In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells

The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells.The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.

Stabilizing perovskite precursors with the reductive natural amino acid for printable mesoscopic perovskite solar cells

Solution processability significantly advances the development of highly-efficient perovskite solar cells.However,the precursor solution tends to undergo irreversible degradation reactions,impairing the device performance and reproducibility.Here,we utilize a reductive natural amino acid,Nacetylcysteine(NALC),to stabilize the precursor solution for printable carbon-based hole-conductorfree mesoscopic perovskite solar cells.We find that I_(2) can be generated in the aged solution containing methylammonium iodide(MI) in an inert atmosphere and speed up the MA-FA^(+)(formamidinium) reaction which produces large-size cations and hinders the formation of perovskite phase.NALC effectively stabilizes the precursor via its sulfhydryl group which reduces I_(2) back to I^(-)and provides H^(+).The NALC-stabilized precursor which is aged for 1440 h leads to devices with a power conversion efficiency equivalent to 98% of that for devices prepared with the fresh precursor.Furthermore,NALC improves the device power conversion efficiency from 16.16% to 18.41% along with enhanced stability under atmospheric conditions by modifying grain boundaries in perovskite films and reducing associated defects.

Precursor engineering enables high-performance all-inorganic CsPbIBr_(2) perovskite solar cells with a record efficiency approaching 13%

All-inorganic CsPbIBr_(2) perovskite has attracted widespread attention in photovoltaic and other optoelectronic devices because of its superior thermal stability.However,the deposition of high-quality solutionprocessed CsPbIBr_(2) perovskite films with large thicknesses remains challenging.Here,we develop a triple-component precursor(TCP) by employing lead bromide,lead iodide,and cesium bromide,to replace the most commonly used double-component precursor(DCP) consisting of lead bromide and cesium iodide.Remarkably,the TCP system significantly increases the solution concentration to 1.3 M,leading to a larger film thickness(~390 nm) and enhanced light absorption.The resultant CsPbIBr_(2) films were evaluated in planar n-i-p structured solar cells,which exhibit a considerably higher optimal photocurrent density of 11.50 mA cm^(-2) in comparison to that of DCP-based devices(10.69 mA cm^(-2)).By adopting an organic surface passivator,the maximum device efficiency using TCP is further boosted to a record efficiency of 12.8% for CsPbIBr_(2) perovskite solar cells.

Schwann cells differentiated from skin-derived precursors provide neuroprotection via autophagy inhibition in a cellular model of Parkinson’s disease

Autophagy has been shown to play an important role in Parkinson’s disease.We hypothesized that skin-derived precursor cells exhibit neuroprotective effects in Parkinson’s disease through affecting autophagy.In this study,6-hydroxydopamine-damaged SH-SY5Y cells were pretreated with a culture medium containing skin-derived precursors differentiated into Schwann cells(SKP-SCs).The results showed that the SKP-SC culture medium remarkably enhanced the activity of SH-SY5Y cells damaged by 6-hydroxydopamine,reduced excessive autophagy,increased tyrosine hydroxylase expression,reducedα-synuclein expression,reduced the autophagosome number,and activated the PI3K/AKT/mTOR pathway.Autophagy activator rapamycin inhibited the effects of SKP-SCs,and autophagy inhibitor 3-methyladenine had the opposite effect.These findings confirm that SKP-SCs modulate the PI3K/AKT/mTOR pathway to inhibit autophagy,thereby exhibiting a neuroprotective effect in a cellular model of Parkinson’s disease.This study was approved by the Animal Ethics Committee of Laboratory Animal Center of Nantong University(approval No.S20181009-205)on October 9,2018.

