Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium

Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K⁺ currents (I_to), at holding potential of −80 mV, Na⁺ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.     

Human Milk Oligosaccharide 2′-Fucosyllactose Induces Neuroprotection from Intracerebral Hemorrhage Stroke

Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFβ) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.     

The Characterization of a Subependymal Giant Astrocytoma-Like Cell Line from Murine Astrocyte with mTORC1 Hyperactivation

Tuberous sclerosis complex (TSC) is a genetic disorder caused by inactivating mutations in TSC1 (hamartin) or TSC2 (tuberin), crucial negative regulators of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. TSC affects multiple organs including the brain. The neurologic manifestation is characterized by cortical tubers, subependymal nodules (SEN), and subependymal giant cell astrocytoma (SEGA) in brain. SEGAs may result in hydrocephalus in TSC patients and mTORC1 inhibitors are the current recommended therapy for SEGA. Nevertheless, a major limitation in the research for SEGA is the lack of cell lines or animal models for mechanistic investigations and development of novel therapy. In this study, we generated TSC1-deficient neural cells from spontaneously immortalized mouse astrocytes in an attempt to mimic human SEGA. The TSC1-deficient cells exhibit mTORC1 hyperactivation and characteristics of transition from astrocytes to neural stem/progenitor cell phenotypes. Rapamycin efficiently decreased mTORC1 activity of these TSC1-deficient cells in vitro. In vivo, TSC1-deficient cells could form SEGA-like tumors and Rapamycin treatment decreased tumor growth. Collectively, our study generates a novel SEGA-like cell line that is invaluable for studying mTORC1-driven molecular and pathological alterations in neurologic tissue. These SEGA-like cells also provide opportunities for the development of novel therapeutic strategy for TSC patients with SEGA.     

Molecular Markers of Adult Neurogenesis in the Telencephalon and Tectum of Rainbow Trout,Oncorhynchus mykiss

In the brain of teleost fish, radial glial cells are the major type of astroglial cells. To answer the question as to how radial glia structures adapt to the continuous growth of the brain, which is characteristic of salmonids, it is necessary to study various types of cells (neuronal precursors, astroglial cells, and cells in a state of neuronal differentiation) in the major integrative centers of the salmon brain (telencephalon and tectum opticum), using rainbow trout, Oncorhynchus mykiss, as a model. A study of the distribution of several molecular markers in the telencephalon and tectum with the identification of neural stem/progenitor cells, neuroblasts, and radial glia was carried out on juvenile (three-year-old) O. mykiss. The presence of all of these cell types provides specific conditions for the adult neurogenesis processes in the trout telencephalon and tectum. The distribution of glutamine synthetase, a molecular marker of neural stem cells, in the trout telencephalon revealed a large population of radial glia (RG) corresponding to adult-type neural stem cells (NSCs). RG dominated the pallial region of the telencephalon, while, in the subpallial region, RG was found in the lateral and ventral zones. In the optic tectum, RG fibers were widespread and localized both in the marginal layer and in the periventricular gray layer. Doublecortin (DC) immunolabeling revealed a large population of neuroblasts formed in the postembryonic period, which is indicative of intense adult neurogenesis in the trout brain. The pallial and subpallial regions of the telencephalon contained numerous DC+ cells and their clusters. In the tectum, DC+ cells were found not only in the stratum griseum periventriculare (SGP) and longitudinal torus (TL) containing proliferating cells, but also in the layers containing differentiated neurons: the central gray layer, the periventricular gray and white layers, and the superficial white layer. A study of the localization patterns of vimentin and nestin in the trout telencephalon and tectum showed the presence of neuroepithelial neural stem cells (eNSCs) and ependymoglial cells in the periventricular matrix zones of the brain. The presence of vimentin and nestin in the functionally heterogeneous cell types of adult trout indicates new functional properties of these proteins and their heterogeneous involvement in intracellular motility and adult neurogenesis. Investigation into the later stages of neuronal development in various regions of the fish brain can substantially elucidate the major mechanisms of adult neurogenesis, but it can also contribute to understanding the patterns of formation of certain brain regions and the involvement of RG in the construction of the definite brain structure.     

