Effects of all-trans and 9-cis retinoic acid on differentiating human neural stem cells in vitro.

Cyanobacterial blooms are known sources of environmentally-occurring retinoid compounds, including all-trans and 9-cis retinoic acids (RAs). The developmental hazard for aquatic organisms has been described, while the implications for human health hazard assessment are not yet sufficiently characterized. Here, we employ a human neural stem cell model that can differentiate in vitro into a mixed culture of neurons and glia. Cells were exposed to non-cytotoxic 8-1000 nM all-trans or 9-cis RA for 9-18 days (DIV13 and DIV22, respectively). Impact on biomarkers was analyzed on gene expression (RT-qPCR) and protein level (western blot and proteomics) at both time points; network patterning (immunofluorescence) on DIV22. RA exposure significantly concentration-dependently increased gene expression of retinoic acid receptors and the metabolizing enzyme CYP26A1, confirming the chemical-specific response of the model. Expression of thyroid hormone signaling-related genes remained mostly unchanged. Markers of neural progenitors/stem cells (PAX6, SOX1, SOX2, NESTIN) were decreased with increasing RA concentrations, though a basal population remained. Neural markers (DCX, TUJ1, MAP2, NeuN, SYP) remained unchanged or were decreased at high concentrations (200-1000 nM). Conversely, (astro-)glial marker S100ß was increased concentration-dependently on DIV22. Together, the biomarker analysis indicates an RA-dependent promotion of glial cell fates over neural differentiation, despite the increased abundance of neural protein biomarkers during differentiation. Interestingly, RA exposure induced substantial changes to the cell culture morphology: while low concentrations resulted in a network-like differentiation pattern, high concentrations (200-1000 nM RA) almost completely prevented such network patterning. After functional confirmation for implications in network function, such morphological features could present a proxy for network formation assessment, an apical key event in (neuro-)developmental Adverse Outcome Pathways. The described application of a human in vitro model for (developmental) neurotoxicity to emerging environmentally-relevant retinoids contributes to the evidence-base for the use of differentiating human in vitro models for human health hazard and risk assessment.

Acute IL-6 exposure triggers canonical IL6Ra signaling in hiPSC microglia, but not neural progenitor cells.

BACKGROUND: Prenatal exposure to elevated interleukin (IL)-6 levels is associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ), autism spectrum condition (ASC) and bipolar disorder (BD). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterized the response of human induced pluripotent stem cell (hiPSC-)derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. RESULTS: We observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both protein and transcript levels due to the absence of IL6R expression and soluble (s)IL6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL6, JMJD3 and IL10 expression in MGL, confirming activation of canonical IL6Ra signaling. Bulk RNAseq identified 156 up-regulated genes (FDR < 0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR, which significantly overlapped with an up-regulated gene set from human post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility, consistent with gene ontology pathways highlighted from the RNAseq data and replicating rodent model indications that IRF8 regulates microglial motility. Finally, IL-6 induces MGLs to secrete CCL1, CXCL1, MIP-1α/ß, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 after 3 h and 24 h. CONCLUSION: Our data provide evidence for cell specific effects of acute IL-6 exposure in a human model system, ultimately suggesting that microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro.

