Notch signaling pathway inhibitor DAPT improves alcohol-induced neuronal differentiation impairment in zebrafish / 南方医科大学学报

OBJECTIVE@#To explore the role of the Notch signaling pathway in regulating neuronal differentiation and sensorimotor ability in a zebrafish model of fetal alcohol spectrum disorder.@*METHODS@#Zebrafish embryos treated with DMSO or 50 μmol/L DAPT (a Notch signaling pathway inhibitor) were examined for mortality rate, hatching rate, malformation rate, and body length at 15 days post fertilization (dpf). The mRNA expression levels of sox2, neurogenin1 and huc in the treated zebrafish embryos were detected using in situ hybridization and qRT-PCR, and their behavioral responses to strong light and vibration stimulation were observed. The zebrafish embryos were then exposed to DMSO, 1.5% ethanol, DAPT, or both ethanol and DAPT, and the changes in mRNA expression levels of sox2, neurogenin1, huc, and the Notch signaling pathway genes as well as behavioral responses were evaluated.@*RESULTS@#Exposure to 50 μmol/L DAPT significantly increased the mortality rate of 1 dpf zebrafish embryos (P < 0.01), decreased the hatching rate of 2 dpf embryos (P < 0.01), increased the malformation rate of 3 dpf embryos (P < 0.001), and reduced the body length of 15 dpf embryos (P < 0.05). DAPT treatment significantly downregulated sox2 mRNA expression (P < 0.01) and increased neurogenin1 (P < 0.05) and huc (P < 0.01) mRNA expressions in zebrafish embryos. The zebrafish with DAPT treatment exhibited significantly shortened movement distance (P < 0.001) and lowered movement speed (P < 0.05) in response to all the stimulation conditions. Compared with treatment with 1.5% ethanol alone, which obviously upregulated notch1a, her8a and NICD mRNA expressions in zebrafish embryos (P < 0.05), the combined treatment with ethanol and DAPT significantly increased neurogenin1 and huc mRNA expression, decreased sox2 mRNA expression (P < 0.01), and increased the moving distance and moving speed of zebrafish embryos in response to strong light stimulation (P < 0.05).@*CONCLUSION@#Ethanol exposure causes upregulation of the Notch signaling pathway and impairs neuronal differentiation and sensorimotor ability of zebrafish embryos, and these detrimental effects can be lessened by inhibiting the Notch signaling pathway.

Characterization of neural stem cells derived from human stem cells from the apical papilla undergoing three-dimensional neurosphere induction

Abstract Objectives The endogenous repairing based on the activation of neural stem cells (NSCs) is impaired by neurodegenerative diseases. The present study aims to characterize human stem cells from the apical papilla (hSCAPs) with features of mesenchymal stem cells (MSCs) and to demonstrate the neuronal differentiation of hSCAPs into NSCs through the formation of three-dimensional (3D) neurospheres, verifying the structural, immunophenotyping, self-renewal, gene expression and neuronal activities of these cells to help further improve NSCs transplantation. Methodology The hSCAPs were isolated from healthy impacted human third molar teeth and characterized as MSCs. They were then induced into 3D-neurospheres using a specific neural induction medium. Subsequently, the intra-neurospheral cells were confirmed to be NSCs by the identification of Nissl substance and the analysis of immunofluorescence staining, self-renewal ability, and gene expression of the cells. Moreover, the neuronal activity was investigated using intracellular calcium oscillation. Results The isolated cells from the human apical papilla expressed many markers of MSCs, such as self-renewal ability and multilineage differentiation. These cells were thus characterized as MSCs, specifically as hSCAPs. The neurospheres induced from hSCAPs exhibited a 3D-floating spheroidal shape and larger neurospheres, and consisted of a heterogeneous population of intra-neurospheral cells. Further investigation showed that these intra-neurospheral cells had Nissl body staining and also expressed both Nestin and SOX2. They presented a self-renewal ability as well, which was observed after their disaggregation. Their gene expression profiling also exhibited a significant amount of NSC markers (NES, SOX1, and PAX6). Lastly, a large and dynamic change of the fluorescent signal that indicated calcium ions (Ca2+) was detected in the intracellular calcium oscillation, which indicated the neuronal activity of NSCs-derived hSCAPs. Conclusions The hSCAPs exhibited properties of MSCs and could differentiate into NSCs under 3D-neurosphere generation. The present findings suggest that NSCs-derived hSCAPs may be used as an alternative candidates for cell-based therapy, which uses stem cell transplantation to further treat neurodegenerative diseases.

