Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

Microglial activation that occurs rapidly after closed head injury may play important and complex roles in neuroinflammation-associated neuronal damage and repair.We previously reported that induced neural stem cells can modulate the behavior of activated microglia via CXCL12/CXCR4 signaling,influencing their activation such that they can promote neurological recovery.However,the mechanism of CXCR4 upregulation in induced neural stem cells remains unclear.In this study,we found that nuclear factor-κB activation induced by closed head injury mouse serum in microglia promoted CXCL12 and tumor necrosis factor-αexpression but suppressed insulin-like growth factor-1 expression.However,recombinant complement receptor 2-conjugated Crry(CR2-Crry)reduced the effects of closed head injury mouse serum-induced nuclear factor-κB activation in microglia and the levels of activated microglia,CXCL12,and tumor necrosis factor-α.Additionally,we observed that,in response to stimulation(including stimulation by CXCL12 secreted by activated microglia),CXCR4 and Crry levels can be upregulated in induced neural stem cells via the interplay among CXCL12/CXCR4,Crry,and Akt signaling to modulate microglial activation.In agreement with these in vitro experimental results,we found that Akt activation enhanced the immunoregulatory effects of induced neural stem cell grafts on microglial activation,leading to the promotion of neurological recovery via insulin-like growth factor-1 secretion and the neuroprotective effects of induced neural stem cell grafts through CXCR4 and Crry upregulation in the injured cortices of closed head injury mice.Notably,these beneficial effects of Akt activation in induced neural stem cells were positively correlated with the therapeutic effects of induced neural stem cells on neuronal injury,cerebral edema,and neurological disorders post–closed head injury.In conclusion,our findings reveal that Akt activation may enhance the immunoregulatory effects of induced neural stem cells on microglial activation via upregulation of CXCR4 and Crry,thereby promoting induced neural stem cell–mediated improvement of neuronal injury,cerebral edema,and neurological disorders following closed head injury.

Long non-coding RNA H19 regulates neurogenesis of induced neural stem cells in a mouse model of closed head injury

Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regeneration via cell replacement.However,the neural regeneration efficiency of induced neural stem cells remains limited.In this study,we explored differentially expressed genes and long non-coding RNAs to clarify the mechanism underlying the neurogenesis of induced neural stem cells.We found that H19 was the most downregulated neurogenesis-associated lnc RNA in induced neural stem cells compared with induced pluripotent stem cells.Additionally,we demonstrated that H19 levels in induced neural stem cells were markedly lower than those in induced pluripotent stem cells and were substantially higher than those in induced neural stem cell-derived neurons.We predicted the target genes of H19 and discovered that H19 directly interacts with mi R-325-3p,which directly interacts with Ctbp2 in induced pluripotent stem cells and induced neural stem cells.Silencing H19 or Ctbp2 impaired induced neural stem cell proliferation,and mi R-325-3p suppression restored the effect of H19 inhibition but not the effect of Ctbp2 inhibition.Furthermore,H19 silencing substantially promoted the neural differentiation of induced neural stem cells and did not induce apoptosis of induced neural stem cells.Notably,silencing H19 in induced neural stem cell grafts markedly accelerated the neurological recovery of closed head injury mice.Our results reveal that H19 regulates the neurogenesis of induced neural stem cells.H19 inhibition may promote the neural differentiation of induced neural stem cells,which is closely associated with neurological recovery following closed head injury.

Transplantation of fibrin-thrombin encapsulated human induced neural stem cells promotes functional recovery of spinal cord injury rats through modulation of the microenvironment

