group had significantly lower protein expression of DCX than the control group (P0.05). According to the results of immunofluorescence assay, the AMA group had significantly lower protein expression of Nestin, DCX, and PSA-NCAM in the hippocampal dentate gyrus (DG) region than the control group (P0.05). The AMA group had significantly higher expression of NeuN in the hippocampal CA1 region than the control group (P0.05). The AMA group had significantly lower expression of GFAP in the hippocampal CA1, CA3, and DG regions than the control group (P<0.05).@*CONCLUSIONS@#AMA may cause inhibition of proliferation, survival, and migration of hippocampal neural stem cells. AMA may also affect their differentiation into neurons and astrocytes, which will eventually lead to developmental disorders of hippocampal neural stem cells in offspring rats.

Isolamento e diferenciação das células-tronco da polpa dentária canina em células progenitoras neurais / Isolation and differentiation of canine dental pulp stem cells in neural progenitor cells

O objetivo deste estudo foi verificar a capacidade de diferenciação das células-tronco da polpa dentária canina em células progenitoras neurais bem como quantificar obtenção e viabilidade celular, durante três passagens em cultura. As células foram extraídas da polpa dentária de dois cadáveres caninos, com aproximadamente dez meses de idade, que foram a óbito em decorrência de traumatismo automotivo. Após três subculturas, realizou-se avaliação da viabilidade celular por quantificação em câmara de Neubauer. A partir disso, induziu-se diferenciação neural em meio de cultura neurobasal (Gibco™), com células aderidas ao plástico ou suspensas em placas tratadas com agarose. Após sete e 14 dias em cultivo indutor, observou-se morfologia e perfil imunofenotípico utilizando citometria de fluxo e imunocitoquímica fluorescente. Aos 14 dias as células apresentaram alto grau de expressão para marcadores anti-nestina e anti-glial fibrillary acidic protein (anti-GFAP). Anteriormente, obteve-se ao 25º dia, média de 18x106 células viáveis indiferenciadas oriundas do tecido pulpar. Sugere-se que as células-tronco indiferenciadas da polpa dentária canina apresentem índices satisfatórios de diferenciação em células progenitoras neurais, aderidas ou suspensas em cultura. A polpa dentária dos dentes decíduos caninos, fornece células indiferenciadas viáveis em quantidade adequada.(AU)

The objective of this study was to verify the differentiation capacity of canine tooth pulp stem cells in neural progenitor cells as well as to quantify the attainment and viability during three culture passages. The cells were extracted from the dental pulp of two canine cadavers, with approximately ten months of age, which died due to automotive trauma. After three subcultures, cell viability evaluation was performed by Neubauer chamber quantification. Neural differentiation was induced in neurobasal culture medium (Gibco ™), with cells adhered to the plastic or suspended in agarose-treated plates. After seven and 14 days in inducer culture, morphology and immunophenotypic profile were observed using flow cytometry and fluorescent immunocytochemistry. At 14 days the cells had a high degree of expression for anti-nestin and anti-glial fibrillary acidic (anti-GFAP) markers. Previously, an average of 18x106 undifferentiated viable cells from the pulp tissue were obtained on the 25th day. It is suggested that the undifferentiated canine pulp stem cells present satisfactory differentiation indices in neural progenitor cells, adhered or suspended in culture. The dental pulp of deciduous canine teeth provides viable undifferentiated cells in adequate quantity.(AU)

Human Neural Stem Cells: Translational Research for Neonatal Hypoxic-Ischemic Brain Injury