Investigation of Naoluoxintong on the neural stem cells by facilitating proliferation and differentiation in vitro and on protecting neurons by up-regulating the expression of nestin in MCAO rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The classic traditional Chinese compound Naoluoxintong (NLXT) has been proven an effective remedy for ischemic stroke (IS). The protective effect of NLXT on neural stem cells (NSCs), however, remains unclear. AIM OF THE STUDY: To investigate the protective effect of NLXT on NSCs in rats with middle cerebral artery occlusion (MCAO) and the effect of Nestin expression in vivo. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: the sham-operated group, the MCAO model group and the NLXT group. The MCAO model in rats was established by modified Longa wire embolization method. The sham-operated group, the model group and the NLXT groups were divided into three subgroups according to the sampling time points of 1 d, 3 d and 7 d after successful model-making. Immunofluorescence staining, including bromodeoxyuridine (BrdU)/glial fibrillary acidic protein (GFAP), ß-tubulinIII/GFAP, BrdU/doublecortin (DCX) and BrdU/neuronal nuclei (NeuN), was used to detect the proliferation and survival of NSCs in the hippocampal after drug administration. Protein expression of Nestin, DCX, GFAP and NeuN in the hippocampal was detected by Western blot (WB). RESULTS: Immunofluorescence experiment of Nestin labeled: on the first day, a few Nestin-positive cells were found in the hippocampal DG area. Afterwards, the number of Nestin-labeled positive cells in the model group increased, while the number of cells in the sham group did not fluctuate significantly. The number of positive cells in each administration group increased more than that in the model and normal group. ß-tubulin III/GFAP double-labeled: a small amount of double labeled cells was expressed in the normal group, and the number subsequently fluctuated little. In the model group, ß-tubulin III/GFAP positive cells increased initially after acute ischemia, and gradually decreased afterwards. In the NLXT-treated group, ß-Tubulin III positive cells were significantly increased on day 1, 3 and 7, while GFAP positive cells had little change. BrdU/DCX double-labeled: initially, a small number of BrdU/DCX-labeled positive cells were observed in the normal group and the model group, but there was no increasing trend over time. The positive cells in the NLXT group increased over time, and those in the seven-day group were significantly higher than those in the one-day and three-day groups. BrdU/NEUN double-labeled: in the normal group, BrdU/NEUN positive cells were enriched and distributed regularly. The number of positive cells in the model group was small and decreased gradually with time, and the decrease was most obvious on the third day. The number of positive cells in the NLXT group was significantly higher than that in the model group, and the number of positive cells in the seven-day group was significantly higher than that in the one-day and three-day groups. WB results reflected those three proteins, Nestin, NeuN and DCX, showed an increase in expression, except GFAP, which showed a decreasing trend. CONCLUSIONS: Preliminarily, NLXT can promote the migration and differentiation of NSCs. It may have a protective effect on the brain by promoting repair of brain tissue damage through upregulation of Nestin after IS.

Adenosine A2A receptor blockade prevents cisplatin-induced impairments in neurogenesis and cognitive function.

Chemotherapy-induced cognitive impairment (CICI) has emerged as a significant medical problem without therapeutic options. Using the platinum-based chemotherapy cisplatin to model CICI, we revealed robust elevations in the adenosine A2A receptor (A2AR) and its downstream effectors, cAMP and CREB, by cisplatin in the adult mouse hippocampus, a critical brain structure for learning and memory. Notably, A2AR inhibition by the Food and Drug Administration-approved A2AR antagonist KW-6002 prevented cisplatin-induced impairments in neural progenitor proliferation and dendrite morphogenesis of adult-born neurons, while improving memory and anxiety-like behavior, without affecting tumor growth or cisplatin's antitumor activity. Collectively, our study identifies A2AR signaling as a key pathway that can be therapeutically targeted to prevent cisplatin-induced cognitive impairments.

The calcium channel inhibitor lacidipine inhibits Zika virus replication in neural progenitor cells.

After decades of being considered non-pathogenic, Zika virus (ZIKV) emerged as an important threat to human health during the epidemic of 2015-2016. ZIKV infections are usually asymptomatic, but can cause Guillain-Barré syndrome in adults and microcephaly in newborns. As there are currently no approved antiviral drugs against ZIKV, we tested anti-ZIKV activity of compounds from the NIH Clinical Collection for which we previously showed antiviral activity against the related dengue virus. One of the top hits from the screen was lacidipine, a 1,4-dihydropyridine calcium antagonist that is approved as an antihypertensive drug. Our data show that lacidipine is antiviral against ZIKV (strain H/PF/2013) in both Vero cells and induced pluripotent stem cell (iPSC)-derived human neural progenitor cells with IC50 values of 3.0 µM and <50 nM, respectively. The antiviral effect was also observed against four other ZIKV strains from the African and Asian lineages. Time-of-addition and replicon assays indicated that lacidipine acts at the post-entry stage of the viral replication cycle, inhibiting viral genome replication. Lacidipine altered the subcellular distribution of free cholesterol and neutral lipids, suggesting that the antiviral effect of lacidipine is mediated by altered trafficking of lipids. Together, these results identify lacidipine as a novel inhibitor of ZIKV replication that likely disturbs trafficking of lipids needed for replication organelle formation.

Astragalus Flavone Induces Proliferation and Differentiation of Neural Stem Cells in a Cerebral Infarction Model.