Recent studies have mostly focused on engraftment of cells at the lesioned spinal cord,with the expectation that differentiated neurons facilitate recovery.Only a few studies have attempted to use transplanted cells and/or biomaterials as major modulators of the spinal cord injury microenvironment.Here,we aimed to investigate the role of microenvironment modulation by cell graft on functional recovery after spinal cord injury.Induced neural stem cells reprogrammed from human peripheral blood mononuclear cells,and/or thrombin plus fibrinogen,were transplanted into the lesion site of an immunosuppressed rat spinal cord injury model.Basso,Beattie and Bresnahan score,electrophysiological function,and immunofluorescence/histological analyses showed that transplantation facilitates motor and electrophysiological function,reduces lesion volume,and promotes axonal neurofilament expression at the lesion core.Examination of the graft and niche components revealed that although the graft only survived for a relatively short period(up to 15 days),it still had a crucial impact on the microenvironment.Altogether,induced neural stem cells and human fibrin reduced the number of infiltrated immune cells,biased microglia towards a regenerative M2 phenotype,and changed the cytokine expression profile at the lesion site.Graft-induced changes of the microenvironment during the acute and subacute stages might have disrupted the inflammatory cascade chain reactions,which may have exerted a long-term impact on the functional recovery of spinal cord injury rats.

Exosomes derived from human induced pluripotent stem cell-derived neural progenitor cells protect neuronal function under ischemic conditions

Compared with other stem cells,human induced pluripotent stem cells-derived neural progenitor cells(iPSC-NPCs)are more similar to cortical neurons in morphology and immunohistochemistry.Thus,they have greater potential for promoting the survival and growth of neurons and alleviating the proliferation of astrocytes.Transplantation of stem cell exosomes and stem cells themselves have both been shown to effectively repair nerve injury.However,there is no study on the protective effects of exosomes derived from iPSC-NPCs on oxygen and glucose deprived neurons.In this study,we established an oxygen-glucose deprivation model in embryonic cortical neurons of the rat by culturing the neurons in an atmosphere of 95%N2 and 5%CO2 for 1 hour and then treated them with iPSC-NPC-derived exosomes for 30 minutes.Our results showed that iPSC-NPC-derived exosomes increased the survival of oxygen-and glucose-deprived neurons and the level of brain-derived neurotrophic factor in the culture medium.Additionally,it attenuated oxygen and glucose deprivation-induced changes in the expression of the PTEN/AKT signaling pathway as well as synaptic plasticity-related proteins in the neurons.Further,it increased the length of the longest neurite in the oxygen-and glucose-deprived neurons.These findings validate the hypothesis that exosomes from iPSCNPCs exhibit a neuroprotective effect on oxygen-and glucose-deprived neurons by regulating the PTEN/AKT signaling pathway and neurite outgrowth.This study was approved by the Animal Ethics Committee of Sir Run Run Shaw Hospital,School of Medicine,Zhejiang University,China(approval No.SRRSH20191010)on October 10,2019.

Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.

Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells-possibilities and current limitations

BACKGROUND Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients.They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells(RGCs)and Müller glia.AIM To refine human-induced pluripotent stem cells(hiPSCs)differentiated into threedimensional(3D)retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses.METHODS In this study we described,evaluated,and refined methods with which to generate Müller glia and RGC progenitors,isolated them via magnetic-activated cell sorting,and assessed their lineage stability after prolonged 2D culture.Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry,and the ultrastructural composition of retinal organoid cells was investigated.RESULTS Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids.Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers.CONCLUSION Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

Biochemical properties of norepinephrine as a kind of neurotransmitter secreted by bone marrow-derived neural stem cells induced and differentiated in vitro