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.

Resveratrol promotes the survival and neuronal differentiation of hypoxia-conditioned neuronal progenitor cells in rats with cerebral ischemia / 医学前沿

Hypoxia conditioning could increase the survival of transplanted neuronal progenitor cells (NPCs) in rats with cerebral ischemia but could also hinder neuronal differentiation partly by suppressing mitochondrial metabolism. In this work, the mitochondrial metabolism of hypoxia-conditioned NPCs (hcNPCs) was upregulated via the additional administration of resveratrol, an herbal compound, to resolve the limitation of hypoxia conditioning on neuronal differentiation. Resveratrol was first applied during the in vitro neuronal differentiation of hcNPCs and concurrently promoted the differentiation, synaptogenesis, and functional development of neurons derived from hcNPCs and restored the mitochondrial metabolism. Furthermore, this herbal compound was used as an adjuvant during hcNPC transplantation in a photothrombotic stroke rat model. Resveratrol promoted neuronal differentiation and increased the long-term survival of transplanted hcNPCs. 18-fluorine fluorodeoxyglucose positron emission tomography and rotarod test showed that resveratrol and hcNPC transplantation synergistically improved the neurological and metabolic recovery of stroke rats. In conclusion, resveratrol promoted the neuronal differentiation and therapeutic efficiency of hcNPCs in stroke rats via restoring mitochondrial metabolism. This work suggested a novel approach to promote the clinical translation of NPC transplantation therapy.

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

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

The system for directional and stable differentiation of amniotic fluid stem cells into neuron-like cells / 中国组织工程研究

BACKGROUND: Neuronal regeneration using stem cell differentiation has gained a lot of attentions from researchers. Although embryonic stem cells and induced pluripotent stem cells have good potential for neuronal differentiation, a high risk of tumor development in vivo limits the further study. OBJECTIVE: To establish a stable system for sorting, culture and neuronal differentiation of amniotic fluid stem cells, and to explore the feasibility as seed cells for neuronal regeneration. METHODS: Amniotic fluid sample (10 mL) was obtained at 19-22 weeks of pregnancy under B-ultrasound guidance, and amniotic fluid stem cells were isolated by c-Kit magnetic beads. The markers Oct-4 and Sox2 of amniotic fluid stem cells were identified by immunofluorescence. The expression levels of c-Kit, Oct-4, Sox2 and Nestin in amniotic fluid stem cells after multiple passages were detected by RT-PCR. Then, the cells were cultured by hanging drop for 4 days to observe the embryoid bodies-like structures. Amniotic fluid stem cells were induced to differentiate into neurons using two-stage method. The expression levels of Neuro D and Tuj1 were observed by immunofluorescence. RESULTS AND CONCLUSION: (1) About 1% of amniotic fluid stem cells were positive for c-Kit. (2) (75.0±4.6)% of amniotic fluid stem cells expressed Oct-4 and (86.0±2.8)% of the cells expressed Sox2. (3) The expression levels of c-Kit, Oct-4, Sox2 and Nestin detected by RT-PCR did not change with passage times. (4) Embryoid bodies-like structures formed after hanging drop culture. (5) Immunofluorescence results showed that amniotic fluid stem cells expressed neuronal marker Tuj1, but without the typical morphological features. RT-PCR detected the expression of Tuj1 in different amniotic fluid stem cell specimens as well as in the same sample after several passages. (6) Amniotic fluid stem cells could have the characteristics of neuron-like cells after induction with basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin factor 3 in two stages, and could express neural stem cell marker Neuro D and neuronal marker Tuj1.