BACKGROUND Ischemic cerebrovascular disease leads to the activation and differentiation of neural stem cells (NSCs) into mature neurons and glia cells to repair nerve damage. Astragalus flavone (ASF) has shown its potential role in proliferation and differentiation into dopamine neurons of NSCs. MATERIAL AND METHODS Cerebral infarction models were constructed to determine the effects of ASF on NSCs in vivo and in vitro. RESULTS ASF therapy had the ability to reduce the neurologic function scores and the cerebral infarction volume of the cerebral infarction model. Moreover, ASF was able to increase BrdU-positive cells and promote the expression of Nestin, ß-Tubulin III, and O4, while decreasing the expression of GFAP. qRT-PCR and western blot assays showed ASF promoted the expression of Mash1, Math1, and Ngn2 mRNA and protein in cerebral infarction rats. Meanwhile, ASF (20 µg/ml) was able to increase EdU-positive cells and promote the expression of Nestin, ß-Tubulin III, and O4 of NSCs at day14 in vitro. In normoxia, ASF obviously promoted the expression of Mash1, Ngn1, and Ngn2 mRNA and proteins, but in hypoxia, ASF promoted the expression of Notch1 and Math1 mRNA and proteins and inhibited the expression of Ngn1 and Ngn2 mRNA and proteins. CONCLUSIONS ASF therapy can improve the neurologic functions and reduce the cerebral infarction volume in a cerebral infarction model. Moreover, ASF promoted the proliferation of NSCs and induced differentiation into neurons and oligodendrocytes, which might be involved in regulating factors in Notch signaling.

Assessing Gq-GPCR-induced human astrocyte reactivity using bioengineered neural organoids.

Astrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor-driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury.

Scaffold materials, neurotrophic factors, and seed cells are three elements of neural tissue engineering. As well-known self-assembling peptide-based hydrogels, RADA16-I and modified peptides are attractive matrices for neural tissue engineering. In addition to its neuroprotective effects, cerebral dopamine neurotrophic factor (CDNF) has been reported to promote the proliferation, migration, and differentiation of neural stem cells (NSCs). However, the role of RADA16-I combined with CDNF on NSCs remains unknown. First, the effect of RADA16-I hydrogel and CDNF on the proliferation and differentiation of cultured NSCs was investigated. Next, RADA16-I hydrogel and CDNF were microinjected into the lateral ventricle (LV) of middle cerebral artery occlusion (MCAO) rats to activate endogenous NSCs. CDNF promoted the proliferation of NSCs, while RADA16-I induced the neural differentiation of NSCs in vitro. Importantly, both RADA16-I and CDNF promoted the proliferation, migration, and differentiation of endogenous NSCs by activating the ERK1/2 and STAT3 pathways, and CDNF exerted an obvious neuroprotective effect on brain ischemia-reperfusion injury. These findings provide new information regarding the application of the scaffold material RADA16-I hydrogel and the neurotrophic factor CDNF in neural tissue engineering and suggest that RADA16-I hydrogel and CDNF microinjection may represent a novel therapeutic strategy for the treatment of stroke.

Conversion of Human Fibroblasts into Induced Neural Stem Cells by Small Molecules.

Induced neural stem cells (iNSCs) reprogrammed from somatic cells hold great potentials for drug discovery, disease modelling and the treatment of neurological diseases. Although studies have shown that human somatic cells can be converted into iNSCs by introducing transcription factors, these iNSCs are unlikely to be used for clinical application due to the safety concern of using exogenous genes and viral transduction vectors. Here, we report the successful conversion of human fibroblasts into iNSCs using a cocktail of small molecules. Furthermore, our results demonstrate that these human iNSCs (hiNSCs) have similar gene expression profiles to bona fide NSCs, can proliferate, and are capable of differentiating into glial cells and functional neurons. This study collectively describes a novel approach based on small molecules to produce hiNSCs from human fibroblasts, which may be useful for both research and therapeutic purposes.

Effect of Progranulin on Proliferation and Differentiation of Neural Stem/Progenitor Cells after Oxygen/Glucose Deprivation.