Neonatal hypoxic-ischemic (HI) brain injury is a major cause of neonatal mortality and long-term neurodevelopmental disabilities. Although promising neuroprotective interventions have been studied, the current management of HI brain injury has been limited to supportive measures and induced hypothermia. In addition to engrafting, migrating toward the damage sites and differentiating into multiple lineages, multipotent neural stem/progenitor cells (NSPCs) also provide trophic/immunomodulatory factors and integrate into the host neurons upon implantation into an HI-injured brain. However, NSPC-based therapies have shown poor cell survival and integration, poor differentiation or restricted differentiation into the glial lineages. Furthermore, to achieve full functional recovery following brain injury, the optimization of cell therapy is needed to recapitulate the precise migration of stem cells to the region of interest and the neural rewiring present in the brain microenvironment. Therefore, the efficacy of NSPCs in the treatment of CNS injury is currently insufficient. Human NSPCs (hNSPCs) were isolated from the forebrain of an aborted fetus at 13 weeks of gestation with full parental consent and the approval of the Institutional Review Board of the Yonsei University College of Medicine. Here, to enhance the regenerative ability of hNSPCs in HI brain injury, cells were either pretreated with pharmacological agents or engineered to serve as vehicles for gene delivery. Furthermore, when combined with a poly (glycolic acid)-based synthetic scaffold, hNSPCs provide a more versatile treatment for neonatal HI brain injury. Finally, hNSPCs transfected with zinc-doped ferrite magnetic nanoparticles for controlling both cell migration and differentiation offer a simple and smart tool for cell-based therapies.

A Potential Therapy Using Engineered Stem Cells Prevented Malignant Melanoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.

Autophagy Mediates Astrogenesis in Adult Hippocampal Neural Stem Cells

Neural stem cells (NSCs) have the ability to self-renew and differentiate into neurons, oligodendrocytes, and astrocytes. Highly dynamic nature of NSC differentiation requires the intimate involvement of catabolic processes such as autophagy. Autophagy is a major intracellular degradation pathway necessary for cellular homeostasis and remodeling. Autophagy is important for mammalian development and its role in neurogenesis has recently drawn much attention. However, little is known about how autophagy is associated with differentiation of NSCs into other neural lineages. Here, we report that autophagy plays a critical role in differentiation of adult rat hippocampal neural stem (HCN) cells into astrocytes. During differentiation, autophagy flux peaked at early time points, and remained high. Pharmacological or genetic suppression of autophagy by stable knockdown of Atg7, LC3 or CRISPR-Cas9-mediated knockout (KO) of p62 impaired astrogenesis, while reintroduction of p62 recovered astrogenesis in p62 KO HCN cells. Taken together, our findings suggest that autophagy plays a key role in astrogenesis in adult NSCs.

Molecular Characterization of Primary Human Astrocytes Using Digital Gene Expression Analysis / 대한신경손상학회지

OBJECTIVE: Astrocyte dysfunctions are related to several central nervous system (CNS) pathologies. Transcriptomic profiling of human mRNAs to investigate astrocyte functions may provide the basic molecular-biological data pertaining to the cellular activities of astrocytes. METHODS: Human Primary astrocytes (HPAs) and human neural stem cell line (HB1.F3) were used for differential digital gene analysis. In this study, a massively parallel sequencing platform, next-generation sequencing (NGS), was used to obtain the digital gene expression (DGE) data from HPAs. A comparative analysis of the DGE from HPA and HB1.F3 cells was performed. Sequencing was performed using NGS platform, and subsequently, bioinformatic analyses were implemented to reveal the identity of the pathways, relatively up- or down-regulated in HPA cells. RESULTS: The top, novel canonical pathways up-regulated in HPA cells than in the HB1.F3 cells were “Cyclins and cell cycle regulation,” “Integrin signaling,” “Regulation of eIF4 and p70S6K signaling,” “Wnt/β-catenin signaling,” “mTOR signaling,” “Aryl hydrocarbon receptor signaling,” “Hippo signaling,” “RhoA signaling,” “Signaling by Rho family GTPases,” and “Glioma signaling” pathways. The down-regulated pathways were “Cell cycle: G1/S checkpoint regulation,” “eIF2 signaling,” “Cell cycle: G2/M DNA damage checkpoint regulation,” “Telomerase signaling,” “RhoGDI signaling,” “NRF2-mediated oxidative stress response,” “ERK/MAPK signaling,” “ATM signaling,” “Pancreatic adenocarcinoma signaling,” “VEGF signaling,” and “Role of CHK proteins in cell cycle checkpoint control” pathways. CONCLUSION: This study would be a good reference to understand astrocyte functions at the molecular level, and to develop a diagnostic test, based on the DGE pattern of astrocytes, as a powerful, new clinical tool in many CNS diseases.