Protein profiling of SH-SY5Y neuroblastoma cells: The effect of rhein

Bone marrow mesenchymal stem cells (BMSCs) play an important role in the process of bone repair. The present studyinvestigated the effect of 5-azacytidine (AZA) and trichostatin A (TSA) on BMSC behaviors in vitro. The role of WNTfamily member 5A (WNT5A)/WNT family member 5A (WNT7A)/b-catenin signaling was also investigated. BMSCs wereisolated from a steroid-induced avascular necrosis of the femoral head (SANFH) rabbit model. The third-generation ofBMSCs was used after identification. The results revealed obvious degeneration and necrosis in the SANFH rabbit model.AZA, TSA and TSA ? AZA increased BMSC proliferation in a time-dependent fashion. AZA, TSA and TSA ? AZAinduced the cell cycle release from the G0/G1 phase and inhibited apoptosis in BMSCs. AZA, TSA and TSA ? AZAtreatment significantly decreased caspase-3 and caspase-9 activities. The treatment obviously increased the activity andrelative mRNA expression of alkaline phosphatase. The treatment also significantly up-regulated the proteins associatedwith osteogenic differentiation, including osteocalcin and runt-related transcription factor 2 (RUNX2), and Wnt/b-cateninsignal transduction pathway-related proteins b-catenin, WNT5A and WNT7A. The relative levels of Dickkopf-relatedprotein 1 (an inhibitor of the canonical Wnt pathway) decreased remarkably. Notably, TSA ? AZA treatment exhibited astronger adjustment ability than either single treatment. Collectively, the present studies suggest that AZA, TSA and TSA ?AZA promote cell proliferation and osteogenic differentiation in BMSCs, and these effects are potentially achieved via upregulation of WNT5A/WNT7A/b-catenin signaling.

Effects of astragaloside IV combined with Panax notoginseng saponins on proliferation, apoptosis, migration and neuronal differentiation of bone marrow mesenchymal stem cells in OGD/R model rats / 中草药

Objective: To observe the effects of astragaloside IV (AST IV) combined with Panax Notoginseng saponins (PNS) on proliferation, apoptosis, migration and neuronal differentiation of oxygen glucosedeprivation/reoxygenation model rat bone marrow mesenchymal stem cells (BMSCs). Methods: BMSCs were isolated, cultured, amplified and purified by the whole bone marrow adherent method. The positive expression rates of BMSCs surface markers, CD29, CD90, CD34, and CD45 were detected by flow cytometry. The third generation of BMSCs was pretreated with AST IV and PNS doses of high (100 μmol/L + 60 μmol/L), medium (50 μmol/L + 30 μmol/L), and low (25 μmol/L + 15 μmol/L) for 24 h. The model of ischemia-reperfusion injury was established by OGD/R. Meanwhile, the normal group (BMSCs were cultured normally) and the model group (OGD/R was used to establish an ischemia reperfusion injury model) were established. The cell increment rate was detected by CCK-8 assay. Cell apoptosis was detected by flow cytometry. Transwell assay was used to detect the migration of BMSCs. The condition of BMSCs differentiation into neurons and astrocytes was observed by Nestin/NSE and Nestin/GFAP immunofluorescence double labeling. Results: BMSCs were successfully cultured and separated, and the positive rates of CD29 and CD90 detected by flow cytometry were 94.23% and 94.69%, while the positive rates of CD34 and CD45 were 5.76% and 5.31%. Compared with the normal group, the survival rate of the model group was reduced significantly and the apoptosis rate was increased significantly (P < 0.05). Compared with the model group, the combination of different doses of AST IV and PNS could promote the proliferation of BMSCs (P < 0.05, 0.01) and inhibit the apoptosis (P < 0.05, 0.01). Compared with the normal group, the model group and the AST IV and PNS group at different doses could promote the migration of BMSCs (P < 0.05). Compared with the model group, the number of migrated cells in the AST IV and PNS groups at different doses was increased significantly (P < 0.05). Compared with the normal group, the model group and the AST IV and PNS groups at different doses could promote the differentiation of BMSCs into neurons and astrocytes (P < 0.01). Compared with the model group, the positive expression rates of Nestin/NSE and Nestin/GFAP in the AST IV and PNS groups at different doses were increased significantly (P < 0.01). Conclusion: AST IV combined with PNS can promote the proliferation and migration of BMSCs of ischemia-reperfusion model in vitro, inhibit the apoptosis, and induce their directional differentiation into neurons and astrocytes.