BACKGROUND： It has been proved by many experimental studies from the aspects of morphology and immunocytochemistry in recent years that bone marrow stromal cells （BMSCs） can in vitro induce and differentiate into the cells possessing the properties of nerve cells. But the functions of BMSCs-derived neural stem cells（NSCs） and the differentiated neuron-like cells are still unclear. OBJECTIVE： To observe whether bone marrow-derived NSCs can secrete norepinephrine （NE） under the condition of in vitro culture, induce and differentiation, and analyze the biochemical properties of BMSCs-derived NSCs. DESIGN： A non-randomized and controlled experimental observation SETTING ： Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University MATERIALS： This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5- month-old healthy New Zealand white rabbits. METHODS： This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5 month-old healthy New Zealand white rabbits. BMSCs of rabbits were isolated and performed in vitro culture, induce and differentiation with culture medium of NSCs and differentiation-inducing factor, then identified with immunocytochemical method. Experimental grouping： ①Negative control group： L-02 hepatic cell and RPMI1640 culture medium were used. ② Background culture group： Only culture medium of NSCs as culture solution was added into BMSCs to perform culture, and 0.1 volume fraction of imported fetal bovine serum was supplemented 72 hours later. ③Differentiation inducing factor group： After culture for 72 hours, retinoic acid and glial cell line-derived neurotrophic factors were added in the culture medium of BMSCs and NSCs as corresponding inducing factors. The level of NE in each group was detected on the day of culture and 5, 7, 14 and 20 days after culture with high performance liquid chromatography （HPLC）. The procedure was conducted 3 times in each group.Standard working curve was made according to the corresponding relationship of NE concentration and peak area. The concentration of NE every 1×10^7 cells was calculated according to standard curve and cell counting. MAIN OUTCOME MEASURES ： The level of NE of cultured cells was detected with HPLC; immunocytochemistrical identification of Nestin and neuron specific nuclear protein was performed. RESULTS： ① On the 14^th day after cell culture, BMSCs turned into magnus and round cells which presented Nestin-positive antigen, then changed into neuron-like cells with long processus and presented neuron specific nuclear protein -positive antigen at the 20^th day following culture. ② The ratio of NE concentration and peak area has good linear relationship, and regression equation was Y=1.168 36＋0.000 272 8X,r=-0.998 4. Coefficient variation （CV） was 〈 5% and the recovery rate was 92.39%（ Y referred to concentration and X was peak area）.③NE was well detached within 10 minutes under the condition of this experiment. ④ NE was detected in NSCs and their culture mediums, which were cultured for 7, 14 and 20 days respectively, but no NE in BMSCs, NSCs-free culture medium and L-02 hepatic cell which were as negative control under the HPLC examination. Analysis of variance showed that the level of NE gradually increased following the elongation of culture time （P 〈 0.01 ）. No significant difference in the level of NE existed at the same time between differentiation inducing factor group and basic culture group（P 〉 0.05）. CONCLUSION ： BMSCs of rabbits can proliferate in vitro and express Nestin antigen; They can differentiate into neuron-like cells, express specific neucleoprotein of mature neurons, synthesize and secrete NE as a kind of neurotransmitter.

Neural regeneration by regionally induced stem cells within poststroke brains: Novel therapy perspectives for stroke patients

Ischemic stroke is a critical disease which causes serious neurological functional loss such as paresis. Hope for novel therapies is based on the increasing evidence of the presence of stem cell populations in the central nervous system (CNS) and the development of stem-cell-based therapies for stroke patients. Although mesenchymal stem cells (MSCs) represented initially a promising cell source, only a few transplanted MSCs were present near the injured areas of the CNS. Thus, regional stem cells that are present and/or induced in the CNS may be ideal when considering a treatment following ischemic stroke. In this context, we have recently showed that injury/ischemia-induced neural stem/progenitor cells (iNSPCs) and injury/ischemia-induced multipotent stem cells (iSCs) are present within post-stroke human brains and post-stroke mouse brains. This indicates that iNSPCs/iSCs could be developed for clinical applications treating patients with stroke. The present study introduces the traits of mouse and human iNSPCs, with a focus on the future perspective for CNS regenerative therapies using novel iNSPCs/iSCs.

Optimal time point for the transplantation of neural stem cells induced to differentiate with retinoic acid

Previous studies have demonstrated that differentiated neural stem cells （NSCs） are more suitable for transplantation than non-differentiated NSCs. In this study, NSCs were expanded in vitro for two passages, induced with retinoic acid to differentiate, and harvested between 1 6 days later. They were subsequently cultured in artificial cerebrospinal fluid for an additional 3 days, dudng which their growth and morphology was monitored. NSCs induced for 4 days exhibited a peak rate of cells differentiating into neurons and robust growth. Our results indicate that the optimal time point for transplanting NSCs is following a 4-day period of induced differentiation.