Physiological Electric Field: A Potential Construction Regulator of Human Brain Organoids

Brain organoids can reproduce the regional three-dimensional (3D) tissue structure of human brains, following the in vivo developmental trajectory at the cellular level; therefore, they are considered to present one of the best brain simulation model systems. By briefly summarizing the latest research concerning brain organoid construction methods, the basic principles, and challenges, this review intends to identify the potential role of the physiological electric field (EF) in the construction of brain organoids because of its important regulatory function in neurogenesis. EFs could initiate neural tissue formation, inducing the neuronal differentiation of NSCs, both of which capabilities make it an important element of the in vitro construction of brain organoids. More importantly, by adjusting the stimulation protocol and special/temporal distributions of EFs, neural organoids might be created following a predesigned 3D framework, particularly a specific neural network, because this promotes the orderly growth of neural processes, coordinate neuronal migration and maturation, and stimulate synapse and myelin sheath formation. Thus, the application of EF for constructing brain organoids in a3D matrix could be a promising future direction in neural tissue engineering.     

体外诱导微环境对骨髓间充质干细胞成神经分化的影响及分化后的自发逆转现象

目的体外定向诱导大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)成神经分化,并探讨诱导微环境对其分化的影响及分化后的自发逆转现象。方法体外分离培养大鼠MSCs,流式细胞仪检测细胞表面标志。采用改良神经元诱导液[Modified neuronal induction media(MNM)]定向诱导MSCs,免疫荧光检测神经细胞表面标志。观察胎牛血清(FBS)浓度、细胞密度、MNM剂量、新鲜与使用过的MNM等不同诱导微环境对MSCs成神经分化的影响。结果 MSCs经MNM诱导后,6h即可见尼氏体,表达神经元特异性表面标志神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)和微管相关蛋白-2(MAP-2)。随着诱导微环境的改变,MSCs成神经分化率及神经元表面标志表达亦发生改变,且分化后的神经元样细胞可自发逆转。结论 MSCs能够在MNM微环境中定向成神经分化,但诱导微环境的改变可以从量和质两个层面影响MSCs定向分化。     

Effect of graphene oxide on undifferentiated and retinoic acid-differentiated SH-SY5Y cells line

Graphene oxide (GO), has created an unprecedented opportunity for development and application in biology, due to its abundant functional groups and well water solubility. Recently, the potential toxicity of GO in the environment and in humans has garnered more and more attention. In this paper, we systematically studied the cytotoxicity of GO nanosheets via examining the effect of GO on the morphology, viability and differentiation of a human neuroblastoma SH-SY5Y cell line, which was an ideal model used to study neuronal disease in vitro. The results suggested that GO had no obvious cytotoxicity at low concentration (<80 μg mL(-1)) for 96 h, but the viability of cells exhibited dose- and time-dependent decreases at high concentration (≥ 80 μg mL(-1)). Moreover, GO did not induce apoptosis. Very interestingly, GO significantly enhanced the differentiation of SH-SY5Y induced-retinoic acid (RA) by evaluating neurite length and the expression of neuronal marker MAP2. These data provide a promising application for neurodegenerative diseases.     

Isolation of a Pluripotent Neural Stem Cell from the Embryonic Bovine Brain

We recently isolated stem cells derived from the brain of a bovine fetus, utilizing a particular mechanical separation method. After improving our experimental conditions, we obtained neural stem cells using an optimized culture medium system. The cells were expanded, established in continuous cell culture and used for immunofluorescence cytochemistry. RT-PCR showed that embryonic neural stem cells (NSCs) not only expresses the protein Sox2, Nestin but also Pax6, Musashi proteins and were differentiated into the three classical neuronal phenotypes (neurons, astrocytes, and oligodendrocytes).     

PSA Depletion Induces the Differentiation of Immature Neurons in the Piriform Cortex of Adult Mice

Immature neurons are maintained in cortical regions of the adult mammalian brain. In rodents, many of these immature neurons can be identified in the piriform cortex based on their high expression of early neuronal markers, such as doublecortin (DCX) and the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). This molecule plays critical roles in different neurodevelopmental events. Taking advantage of a DCX-CreERT2/Flox-EGFP reporter mice, we investigated the impact of targeted PSA enzymatic depletion in the piriform cortex on the fate of immature neurons. We report here that the removal of PSA accelerated the final development of immature neurons. This was revealed by a higher frequency of NeuN expression, an increase in the number of cells carrying an axon initial segment (AIS), and an increase in the number of dendrites and dendritic spines on the immature neurons. Taken together, our results demonstrated the crucial role of the PSA moiety in the protracted development of immature neurons residing outside of the neurogenic niches. More studies will be required to understand the intrinsic and extrinsic factors affecting PSA-NCAM expression to understand how the brain regulates the incorporation of these immature neurons to the established neuronal circuits of the adult brain.     

Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFβ signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.     