Aucubin Promotes Differentiation of Neural Precursor Cells into GABAergic Neurons

Aucubin is a small compound naturally found in traditional medicinal herbs with primarily anti-inflammatory and protective effects. In the nervous system, aucubin is reported to be neuroprotective by enhancing neuronal survival and inhibiting apoptotic cell death in cultures and disease models. Our previous data, however, suggest that aucubin facilitates neurite elongation in cultured hippocampal neurons and axonal regrowth in regenerating sciatic nerves. Here, we investigated whether aucubin facilitates the differentiation of neural precursor cells (NPCs) into specific types of neurons. In NPCs cultured primarily from the rat embryonic hippocampus, aucubin significantly elevated the number of GAD65/67 immunoreactive cells and the expression of GAD65/67 proteins was upregulated dramatically by more than three-fold at relatively low concentrations of aucubin (0.01 µM to 10 µM). The expression of both NeuN and vGluT1 of NPCs, the markers for neurons and glutamatergic cells, respectively, and the number of vGluT1 immunoreactive cells also increased with higher concentrations of aucubin (1 µM and 10 µM), but the ratio of the increases was largely lower than GAD expression and GAD immunoreactive cells. The GABAergic differentiation of pax6-expressing late NPCs into GABA-producing cells was further supported in cortical NPCs primarily cultured from transgenic mouse brains, which express recombinant GFP under the control of pax6 promoter. The results suggest that aucubin can be developed as a therapeutic candidate for neurodegenerative disorders caused by the loss of inhibitory GABAergic neurons.

Laminin and Platelet-Derived Growth Factor-BB Promote Neuronal Differentiation of Human Urine-Derived Stem Cells

Urine-derived stem cells (USCs) are considered as a promising cell source capable of neuronal differentiation. In addition, specific growth factors and extracellular matrix are essential for enhancing their neuronal differentiation efficiency. In this study, we investigated the possibility of neuronal differentiation of USCs and the role of laminin and platelet-derived growth factor BB (PDGF-BB) as promoting factors. USCs were isolated from fresh urine of healthy donors. Cultured USCs were adherent to the plate and their morphology was similar to the cobblestone. In addition, they showed chromosome stability, rapid proliferation rate, colony forming capacity, and mesenchymal stem cell characteristics. For inducing the neuronal differentiation, USCs were cultured for 14 days in neuronal differentiation media supplemented with/without laminin and/or PDGF-BB. To identify the expression of neuronal markers, RT-PCR, flow cytometry analysis and immunocytochemistry were used. After neuronal induction, the cells showed neuron-like morphological change and high expression level of neuronal markers. In addition, laminin and PDGF-BB respectively promoted the neuronal differentiation of USCs and the combination of laminin and PDGF-BB showed a synergistic effect for the neuronal differentiation of USCs. In conclusion, USCs are noteworthy cell source in the field of neuronal regeneration and laminin and PDGF-BB promote their neuronal differentiation efficiency.

M2 Phenotype Microglia-derived Cytokine Stimulates Proliferation and Neuronal Differentiation of Endogenous Stem Cells in Ischemic Brain

Microglia play a key role in the immune response and inflammatory reaction that occurs in response to ischemic stroke. Activated microglia promote neuronal damage or protection in injured brain tissue. Extracellular signals polarize the microglia towards the M1/M2 phenotype. The M1/M2 phenotype microglia released pro- and anti-inflammatory cytokines which induce the activation of neural stem/progenitor cells (NSPCs). In this study, we investigated how the cytokines released by microglia affect the activation of NSPCs. First, we treated BV2 cells with a lipopolysaccharide (LPS; 20 ng/ml) for M1 phenotype microglia and interleukin-4 (IL-4; 20 ng/ml) for M2 phenotype microglia in BV2 cells. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 1 h. In ex vivo, brain sections containing the subventricular zone (SVZ) were cultured in conditioned media of M1 and M2 phenotype-conditioned media for 3 d. We measured the expression of cytokines in the conditioned media by RT-PCR and ELISA. The M2 phenotype microglia-conditioned media led to the proliferation and neural differentiation of NSPCs in the ipsilateral SVZ after ischemic stroke. The RT-PCR and ELISA results showed that the expression of TGF-α mRNA was significantly higher in the M2 phenotype microglia-conditioned media. These data support that M2 phenotype microglia-derived TGF-α is one of the key factors to enhance proliferation and neural differntiation of NSPCs after ischemic stroke.

Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development

The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both Pax6 and Tert genes. Interestingly, the VPA-induced TERT overexpression resulted to sequential upregulations of glutamatergic markers such as Ngn2 and NeuroD1, and inter-synaptic markers such as PSD-95, α-CaMKII, vGluT1 and synaptophysin. Transfection of Tert siRNA reversed the effects of VPA in cultured NPCs confirming the direct involvement of TERT in the expression of those markers. This study suggests the involvement of TERT in the VPA-induced autistic phenotypes and has important implications for the role of TERT as a modulator of balanced neuronal development and transmission in the brain.

Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells

The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.

Screening of natural compounds with neuronal differentiation promoting effects in a cell-based model / 中国天然药物

The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines (TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid (pTα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, qT-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.

Effects of Gastrodiae rhizoma on proliferation and differentiation of human embryonic neural stem cells

OBJECTIVE@#To investigate the effects of Gastrodiae rhizoma, a dried root of Gastrodia elata Blume, on proliferation and differentiation of human NSCs derived from embryonic stem cells.@*METHODS@#A 70% ethanol extract of Gastrodiae rhizoma (EEGR) was estimated with 4-hydroxybenzyl alcohol as a representative constituent by HPLC.@*RESULTS@#MTT assay showed that the treatment with EEGR increased the viability of NSCs in growth media. Compared to control, EEGR increased the number of dendrites and denritic spines extended from a differentiated NSC. Whereas EEGR decreased the mRNA expression of Nestin, it increased that of Tuj1 and MAP2 in NSCs grown in differentiation media. Immunocytochemical analysis using confocal microscopy also revealed the increased expression of MAP2 in dendrites of EEGR-treated NSCs. Furthermore, EEGR decreased mRNA expression of Sox2 in NSCs grown even in growth media.@*CONCLUSIONS@#In conclusion, our study demonstrates for the first time that EEGR induced proliferation and neuronal differentiation of NSCs, suggesting its potential benefits on NSC-based therapies and neuroregeneration in various neurodegenerative diseases and brain injuries.

Effects of Gastrodiae rhizoma on proliferation and differentiation of human embryonic neural stem cells

Objective: To investigate the effects of Gastrodiae rhizoma, a dried root of Gastrodia elata Blume, on proliferation and differentiation of human NSCs derived from embryonic stem cells. Methods: A 70% ethanol extract of Gastrodiae rhizoma (EEGR) was estimated with 4-hydroxybenzyl alcohol as a representative constituent by HPLC. Results: MTT assay showed that the treatment with EEGR increased the viability of NSCs in growth media. Compared to control, EEGR increased the number of dendrites and denritic spines extended from a differentiated NSC. Whereas EEGR decreased the mRNA expression of Nestin, it increased that of Tuj1 and MAP2 in NSCs grown in differentiation media. Immunocytochemical analysis using confocal microscopy also revealed the increased expression of MAP2 in dendrites of EEGR-treated NSCs. Furthermore, EEGR decreased mRNA expression of Sox2 in NSCs grown even in growth media. Conclusions: In conclusion, our study demonstrates for the first time that EEGR induced proliferation and neuronal differentiation of NSCs, suggesting its potential benefits on NSC-based therapies and neuroregeneration in various neurodegenerative diseases and brain injuries.

NgR1 Expressed in P19 Embryonal Carcinoma Cells Differentiated by Retinoic Acid Can Activate STAT3

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

In vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources.