Reprogramming of mouse neural stem cells to induced pluripotent stem cells using Oct4 combined with microRNA

microRNA is important for maintaining characteristics of embryonic stem cells, and microRNA302a （MiR-302a） has been shown to exert important effects on cell reprogramming. Therefore, the present study used miR294 and miR302a, in combination with Oct4, to induce mouse neural stem cells （NSCs） into induced pluripotent stem （iPS） cells. Following identification of iPS cells, the effects of microRNA on cell reprogramming were analyzed. Results suggested that reprogramming efficiency with Oct4 ＋ miR-294 ＋ miR-302a was 7-fold greater than Oct4 alone （0.1% vs. 0.014%）. The iPS cells were undifferentiated and positive for alkaline phosphatase, SSEA-1, and Oct4. These findings demonstrated that microRNAs play an important role in cell reprogramming and provide a safe and efficient induction system for cellular reprogramming.

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human-induced neural stem cells(iNSCs)transplantation is a potential treatment of neurodegeneration diseases.However,whether the reprogrammed cells have the same characterizations as human fetal neural stem cells needs further exploration.Here we isolated human fetal neural stem cells from aborted 12-week fetal brains and compared with iNSCs reprogrammed from human peripheral blood mononuclear cells in gene expression,proliferation ability,differentiation capacity,and the responses to tumor necrosis factor-α.We found that iNSCs and NSCs both expressed neural stem cell markers Nestin,SOX1,and SOX2.However,only iNSCs can be patterned into dopaminergic neurons and motor neurons.Furthermore,both iNSCs and NSCs can differentiate into oligodendrocyte progenitor cells.In addition,a low dose of tumor necrosis factor-αdid not inhibit the proliferation and differentiation of iNSCs and NSCs.In conclusion,iNSCs have properties similar to,and even better than,fetal neural stem cells and may be suitable for disease modeling and transplantation.

Transplantation of vascular endothelial growth factor-modified neural stem/progenitor cells promotes the recovery of neurological function following hypoxic-ischemic brain damage

Neural stem/progenitor cell （NSC） transplantation has been shown to effectively improve neurological function in rats with hypoxic-isch- emic brain damage. Vascular endothelial growth factor （VEGF） is a signaling protein that stimulates angiogenesis and improves neural regeneration. We hypothesized that transplantation of VEGF-transfected NSCs would alleviate hypoxic-ischemic brain damage in neo- natal rats. We produced and transfected a recombinant lentiviral vector containing the VEGF165gene into cultured NSCs. The transfected NSCs were transplanted into the left sensorimotor cortex of rats 3 days after hypoxic-ischemic brain damage. Compared with the NSCs group, VEGF mRNA and protein expression levels were increased in the transgene NSCs group, and learning and memory abilities were significantly improved at 30 days. Furthermore, histopathological changes were alleviated in these animals. Our findings indicate that transplantation of VEGF-transfected NSCs may facilitate the recovery of neurological function, and that its therapeutic effectiveness is better than that of unmodified NSCs.

Comparison of phenotypic markers and neural differentiation potential of multipotent adult progenitor cells and mesenchymal stem cells

AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reversetranscription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 andhuman leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82%vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34),GFAP (1.12),Tau (1.08),MAP-1B (0.92), MAP-2 (1.14) andNSE (0.4) (P < 0.001 for all). CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.

Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells

Propofol and remifentanil alter intracellular Ca^2＋ concentration （[Ca^2＋]i） in neural stem/progen-itor cells by activating γ-aminobutyric acid type A receptors and by reducing testosterone levels. However, whether this process affects neural stem/progenitor cell proliferation and differenti-ation remains unknown. In the present study, we applied propofol and remifentanil, alone or in combination, at low, moderate or high concentrations （1, 2–2.5 and 4–5 times the clinically effective blood drug concentration）, to neural stem/progenitor cells from the hippocampi of newborn rat pups. Low concentrations of propofol, remifentanil or both had no noticeable effect on cell proliferation or differentiation; however, moderate and high concentrations of propofol and/or remifentanil markedly suppressed neural stem/progenitor cell proliferation and differen-tiation, and induced a decrease in [Ca^2＋]i during the initial stage of neural stem/progenitor cell differentiation. We therefore propose that propofol and remifentanil interfere with the prolifer-ation and differentiation of neural stem/progenitor cells by altering [Ca^2＋]i. Our ifndings suggest that propofol and/or remifentanil should be used with caution in pediatric anesthesia.