We previously demonstrated that sivelestat, a selective neutrophil elastase inhibitor, attenuates the cleavage of progranulin (PGRN) and ischemia-induced cell injury in the brain. To obtain further insight into the role of PGRN, in the present study we evaluated the direct effects of sivelestat and recombinant PGRN (rPGRN) on the proliferation and differentiation of neural stem cells in cultures of neural stem/progenitor cells (NS/PC) under the ischemic condition in vitro. We demonstrated that oxygen/glucose deprivation (OGD)-induced cell proliferation of NS/PC was increased by rPGRN treatment. In addition, this increase was accompanied by increased phosphorylation of Akt and GSK-3ß (Ser9) after OGD. But none of these responses occurred by treatment with sivelestat. Therefore, activation of the Akt/GSK-3ß pathway could well be involved in this proliferative effect of rPGRN. Although OGD and reoxygenation-induced changes in the differentiation of NS/PC into neurons or astrocytes was not affected by treatment with rPGRN or sivelestat, it is noteworthy that rPGRN enhanced neurite outgrowth of ß3-tubulin-positive neurons that had differentiated from the NS/PC. These findings suggest that enhancement of proliferation of endogenous NS/PC and neurite outgrowth of differentiated neurons from NS/PC by PGRN could be useful for a new therapeutic approach for cerebral ischemia.

Modified Industrial Three-Dimensional Polylactic Acid Scaffold Cell Chip Promotes the Proliferation and Differentiation of Human Neural Stem Cells.

In this study, we fabricated a three-dimensional (3D) scaffold using industrial polylactic acid (PLA), which promoted the proliferation and differentiation of human neural stem cells. An industrial PLA 3D scaffold (IPTS) cell chip with a square-shaped pattern was fabricated via computer-aided design and printed using a fused deposition modeling technique. To improve cell adhesion and cell differentiation, we coated the IPTS cell chip with gold nanoparticles (Au-NPs), nerve growth factor (NGF) protein, an NGF peptide fragment, and sonic hedgehog (SHH) protein. The proliferation of F3.Olig2 neural stem cells was increased in the IPTS cell chips coated with Au-NPs and NGF peptide fragments when compared with that of the cells cultured on non-coated IPTS cell chips. Cells cultured on the IPTS-SHH cell chip also showed high expression of motor neuron cell-specific markers, such as HB9 and TUJ-1. Therefore, we suggest that the newly engineered industrial PLA scaffold is an innovative tool for cell proliferation and motor neuron differentiation.

Methylxanthines Induce a Change in the AD/Neurodegeneration-Linked Lipid Profile in Neuroblastoma Cells.

Alzheimer's disease (AD) is characterized by an increased plaque burden and tangle accumulation in the brain accompanied by extensive lipid alterations. Methylxanthines (MTXs) are alkaloids frequently consumed by dietary intake known to interfere with the molecular mechanisms leading to AD. Besides the fact that MTX consumption is associated with changes in triglycerides and cholesterol in serum and liver, little is known about the effect of MTXs on other lipid classes, which raises the question of whether MTX can alter lipids in a way that may be relevant in AD. Here we have analyzed naturally occurring MTXs caffeine, theobromine, theophylline, and the synthetic MTXs pentoxifylline and propentofylline also used as drugs in different neuroblastoma cell lines. Our results show that lipid alterations are not limited to triglycerides and cholesterol in the liver and serum, but also include changes in sphingomyelins, ceramides, phosphatidylcholine, and plasmalogens in neuroblastoma cells. These changes comprise alterations known to be beneficial, but also adverse effects regarding AD were observed. Our results give an additional perspective of the complex link between MTX and AD, and suggest combining MTX with a lipid-altering diet compensating the adverse effects of MTX rather than using MTX alone to prevent or treat AD.

Propofol induces the apoptosis of neural stem cells via microRNA-9-5p / chemokine CXC receptor 4 signaling pathway.

