The Antioxidant Phytochemical Schisandrin A Promotes Neural Cell Proliferation and Differentiation after Ischemic Brain Injury

Schisandrin A (SCH) is a natural bioactive phytonutrient that belongs to the lignan derivatives found in Schisandra chinensis fruit. This study aims to investigate the impact of SCH on promoting neural progenitor cell (NPC) regeneration for avoiding stroke ischemic injury. The promoting effect of SCH on NPCs was evaluated by photothrombotic model, immunofluorescence, cell line culture of NPCs, and Western blot assay. The results showed that neuron-specific class III beta-tubulin (Tuj1) was positive with Map2 positive nerve fibers in the ischemic area after using SCH. In addition, Nestin and SOX2 positive NPCs were significantly (p < 0.05) increased in the penumbra and core. Further analysis identified that SCH can regulate the expression level of cell division control protein 42 (Cdc42). In conclusion, our findings suggest that SCH enhanced NPCs proliferation and differentiation possible by Cdc42 to regulated cytoskeletal rearrangement and polarization of cells, which provides new hope for the late recovery of stroke.     

Subdominant H60 antigen-specific CD8 T-cell response precedes dominant H4 antigen-specific response during the initial phase of allogenic skin graft rejection

In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.     

Induction of Nanog in neural progenitor cells for adaptive regeneration of ischemic brain

NANOG plays a key role in cellular plasticity and the acquisition of the stem cell state during reprogramming, but its role in the regenerative process remains unclear. Here, we show that the induction of NANOG in neuronal cells is necessary for the physiological initiation of neuronal regeneration in response to ischemic stress. Specifically, we found that NANOG was preferentially expressed in undifferentiated neuronal cells, and forced expression of Nanog in neural progenitor cells (NPCs) promoted their self-renewing expansion both in ex-vivo slice cultures and in vitro limiting dilution analysis. Notably, the upstream region of the Nanog gene contains sequence motifs for hypoxia-inducible factor-1 alpha (HIF-1α). Therefore, cerebral neurons exposed to hypoxia significantly upregulated NANOG expression selectively in primitive (CD133⁺) cells, but not in mature cells, leading to the expansion of NPCs. Notably, up to 80% of the neuronal expansion induced by hypoxia was attributed to NANOG-expressing neuronal cells, whereas knockdown during hypoxia abolished this expansion and was accompanied by the downregulation of other pluripotency-related genes. Moreover, the number of NANOG-expressing neuronal cells were transiently increased in response to ischemic insult, predominantly in the infarct area of brain regions undergoing neurogenesis, but not in non-neurogenic loci. Together, these findings reveal a functional effect of NANOG-induction for the initiation of adaptive neuronal regeneration among heterogeneous NPC subsets, pointing to cellular plasticity as a potential link between regeneration and reprogramming processes.Subject terms: Stem-cell research, Experimental models of disease     

Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model

Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model. Bone marrow-derived MSCs were transplanted into the brain parenchyma 3 days after induction of stroke by occluding middle cerebral artery for 2 h. Stoke induced proliferation of resident neural stem cells in subventricular zone. However, most of new born cells underwent cell death and had a limited impact on functional recovery after stroke. Transplantation of MSCs enhanced proliferation of endogenous neural stem cells while suppressing the cell death of newly generated cells. Thereby, newborn cells migrated toward ischemic territory and differentiated in ischemic boundaries into doublecortin+ neuroblasts at higher rates in animals with MSCs compared to control group. The present study indicates that therapeutic effects of MSCs are at least partly ascribed to dual functions of MSCs by enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. The results reinforce the prospects of clinical application using MSCs in the treatment of neurological disorders.     

Migration of human neural stem cells toward an intracranial glioma

Many in vivo and in vitro studies have demonstrated the targeted migration of neural stem cells (NSC) to infiltrating brain tumors, including malignant glioma, highlighting a potential therapeutic approach. However, there is not enough information to apply this approach to clinical therapy. The most important things in stem cell therapy for brain tumors involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. By histological analysis using two different live-dyes, human NSCs were shown to migrate away from the transplanted site in the direction of the expanding C6 glioma and to intermix with the tumor bed, especially with the tumor core. This intermixing occurred within 7 days when NSCs were implanted into glioma model. The time course of migratory HB1.F5 with the greatest mobility of three NSC lines was as follows. As early as 3 days after transplantation, several NSCs were found leaving the implant site, primarily approaching microsatellites and frontier cells located near the site of NSC implantation. Through 7 days post-transplantation, massive numbers of NSCs continued to be attracted to and interspersed with C6 glioma, and were finally distributed extensively throughout the whole tumor bed, including the core and penumbra of the tumor mass. However, NSCs appeared to penetrate into the tumor mass very well, whereas normal fibroblast cells could not migrate. These findings strengthen the potential for human NSCs as attractive vehicles to improve therapeutic gene delivery to cancer or glioma if they are optimized to selectively kill neoplastic cells.     