Small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells improve postoperative cognitive dysfunction in mice with diabetes

Postoperative cognitive dysfunction(POCD)is a common surgical complication.Diabetes mellitus(DM)increases risk of developing POCD after surgery.DM patients with POCD seriously threaten the quality of patients’life,however,the intrinsic mechanism is unclear,and the effective treatment is deficiency.Previous studies have demonstrated neuronal loss and reduced neurogenesis in the hippocampus in mouse models of POCD.In this study,we constructed a mouse model of DM by intraperitoneal injection of streptozotocin,and then induced postoperative cognitive dysfunction by transient bilateral common carotid artery occlusion.We found that mouse models of DM-POCD exhibited the most serious cognitive impairment,as well as the most hippocampal neural stem cells(H-NSCs)loss and neurogenesis decline.Subsequently,we hypothesized that small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells(iMSC-sEVs)might promote neurogenesis and restore cognitive function in patients with DM-POCD.iMSC-sEVs were administered via the tail vein beginning on day 2 after surgery,and then once every 3 days for 1 month thereafter.Our results showed that iMSC-sEVs treatment significantly recovered compromised proliferation and neuronal-differentiation capacity in H-NSCs,and reversed cognitive impairment in mouse models of DM-POCD.Furthermore,miRNA sequencing and qPCR showed miR-21-5p and miR-486-5p were the highest expression in iMSC-sEVs.We found iMSC-sEVs mainly transferred miR-21-5p and miR-486-5p to promote H-NSCs proliferation and neurogenesis.As miR-21-5p was demonstrated to directly targete Epha4 and CDKN2C,while miR-486-5p can inhibit FoxO1 in NSCs.We then demonstrated iMSC-sEVs can transfer miR-21-5p and miR-486-5p to inhibit EphA4,CDKN2C,and FoxO1 expression in H-NSCs.Collectively,these results indicate significant H-NSC loss and neurogenesis reduction lead to DM-POCD,the application of iMSC-sEVs may represent a novel cell-free therapeutic tool for diabetic patients with postoperative cognitive dysfunction.

Transcriptional regulation of adult neural stem/progenitor cells: tales from the subventricular zone

In rodents,well characterized neurogenic niches of the adult brain,such as the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus,support the maintenance of neural/stem progenitor cells(NSPCs)and the production of new neurons throughout the lifespan.The adult neurogenic process is dependent on the intrinsic gene expression signatures of NSPCs that make them competent for self-renewal and neuronal differentiation.At the same time,it is receptive to regulation by various extracellular signals that allow the modulation of neuronal production and integration into brain circuitries by various physiological stimuli.A drawback of this plasticity is the sensitivity of adult neurogenesis to alterations of the niche environment that can occur due to aging,injury or disease.At the core of the molecular mechanisms regulating neurogenesis,several transcription factors have been identified that maintain NSPC identity and mediate NSPC response to extrinsic cues.Here,we focus on REST,Egr1 and Dbx2 and their roles in adult neurogenesis,especially in the subventricular zone.We review recent work from our and other laboratories implicating these transcription factors in the control of NSPC proliferation and differentiation and in the response of NSPCs to extrinsic influences from the niche.We also discuss how their altered regulation may affect the neurogenic process in the aged and in the diseased brain.Finally,we highlight key open questions that need to be addressed to foster our understanding of the transcriptional mechanisms controlling adult neurogenesis.

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become ＂activated＂ after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine（BrdU） incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.