Recent studies suggested that propofol, one of the most widely used anesthetics, may cause neurotoxicity in the developing brain, leading to cognitive deficits in adults. However, the underlying mechanisms remain unclear. In this study, we aimed to evaluate the mechanisms of propofol neurotoxicity in the neural stem cells (NSCs). The mRNA and protein expression levels of microRNA-9-5p (miR-9-5p) and chemokine CXC receptor 4 (CXCR4) were determined by quantitative reverse transcription-polymerase chain reaction and Western blotting analyses. Cell viability and apoptosis were evaluated using the cell counting kit-8 and Hoechst staining kits. The levels of apoptosis-related proteins B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein, and caspase-3 were detected by Western blotting analysis. These results confirmed that propofol activated cell apoptosis in a dose-dependent manner. A significant increase in the miR-9-5p and CXCR4 expression was observed in the propofol-treated cells. The overexpression of miR-9-5p induced apoptosis in NSCs, accompanied by elevated apoptosis-related protein activity. Furthermore, mitigated CXCR4 expression reduced propofol-induced cell apoptosis. We conclude that propofol induces cell death in NSCs, and overexpression of miR-9-5p/CXCR4 contributes to propofol-induced cell apoptosis, which might be a target for developing novel strategies to treat propofol neurotoxicity.

iPS-derived neural stem cells for disease modeling and evaluation of therapeutics for mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare, lysosomal disorder caused by mutations in a gene encoding iduronate-2-sulfatase (IDS). IDS deficiency results in an accumulation of glycosaminoglycans (GAGs) and secondary accumulations of other lipids in lysosomes. Symptoms of MPS II include a variety of soft and hard tissue problems, developmental delay, and deterioration of multiple organs. Enzyme replacement therapy is an approved treatment for MPS II, but fails to improve neuronal symptoms. Cell-based neuronal models of MPS II disease are needed for compound screening and drug development for the treatment of the neuronal symptoms in MPS II. In this study, three induced pluripotent stem cell (iPSC) lines were generated from three MPS II patient-derived dermal fibroblast cell lines that were differentiated into neural stem cells and neurons. The disease phenotypes were measured using immunofluorescence staining and Nile red dye staining. In addition, the therapeutic effects of recombinant human IDS enzyme, delta-tocopherol (DT), and hydroxypropyl-beta-cyclodextrin (HPBCD) were determined in the MPS II disease cells. Finally, the neural stem cells from two of the MPS II iPSC lines exhibited typical disease features including a deficiency of IDS activity, abnormal glycosaminoglycan storage, and secondary lipid accumulation. Enzyme replacement therapy partially rescued the disease phenotypes in these cells. DT showed a significant effect in reducing the secondary accumulation of lipids in the MPS II neural stem cells. In contrast, HPBCD displayed limited or no effect in these cells. Our data indicate that these MPS II cells can be used as a cell-based disease model to study disease pathogenesis, evaluate drug efficacy, and screen compounds for drug development.

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1ß (IL-1ß), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

Fluoxetine regulates the neuronal differentiation of neural stem cells transplanted into rat brains after stroke by increasing the 5HT level.

Fluoxetine, a 5-HT uptake inhibitor, has been adopted for the treatment of post-stroke depression in recent years. It has been confirmed to induce neuronal regeneration in vivo, but its effect on inducing stem cell differentiation after transplantation has not yet been verified. To evaluate its regulatory effect on stem cell differentiation, fluoxetine was used in this study to treat rats with cerebral ischemia after neural stem cell (NSC) transplantation. The results showed that the proportion of NSCs differentiating into neurons significantly increased after fluoxetine treatment. In NSC adherent culture, the addition of 5-HT but not of fluoxetine significantly increased the neuronal differentiation ratio of NSCs. Moreover, the addition of 5-HT2A or 5-HT3A antagonists inhibited this effect. In addition, Western blotting revealed that the increase in 5-HT inhibited ERK2 phosphorylation and upregulated neurogenin1 expression. In conclusion, fluoxetine increased the 5-HT level and promoted neuronal differentiation, thereby upregulating neurogenin1 expression and downregulating ERK2 phosphorylation.

Jujuboside a promotes proliferation and neuronal differentiation of APPswe-overexpressing neural stem cells by activating Wnt/ß-catenin signaling pathway.