Neuroprotective effects of Tilia americana var. mexicana on damage induced by cerebral ischaemia in mice

Tilia americana var. mexicana (T. americana) is a plant widely used in Mexico for its medicinal properties on the central nervous system. In the present study, we designed a protocol to investigate the neuroprotective effects of non-polar and polar extracts of T. americana on damage induced by cerebral ischaemia in mice. Vehicle or extracts were administered immediately after ischaemia. Functional neurological deficit, survival percentage and infarct area were determined in each experimental group. Results showed that groups treated with non-polar or polar extracts of T. americana had increased survival rate, improved neurological deficits and diminished the infarct area in relation to the ischaemic group. In conclusion, this study confirms the neuroprotective activity of T. americana, suggests a possible synergism between non-polar and polar constituents and supports its potential as a useful aid in the clinical management of stroke.     

Hydrogel-mediated drug delivery for treating stroke

Stroke is a common disease and is the major cause of death and disability. It occurs and generates devastating neurological deficits when cerebral blood vessel is blocked(ischemic stroke, IS) or ruptured(hemorrhagic stroke, HS). Hydrogel, being biodegradable and biocompatible, have shown attractive advantages in stroke therapy as a new biomaterial with desirable mechanical properties and tunability of structure,owing to special ability to load different cargoes for multiple treatment strategies,...     

Neural cell adhesion molecule (NCAM) induces neuronal phenotype acquisition in dominant negative MEK1-expressing hippocampal neural progenitor cells

It has been shown that neural cell adhesion molecule (NCAM)-induced neuronal differentiation is extracellular signal-regulated kinase (ERK)-dependent. However, an involvement of the mitogen activated protein kinase (MAPK) kinase (MEK), an upstream kinase of ERK, has not been directly demonstrated in this process. Therefore, we investigated whether the MEK1 plays a critical role in the NCAM-induced neuronal differentiation of hippocampal neural progenitor cells (NPCs). NPCs were transiently transfected with expression plasmids encoding activated or dominant negative (DN) forms of MEK1. The expression of DN MEK1 inhibited neuronal phenotype acquisition and soluble NCAM rescued the defect in the neuronal phenotype acquisition in DN-MEK1-transfected cells, suggesting that NCAM might contribute to the neuronal differentiation via distinct, parallel pathways including the MEK pathway. In cells expressing wild type MEK1 or constitutively active MEK1 on the other hand, the percentage of cells positive for beta-tubulin type III (Tuj1), a marker for early postmitotic neurons, was higher than seen in vector-transfected cells. These results suggest that the activation of MEK1 is required for obtaining neuronal phenotype in NPCs.     

Size‐Controlled Construction of Magnetic Nanoparticle Clusters Using DNA‐Binding Zinc Finger Protein

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well‐defined and size‐controllable manner remains a challenge. Here we demonstrate a strategy to construct size‐controlled NPCs using the DNA‐binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin‐conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size‐controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three‐fold increase in the spin–spin relaxivity in MRI compared with Feridex. Furthermore, folate‐conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes.     

Oil phase evaporation-induced self-assembly of hydrophobic nanoparticles into spherical clusters with controlled surface chemistry in an oil-in-water dispersion and comparison of behaviors of individual and clustered iron oxide nanoparticles

We report a general method for preparing nanoparticle clusters (NPCs) in an oil-in-water emulsion system mediated by cetyl trimethylammonium bromide (CTAB), where previously only individual nanoparticles were obtained. NPCs of magnetic, metallic, and semiconductor nanoparticles have been prepared to demonstrate the generality of the method. The NPCs were spherical and composed of densely packed individual nanoparticles. The number density of nanoparticles in the oil phase was found to be critical for the formation, morphology, and yield of NPCs. The method developed here is scalable and can produce NPCs in nearly 100% yield at a concentration of 5 mg/mL in water, which is approximately 5 times higher than the highest value reported in the literature. The surface chemistry of NPCs can also be controlled by replacing CTAB with polymers containing different functional groups via a similar procedure. The reproducible production of NPCs with well-defined shapes has allowed us to compare the properties of individual and clustered iron oxide nanoparticles, including magnetization, magnetic moments, and contrast enhancement in magnetic resonance imaging (MRI). We found that, due to their collective properties, NPCs are more responsive to an external magnetic field and can potentially serve as better contrast enhancement agents than individually dispersed magnetic NPs in MRI.     

Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats

Retinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.Subject terms: Translational research, Stem-cell research, Retina     

Monitoring of Altered Free Fatty Acid Metabolic Patterns in Rat Plasma Following Hemorrhagic Stroke

To uncover the physiological changes occurring after hemorrhagic stroke, we analyzed 24 free fatty acids in plasma as tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry from control and intracerebral hemorrhage rats. Hematoma volume decreased, and hemorrhage-induced behavioral abnormalities recovered overtime. Altered fatty acid metabolism at 7 days after hemorrhagic stroke spontaneously recovered to normal control levels at 14 days. The deformed star patterns of hemorrhagic stroke group were readily distinguished from the tetracosagonal shape of the control mean. Thus, the present method may be used for monitoring of biochemical and physiological events in hemorrhagic stroke.     

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 μg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.     

干细胞迁移机理的近场扫描光学显微术研究

将内皮细胞生长因子(VEGF)置于甲基纤维素碟中形成VEGF的浓度梯度分布,并将人脐带间充质干细胞(Mesenchymal stem cells,MSCs)于此浓度梯度中培养,观察VEGF能否诱导MSCs定向迁移。应用近场扫描光学显微术(Near-field scanning optical microscopy,NSOM)同时获取了VEGF诱导前后的MSCs的形貌和光学信息。结果表明,近场光学图观测到形貌图上所没有的黑色斑点,分析认为这些黑斑为细胞的黏着斑。近场光学图显示经过VEGF诱导后细胞的黏着斑数量明显增加。同时,对诱导前后干细胞的骨架蛋白进行免疫荧光标记并用共聚焦显微镜进行观察,结果表明细胞骨架由诱导前的无序状态转变为诱导后的有序状态,说明诱导后的干细胞处于迁移状态。光学超微结构图则显示了诱导后干细胞表面的光学细节比诱导前细胞大量增加,出现了大量直径约200 nm的光斑,这是由于细胞表面大量分泌黏附分子等蛋白分子引起的,这些结果为VEGF能够诱导MSCs进行定向迁移提供了实验依据和可视化证明。也表明NSOM不但能提供高分辨的光学分辨率,还可提供生物细胞精细结构的更深层次的光学信息。     

Characterization of a brain tumor cell line established from transgenic mice expressing the vasopressin SV-40 T antigen

We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.     

超声双重造影及MRI对胃癌诊断及术前T分期的对比研究

选取2017年8月~2019年2月我院收治的胃癌患者130例作为研究对象,依据患者病理诊断结果作为金标准,分析磁共振成像(magnetic resonance imaging,MRI)与超声双重造影对胃癌的诊断价值及术前T分期的价值。结果显示,超声双重造影共检出120例胃癌患者,出现10例漏诊,MRI共检出109例胃癌患者,出现21例漏诊。金标准共检出52例T1期、32例T2期、26例T3期、20例T4期患者,超声双重造影诊断出T1期48例,符合率92.31%;T2期30例,符合率93.75%;T3期24例,符合率92.31%;T4期18例,符合率90.00%,进一步分析显示,超声双重造影对不同胃癌T分期的诊断价值均高于MRI。因此,超声双重造影对胃癌的诊断价值及对术前T分期诊断价值均高于MRI。     

Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices

Immune cell migration is a fundamental process that enables immunosurveillance and immune responses. Understanding the mechanism of immune cell migration is not only of importance to the biology of cells, but also has high relevance to cell trafficking mediated physiological processes and diseases such as embryogenesis, wound healing, autoimmune diseases and cancers. In addition to the well-known chemical concentration gradient based guiding mechanism (i.e. chemotaxis), recent studies have shown that lymphocytes can respond to applied physiologically relevant direct current (DC) electric fields by migrating toward the cathode of the fields (i.e. electrotaxis) in both in vitro and in vivo settings. In the present study, we employed two microfluidic devices allowing controlled application of electric fields inside the microfluidic channel for quantitative studies of lymphocyte electrotaxis in vitro at the single cell level. The first device is fabricated by soft-lithography and the second device is made in glass with integrated on-chip electrodes. Using both devices, we for the first time showed that anti-CD3/CD28 antibodies activated human blood T cells migrate to the cathode of the applied DC electric field. This finding is consistent with previous electrotaxis studies on other lymphocyte subsets suggesting electrotaxis is a novel guiding mechanism for immune cell migration. Furthermore, the characteristics of electrotaxis and chemotaxis of activated T cells in PDMS microfluidic devices are compared.     

Ce(III)在辣根体中的迁移

应用放射性同位素示踪法, 即结合放射自显影和液体闪烁器检测及电镜放射自显影技术, 首次定性、定量地研究Ce(III)离子在辣根植物中的迁移. 结果表明: (1) Ce(III)离子能被辣根植物吸收并随时间发生迁移; (2)电镜放射自显影结果显示, Ce(III)离子迁移是通过质外体至共质体进入细胞的. Ce(III)离子在辣根植物体中的行为, 为稀土离子植物环境效应机理及辣根植物安全防护提供借鉴.     

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.