Regenerative medicine is an evolving interdisciplinary topic of research involving numerous technological methods that utilize stem cells to repair damaged tissues. Particularly, mesenchymal stem cells (MSCs) are a great tool in regenerative medicine because of their lack of tumorogenicity, immunogenicity and ability to perform immunomodulatory as well as anti-inflammatory functions. Numerous studies have investigated the role of MSCs in tissue repair and modulation of allogeneic immune responses. MSCs derived from different sources hold unique regenerative potential as they are selfrenewing and can differentiate into chondrocytes, osteoblasts, adipocytes, cardiomyocytes, hepatocytes, endothelial and neuronal cells, among which neuronal-like cells have gained special interest. MSCs also have the ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation. In this review we focus on neural differentiation potential ofMSCs isolated from different sources and how certain growth factors/small molecules can be used to derive neuronal phenotypes from MSCs. We also discuss the efficacy of MSCs when transplanted in vivo and how they can generate certain neurons and lead to relief or recovery of the diseased condition. Furthermore, we have tried to evaluate the appropriatemerits of different sources ofMSCs with respect to their propensity towards neurological differentiation as well as their effectiveness in preclinical studies.

Purificação de células troco de lipoaspirado humano por aptâmeros de DNA, seguida da caracterização dos fenótipos obtidos da diferenciação neuronal / Human adipose mesechymal stem cell separation by DNA aptamers followed by the characterization of the obtained phenotypes from neuronal differentiation

Células tronco mesenquimais de tecido adiposo, são uma promissora ferramenta para aplicações clínicas em terapias celular e regenerativa, em vista da facilidade de sua extração e da maior quantidade de células por unidade de massa de tecido quando comparado a outras fontes clássicas de células mesenquimais como medula óssea. O protocolo clássico de extração e purificação dessas células, depende de sua adesão em plástico e xeno-materiais demandando muito tempo para ser utilizado por médicos para auxiliar pacientes em procedimentos de emergência. Estas células são capazes se diferenciar em diversos tipos celulares, o que as torna boas candidatas para terapia celular, embora sua capacidade de transdiferenciação para fenótipos neuronais seja ainda discutida. Neste trabalho demonstramos um novo processo para isolar essas células na base de epitopos específicos expressos (assinatura molecular de superfície) utilizando aptâmeros como ligantes de alta afinidade para estes sitios. Aptâmeros, moléculas de DNA simples fita identificadas a partir de uma biblioteca combinatória de sequencias de DNA simples-fita foram identificados por ciclos reiterativos de seleção in vitro (SELEX) utilizando células tronco do lipoaspirado como alvo. Dois aptâmeros isolados, denominados APT9 e APT11, foram capazes de identificar subpopulações (15,8 e 23,7% respectivamente) dentre as células tronco mesenquimais (classicamente CD29+/CD90+/CD45-) e separá-las usando nano-partículas magnéticas acopladas aos aptâmeros. Além disso, seguindo uma indução para diferenciação neuronal, as células tronco mesenquimais passam a apresentar morfologia neuronal e apresentam expressão e atividade de diversos receptores de neurotransmissores, avaliados por PCR real-time e imageamento de variações da concentração de cálcio intracelular ápos stimulação com vários agonistas de receptores metatrópicos e ionotrópicos. Ao longo da diferenciação, os níveis transcricionais de mRNA de receptores de cininas (B1 e B2), nicotínicos (alfa 7), muscarínicos (M1, M3 e M4), glutamatérgicos (AMPA2 e mGluR2), purinérgicos (P2Y1 e P2Y4) e GABAergicos (GABA-A, subunidade 3) e da óxido nítrico sintase neural aumentaram quando comparados aos níveis das células não diferenciadas, enquanto que os níveis de expressão de outros receptores incluindo purinérgicos P2X1, P3X4, P2X7 e P2Y6 e muscarínico M5 diminuíram. Os níveis de atividade das classes dos receptores estudados, por imageamento de variações da concentração de cálcio intrac, aumentaram para a maioria dos agonistas analisados durante a diferenciação neuronal com exceção para respostas induzidas por glutamato e NMDA. Células diferenciadas expressavam altos níveis de antígenos específicos de neurônios como ß3-tubulina, NF-H, NeuN e MAP-2 indicando uma diferenciação em fenótipo neuronal bem sucedida. Desta maneira, esta tese, ao identificar aptâmeros, prove uma inovadora solução para médicos usarem as células tronco mesenquimais dentro de uma sala de cirurgia, através de um método que é capaz de purificar essas células em um tempo clínico viável, com pureza e sem contato com contaminantes. Além disso, nós mostramos aqui que com um protocolo como o proposto para diferenciação neuronal, nós poderíamos induzir essas células para se diferenciar em neurônios, através da ativação de fatores de transcrição específicos, levando às células tronco mesenquimais a serem possivelmente utilizadas em terapias celulares de reparo neuronal