Ultrastructure of human neural stem/progenitor cells and neurospheres

BACKGROUND： Biological and morphological characteristics of neural stem/progenitor cells （NSPCs） have been widely investigated. OBJECTIVE： To explore the ultrastructure of human embryo-derived NSPCs and neurospheres cultivated in vitro using electron microscopy. DESIGN, TIME AND SETTING： A cell biology experiment was performed at the Brain Tumor Laboratory of Soochow University, and Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University between August 2007 and April 2008. MATERIALS： Human fetal brain tissue was obtained from an 8-week-old aborted fetus; serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium was provided by Gibco, USA; scanning electron microscope was provided by Hitachi Instruments, Japan; transmission electron microscope was provided by JEOL, Japan. METHODS： NSPCs were isolated from human fetal brain tissue and cultivated in serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium. Cells were passaged every 5-7 days. After three passages, NSPCs were harvested and used for ultrastructural examination. MAIN OUTCOME MEASURES： Ultrastructural examination of human NSPCs and adjacent cells in neurospheres. RESULTS： Individual NSPCs were visible as spherical morphologies with rough surfaces under scanning electron microscope. Generally, they had large nuclei and little cytoplasm. Nuclei were frequently globular with large amounts of euchromatin and a small quantity of heterochromatin, and most NSPCs had only one nucleolus. The Golgi apparatus and endoplasmic reticulum were underdeveloped; however, autophagosomes were clearly visible. The neurospheres were made up of NSPCs and non-fixiform material inside. Between adjacent cells and at the cytoplasmic surface of apposed plasma membranes, there were vesicle-like structures. Some membrane boundaries with high permeabilities were observed between some contiguous NSPCs in neurospheres, possibly attributable to plasmalemmal fusion between adjacent cells. CONCLUSION： A large number of autophagosomes were observed in NSPCs and gap junctions were visible between adjacent NSPCs.

Skeletal muscle generated from induced pluripotent stem cells-induction and application

Human induced pluripotent stem cells(hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patientiPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle diseaseiPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage:(1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease;(2) setting up a highthroughput drug screening system; and(3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.

Neuron-specific enolase expression in a rat model of radiation-induced brain injury following vascular endothelial growth factor-modified neural stem cell transplantation

BACKGROUND： Previous studies have shown that transplantation of vascular endothelial growth factor （VEGF）-modified neural stem cells （NSC） provides better outcomes, compared with neural stem cells, in the treatment of brain damage. OBJECTIVE： To compare the effects of VEGF-modified NSC transplantation and NSC transplantation on radiation-induced brain injury, and to determine neuron-specific enolase （NSE） expression in the brain. DESIGN, TIME, AND SETTING： The randomized, controlled study was performed at the Linbaixin Experimental Center, Second Affiliated Hospital, Sun Yat-sen University, China from November 2007 to October 2008. MATERIALS： VEGF-modified C17.2 NSCs were supplied by Harvard Medical School, USA. Streptavidin-biotin-peroxidase-complex kit （Boster, China） and 5, 6-carboxyfluorescein diacetate succinimidyl ester （Fluka, USA） were used in this study. METHODS： A total of 84 Sprague Dawley rats were randomly assigned to a blank control group （n = 20）, model group （n = 20）, NSC group （n = 20）, and a VEGF-modified NSC group （n = 24）. Rat models of radiation-induced brain injury were established in the model, NSC, and VEGF-modified NSC groups. At 1 week following model induction, 10 pL （5 ×10^4 cells/μL） VEGF-modified NSCs or NSCs were respectively infused into the striatum and cerebral cortex of rats from the VEGF-modified NSC and NSC groups. A total of 10μL saline was injected into rats from the blank control and model groups. MAIN OUTCOME MEASURES： NSE expression in the brain was detected by immunohistochemistry following VEGF-modified NSC transplantation. RESULTS： NSE expression was significantly decreased in the brains of radiation-induced brain injury rats （P 〈 0.05）. The number of NSE-positive neurons significantly increased in the NSC and VEGF-modified NSC groups, compared with the model group （P 〈 0.05）. NSE expression significantly increased in the VEGF-modified NSC group, compared with the NSC group, at 6 weeks following transplantation （P 〈 0.05）. CONCLUSION： VEGF-modified NSC transplantation increased NSE expression in rats with radiation-induced brain injury, and the outcomes were superior to NSC transplantation.