Effects of different melatonin treatment regimens on the proliferation of endogenous neural stem cells in neonatal rats with hypoxic-ischemic brain damage / 中国当代儿科杂志

OBJECTIVE@#To study the effects of different melatonin treatment regimens on the proliferation of neural stem cells (NSCs) and long-term histopathology in neonatal rats with hypoxic-ischemic brain damage (HIBD), and to identify better melatonin treatment regimens.@*METHODS@#A total of 96 Sprague-Dawley rats aged 7 days were randomly divided into normal control, HIBD, single-dose immediate melatonin treatment (SDIT), and 7-day continuous melatonin treatment (7DCT) groups, with 24 rats in each group. The rat model of HIBD was prepared by isolation and electrocoagulation of the right common carotid artery as well as hypoxic treatment in a hypoxic chamber (oxygen concentration 8.00% ± 0.01%) for 2 hours. On day 7 after modeling, proliferating cell nuclear antigen/Nestin double-labeling immunofluorescence was used to measure the proliferation of endogenous NSCs in the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG) region in 8 rats in each group, and Western blot was used to measure the protein expression of Nestin in brain. On day 28 after modeling, hematoxylin-eosin (HE) staining and Nissl staining were used to observe the changes in the histopathology and the number of pyramidal cells in the hippocampal CA1 region in 8 rats in each group.@*RESULTS@#Immunofluorescent staining showed that compared with the HIBD group, the SDIT and 7DCT groups had a significant increase in the number of PCNA+Nestin+DAPI+ cells, and the 7DCT group had a significantly higher number than the SDIT group (P<0.01). Western blot showed that the SDIT and 7DCT groups had significantly higher protein expression of Nestin than the HIBD group, and the 7DCT group had significantly higher expression than the SDIT group (P<0.05). HE staining showed that the SDIT and 7DCT groups had alleviated cell injury, and Nissl staining showed that compared with the HIBD group, the SDIT and 7DCT groups had a significant increase in the number of pyramidal cells, and the 7DCT group had a significantly higher number than the SDIT group (P<0.01).@*CONCLUSIONS@#Both single-dose immediate melatonin treatment and 7-day continuous melatonin treatment can promote the proliferation of endogenous NSCs and alleviate long-term histological injury in the brain of neonatal rats with HIBD. A 7-day continuous melatonin treatment has a better effect than single-dose immediate melatonin treatment.

MicroRNA-132 in the Adult Dentate Gyrus is Involved in Opioid Addiction Via Modifying the Differentiation of Neural Stem Cells / 神经科学通报·英文版

MicroRNA-132 (miR-132), a small RNA that regulates gene expression, is known to promote neurogenesis in the embryonic nervous system and adult brain. Although exposure to psychoactive substances can increase miR-132 expression in cultured neural stem cells (NSCs) and the adult brain of rodents, little is known about its role in opioid addiction. So, we set out to determine the effect of miR-132 on differentiation of the NSCs and whether this effect is involved in opioid addiction using the rat morphine self-administration (MSA) model. We found that miR-132 overexpression enhanced the differentiation of NSCs in vivo and in vitro. Similarly, specific overexpression of miR-132 in NSCs of the adult hippocampal dentate gyrus (DG) during the acquisition stage of MSA potentiated morphine-seeking behavior. These findings indicate that miR-132 is involved in opioid addiction, probably by promoting the differentiation of NSCs in the adult DG.