稳定表达绿色荧光蛋白标记的人单纯疱疹病毒2型潜伏相关转录体ORF2融合蛋白的Vero细胞株的建立

目的建立稳定表达绿色荧光蛋白(EGFP)标记的人单纯疱疹病毒2型(HSV-2)潜伏相关转录体(LAT)开放读码框2(ORF2)融合蛋白的Vero细胞株。方法构建重组真核表达质粒pEGFP-ORF2,经酶切及测序鉴定正确后,体外转染Vero细胞,经G418筛选稳定表达融合蛋白的克隆,扩大培养后,荧光显微镜观察EGFP的表达,RT-PCR检测目的基因的转录。结果重组真核表达质粒pEGFP-ORF2经酶切及测序鉴定构建正确,经G418培养20d筛选出的Vero细胞株荧光显微镜下可见融合蛋白表达,RT-PCR检测显示,转染了重组表达质粒的Vero细胞内有目的基因的表达。结论已成功建立了稳定表达EGFP-ORF2的Vero细胞株,为进一步研究HSV-2LATORF2的功能奠定了基础。     

Blood–Nerve Barrier (BNB) Pathology in Diabetic Peripheral Neuropathy and In Vitro Human BNB Model

In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of peripheral nerves is disrupted by dysfunction of the blood–nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.     

CD133-Positive Cells Might Be Responsible for Efficient Proliferation of Human Meningioma Cells

Owing to lack of appropriate model systems, investigations of meningioma biology have come to a stop. In this study, we developed a comprehensive digestion method and defined a culture system. Using this method and system, primary meningioma cells in conditioned suspension medium and a hypoxic environment could be amplified in spheres and were passaged for more than ten generations. Meningioma sphere cells were positive for meningioma cell markers and negative for markers of neural cell types. Importantly, we found the cells expressed the stem cell marker, CD133, but not nestin. All of the tumor sphere cell populations showed a slower degree of cell proliferation than that of human glioma cells and fetal neural stem cells (NSCs). Further studies showed that the proliferative rate was positively correlated with CD133 expression. The higher the CD133 expression, the faster the cell proliferation. With the increase in cell generations, the cell proliferation rate gradually slowed down, and CD133 expression also decreased. Single CD133(+) cells rather than CD133(-) cells could form spheres. Thus, the results above indicated that those cells expressing CD133 in spheres might be stem-like cells, which may be responsible for efficient amplification of human meningioma cells. Decreased expression of CD133 may lead to the failure of long-term passaging.     

Complement C4 Is Reduced in iPSC-Derived Astrocytes of Autism Spectrum Disorder Subjects

In recent years, accumulating evidence has shown that the innate immune complement system is involved in several aspects of normal brain development and in neurodevelopmental disorders, including autism spectrum disorder (ASD). Although abnormal expression of complement components was observed in post-mortem brain samples from individuals with ASD, little is known about the expression patterns of complement molecules in distinct cell types in the developing autistic brain. In the present study, we characterized the mRNA and protein expression profiles of a wide range of complement system components, receptors and regulators in induced pluripotent stem cell (iPSC)-derived neural progenitor cells, neurons and astrocytes of individuals with ASD and neurotypical controls, which constitute in vitro cellular models that recapitulate certain features of both human brain development and ASD pathophysiology. We observed that all the analyzed cell lines constitutively express several key complement molecules. Interestingly, using different quantification strategies, we found that complement C4 mRNA and protein are expressed in significantly lower levels by astrocytes derived from ASD individuals compared to control astrocytes. As astrocytes participate in synapse elimination, and diminished C4 levels have been linked to defective synaptic pruning, our findings may contribute to an increased understanding of the atypically enhanced brain connectivity in ASD.     

Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation

Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.     

Nanotechnology Facilitated Cultured Neuronal Network and Its Applications

The development of a biomimetic neuronal network from neural cells is a big challenge for researchers. Recent advances in nanotechnology, on the other hand, have enabled unprecedented tools and techniques for guiding and directing neural stem cell proliferation and differentiation in vitro to construct an in vivo-like neuronal network. Nanotechnology allows control over neural stem cells by means of scaffolds that guide neurons to reform synaptic networks in suitable directions in 3D architecture, surface modification/nanopatterning to decide cell fate and stimulate/record signals from neurons to find out the relationships between neuronal circuit connectivity and their pathophysiological functions. Overall, nanotechnology-mediated methods facilitate precise physiochemical controls essential to develop tools appropriate for applications in neuroscience. This review emphasizes the newest applications of nanotechnology for examining central nervous system (CNS) roles and, therefore, provides an insight into how these technologies can be tested in vitro before being used in preclinical and clinical research and their potential role in regenerative medicine and tissue engineering.