Adipose mesenchymal stem cells are promising tools for clinical applications in cellular and regeneration therapies, in view of easiness of extraction and higher amount of isolated stem cells per mass of tissue when compared to other classical mesenchymal stem cell sources including bone marrow. The classical protocol to extract and purify these cells, depending on plastic adherence and xeno-materials, is too time consuming to be used by physicians to help patients at emergency procedures. These cells are able to differentiate into various cell types, making them good candidates for cell therapy, however their capability for transdifferentiation into neural phenotypes is yet discussed. Here we show a novel process to isolate these cells using their surface molecular signature and aptamers, ssDNA molecules identified through the SELEX technique, denominated APT9 and APT11 that are able to identify subpopulations (15,8 and 23,7% respectively) within the mesenchymal stem cells (classically CD29+/CD90+/CD45-) and separate them using magnetic nano-particles attached to the aptamers. Moreover, following induction to neural differentiation, mesenchymal cells presents neuronal morphology and present expression and activity of several neurotransmitter receptors, as evaluated by real-time PCR and calcium imaging. During this process, mRNA transcription levels of bradykinin (B1 and B2), cholinergic (alpha 7), muscarinic (M1, M3 and M4), glutamatergic (AMPA2 and mGlu2), purinergic (P2Y1 and P2Y4) and GABAergic (GABA-A, subunit 3) receptors and neuronal nitric oxide synthase were augmented when compared to levels of undifferentiated cells, while the expression levels of other receptors including purinergic P2X1, P2X4, P2X7 and P2Y6 and muscarinic M5 receptors were down-regulated. Activity levels of the studied receptor classes, as studied by calcium imaging, increased for most of the agonists analyzed during the neuronal differentiation with the exception for glutamate- and NMDA-induced receptor responses. Differentiated cells expressed high levels of neuron-specific antigens such as ß3-tubulin, NF-H, NeuN and MAP-2, indicating a successful differentiation into neuronal phenotypes. This thesis, by identifying aptamers, provides a novel solution for physicians to use mesenchymal stem cells inside a surgery room, by using a method that are able to purify the cells in a clinical viable time, with purity and no contact with contaminats. Furthermore, we show here that with a protocol as provided for neuronal differentiation, we could induce these cells to differentiate into neurons, by activating specific transcription factors,making mesenchymal stem cells to possibly be used in neuronal repair cell therapies

Study on expression of Hes1 mRNA during the differentiation of neural stem cells toward neurons / 重庆医学

Objective To explore the expression of Hes1 mRNA during neural stem cells(NSC) differentiation toward neurons . Methods To establish the model of cultivation NSC in the hippocampal of newborn (24 h) SD rats ,and then to observe the mor-phology of NSC in the course of proliferation and differentiation .Before and after cellular induction ,the expression of Nestin and NSE were respectively measured to detect cell types by immunochemistry method .And flow cytometry was used to determine cell cycle phases ,so as to detect proliferative activity of these cells .Meanwhile ,the expression of Hes1 mRNA in NSC was determined by reverse transcription-PCR(RT-PCR) .Results The results demonstrated that NSC isolated from hippocampal showed vigorously clonal proliferation in vitro ,and positive Nestin expression .In addition ,the differentiated cells demonstrated positive NSE expres-sion .Flow cytometry analysis showed that the percentage of NSC in S phase was obviously higher than that of induced differentia-tion of all time(P0 .05) .Conclusion The high level of Hes1 mRNA was probably involved proliferation of NSC .How-ever ,low level of Hes1 mRNA might contribute to neuronal differentiation .