Mobilization of hippocampal neurogenesis has been considered as a potential strategy for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD). In present study, we evaluated both the neuroprotective effects and the effects on the proliferation and differentiation of APP-overexpressing neural stem cells (APP-NSCs) by Jujuboside A (JuA) in vitro. Our results demonstrated that JuA (50 µM) decreased apoptosis and suppressed oxidative stress damage of APP-NSCs. JuA (50 µM) upregulated the secretion of brain-derived neurotrophic factor and promoted the proliferation and neuronal differentiation of APP-NSCs. Moreover, JuA (50 µM) upregulated Wnt-3a and ß-catenin protein expression, and enhanced the expression of downstream genes Ccnd1, Neurod1 and Prox1. However, XAV-939, an inhibitor of the Wnt/ß-catenin signaling pathway, inhibited these positive effects of JuA. Taken together, these findings suggest that JuA promote proliferation and neuronal differentiation of APP-NSCs partly by activating the Wnt/ß-catenin signaling pathway. We hope that this study will provide a viable strategy for the treatment of AD.

Silk fibroin nanofibrous scaffolds incorporated with microRNA-222 loaded chitosan nanoparticles for enhanced neuronal differentiation of neural stem cells.

Neural stem cells (NSCs) transplantation therapy is a promising method for neural tissue regeneration. How to enhance the neuronal differentiation of NSCs has been the most challenging aspect of NSCs application. Herein, the microRNA-222 loaded chitosan nanoparticles (miR-222/CS NPs) were incorporated with silk fibroin (SF) nanofibrous scaffolds to enhance neuronal differentiation of NSCs. The encapsulation efficiency of miR-222 in the miR-222/CS NPs was (96.4 ± 0.3) %. The results of the electrophoretic assay and cellular uptake assay confirmed that miR-222 was stable in the miR-222/CS NPs and can be effectively delivered into NSCs. The water contact angle decreased from (89 ± 3.05)° for the SF scaffolds to (14 ± 1.00)° for the composite scaffolds. The Western blot and RT-PCR results confirmed that the composite scaffolds could enhance neuronal differentiation of NSCs. In conclusion, the SF nanofibrous scaffolds in combination with miR-222/CS NPs are a promising approach for neural tissue regeneration.

Modulation of gene expression on a transcriptome-wide level following human neural stem cell transplantation in aged mouse stroke brains.

INTRODUCTION: Neural stem cell (NSC) transplantation offers great potential for treating ischemic stroke. Clinically, ischemia followed by reperfusion results in robust cerebrovascular injury that upregulates proinflammatory factors, disrupts neurovascular units, and causes brain cell death. NSCs possess multiple actions that can be exploited for reducing the severity of neurovascular injury. Our previous studies in young adult mice showed that human NSC transplantation during the subacute stage diminishes stroke pathophysiology and improves behavioral outcome. METHODS: We employed a well-established and commonly used stroke model, middle cerebral artery occlusion with subsequent reperfusion (MCAO/R). Here, we assessed the outcomes of hNSC transplantation 48 h post-MCAO (24 h post-transplant) in aged mouse brains in response to stroke because aging is a crucial risk factor for cerebral ischemia. Next, we tested whether administration of the integrin α5ß1 inhibitor, ATN-161, prior to hNSC transplantation further affects stoke outcome as compared with NSCs alone. RNA sequencing (RNA-seq) was used to assess the impact of hNSC transplantation on differentially expressed genes (DEGs) on a transcriptome-wide level. RESULTS: Here, we report that hNSC-engrafted brains with or without ATN-161 showed significantly reduced infarct size, and attenuated the induction of proinflammatory factors and matrix metalloproteases. RNA-seq analysis revealed DEGs and molecular pathways by which hNSCs induce a beneficial post-stroke outcome in aged stroke brains. 811 genes were differentially expressed (651 downregulated and 160 upregulated) in hNSC-engrafted stroke brains. Functional pathway analysis identified enriched and depleted pathways in hNSC-engrafted aged mouse stroke brains. Depletion of pathways following hNSC-engraftment included signaling involving neuroinflammation, acute phase response, leukocyte extravasation, and phagosome formation. On the other hand, enrichment of pathways in hNSC-engrafted brains was associated with PPAR signaling, LXR/RXR activation, and inhibition of matrix metalloproteases. Hierarchical cluster analysis of DEGs in hNSC-engrafted brains indicate decreased expression of genes encoding TNF receptors, proinflammatory factors, apoptosis factors, adhesion and leukocyte extravasation, and Toll-like receptors. CONCLUSIONS: Our study is the first to show global transcripts differentially expressed following hNSC transplantation in the subacute phase of stroke in aged mice. The outcome of our transcriptome study would be useful to develop new therapies ameliorating early-stage stroke injury.