Schwann cells originating from skin-derived precursors promote peripheral nerve regeneration in rats

Artificial guidance channels containing Schwann cells can promote the regeneration of injured peripheral nerve over long distances. However, primary Schwann cells are not suitable for autotransplantation. Under specific conditions, skin-derived progenitors can be induced to dif- ferentiate into Schwann cells. Therefore, adult rat dorsal skin （dermis）-derived progenitors were isolated and induced to differentiate with DMEM/F12 containing B27, neuregulin 1, and for- skolin. Immunofluorescence staining and reverse transcription polymerase chain reaction （RT- PCR） confirmed that the resultant cells were indeed Schwann cells. Artificial guidance channels containing skin-derived progenitors, Schwann cells originating from skin-derived progenitors, or primary Schwann cells were used to bridge 5 mm sciatic nerve defects. Schwann cells originating from skin-derived progenitors significantly promoted sciatic nerve axonal regeneration. The sig- nificant recovery of injured rat sciatic nerve function after the transplantation of Schwann cells originating from skin-derived progenitors was confirmed by electromyogram. The therapeutic effect of Schwann cells originating from skin-derived progenitors was better than that of skin-de- rived progenitors. These findings indicate that Schwann cells originating from skin-derived precursors can promote peripheral nerve regeneration in rats.

Changes of the Transcriptional Levels of Molecules Associated with Endogenous Antigen Processing and Presentation in Porcine Skin-derived Dendritic Cells Infected with PCV2 in vivo

[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.

miRNA-21-5p is an important contributor to the promotion of injured peripheral nerve regeneration using hypoxia-pretreated bone marrow-derived neural crest cells

Our previous study found that rat bone marrow–derived neural crest cells(acting as Schwann cell progenitors)have the potential to promote long-distance nerve repair.Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication.Nevertheless,the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear.To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves,we collected conditioned culture medium from hypoxia-pretreated neural crest cells,and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation.The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells.We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells.Subsequently,to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons,we used a microfluidic axonal dissociation model of sensory neurons in vitro,and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons,which was greatly dependent on loaded miR-21-5p.Finally,we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb,as well as muscle tissue morphology of the hind limbs,were obviously restored.These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p.miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome.This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves,and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.

Functional electrical stimulation-facilitated proliferation and regeneration of neural precursor cells in the brains of rats with cerebral infarction

Previous studies have shown that proliferation of endogenous neural precursor cells cannot alone compensate for the damage to neurons and axons. From the perspective of neural plastici- ty, we observed the effects of functional electrical stimulation treatment on endogenous neural precursor cell proliferation and expression of basic fibroblast growth factor and epidermal growth factor in the rat brain on the infarct side. Functional electrical stimulation was performed in rat models of acute middle cerebral artery occlusion. Simultaneously, we set up a placebo stimulation group and a sham-operated group. Immunohistochemical staining showed that, at 7 and 14 days, compared with the placebo group, the numbers of nestin （a neural precursor cell marker）-positive cells in the subgranular zone and subventricular zone were increased in the functional electrical stimulation treatment group. Western blot assays and reverse-transcription PCR showed that total protein levels and gene expression of epidermal growth factor and basic fibroblast growth factor were also upregulated on the infarct side. Prehensile traction test results showed that, at 14 days, prehension function of rats in the functional electrical stimulation group was significantly better than in the placebo group. These results suggest that functional electrical stimulation can promote endogenous neural precursor cell proliferation in the brains of acute cerebral infarction rats, enhance expression of basic fibroblast growth factor and epidermal growth factor, and improve the motor function of rats.

Migration of R28 Retinal Precursor Cells into Cochlear and Vestibular Organs

Damaged hair cells and neurons in the inner ear generally can not be replaced in mammals. The loss of these cells causes permanent functional disorders in both the cochlear and vestibular systems. Transplantation of retinal precursor cells, R28 cells, into inner ear tissue may help replace missing cells. The aim of the current project was to induce R28 cell transdifferentiation into cochlear and vestibular cell types under culture conditions. The first part was related to R28 cell labeling with DiI fluorescence that would help identify and track R28 cells. The second part involved co-culturing R28 cells in cochlear and vestibular organotropic cultures or isolated spiral ganglion neurons. The results suggest that R28 cells have the potential to differentiate into supporting cell types and spiral ganglion neurons in serum free medium, probably under the influence of diffusible signals from inner ear tissues. This information is useful for future efforts in inducing stem cell differentiation in the inner ear to replace lost sensory and neural cells.

Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

BACKGROUND： Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy （CRT） for treating Parkinson disease （PD）. Embryonic mesencephalic precursor cells （MPCs） can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far. OBJECTIVE： To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats. DESIGN ： A randomized and controlled trial taking SD rats as experimental animals.SETTING： Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS： This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats, with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. METHODS ： Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1 out of 20 model rats after execution, and the other 19 rats were randomly divided into control group （n=5）, sham transplantation group （n=5）and cell grafted group （n=9）. Primary cultured E12 MPC cell suspension （1.2×10^11 L^-1）were used as donor cells. 4μL primary cultured E12 MPC cell suspension prepared freshly was injected into the lesioned corpus striatum of rats in cell grafted group, and 4μL D-Hank＇s solution was injected in sham transplantation group in the same way. There was no injection in control group. Apomorphine-induced rotation rate of PD rats were recorded respectively in cell grafted group and sham transplantation group pre-operation （initial value） and at postoperative 2, 4, 6 and 16 weeks. Apomorphine-induced rotation rate of PD rats was recorded in control group at postoperative 2 months （initial value） and following 2,4,6 and 16 weeks. To determine TH antigen with immunohistological ABC method （DAB developing） at 6 months post-transplantation to investigate the differentiation and survival of donor cells in the host body.MAIN OUTCOME MEASURES： Apomorphine-induced rotation behavior before and after transplantation and the survival and differentiation of implanted cells in the host body at 6 months post-transplantation. RESULTS： Among 19 model rats, one rat died after transplantation respectively in the cell grafted group and sham transplantation group; finally 17 model rats entered the stage of result analysis. Relative apomorphine-induced rotation rate was significantly decreased in the cell grafted group as compared with that before transplantation , with significant difference （P 〈 0.01 .P 〈 0.05）;the mean value of relative apomorphine-induced rotation rate was significantly decreased at postoperative 16 weeks in cell grafted group as compared with that of corresponding relative rotation rate in control group , also with significant difference （P 〈 0.05）.Immunohistological results showed that donor cells could differentiate into large and multi-polar dopaminergic neurons in the host body. CONCLUSION ： Primary cultured embryonic MPCs can be used as the donor cells in CRT for treating PD.

Do neural precursor cells exist in a distal neurogenic region following cerebral hemorrhage?

BACKGROUND： Cerebral injury in adult mammals can induce neural precursor cells （NPCs） to proliferate and migrate towards the focal zone, but it is unclear whether endogenous NPCs can migrate towards regions distal to the hemorrhagic focus or whether NPCs differentiate in the peripheral hemorrhagic region. OBJECTIVE： To investigate the distribution of endogenous NPCs in different brain regions of rats with experimental cerebral hemorrhage, as well as NPC proliferation and differentiation with time. DESIGN, TIME AND SE＇B＇ING： A randomized, controlled animal experiment was performed at the Department of Neurobiology, Luzhou Medical College, between January 2007 and October 2008. MATERIALS： Bromodeoxyuridine （BrdU） was purchased from Roche, Germany. Mouse anti-rat BrdU monoclonal antibody, rabbit anti-nestin polyclonal antibody, rabbit anti-neuron specific enolase （NSE） polyclonal antibody were purchased from Wuhan Boster, China. Rabbit anti-glial fibrillary acidic protein （GFAP） polyclonal antibody was purchased from Sigma, USA. METHODS： Thirty-five adult Sprague Dawley rats were randomly divided into three groups： （1） cerebral hemorrhage group （n = 25）, rats were stereotaxically administered 50 p L autologous arterial blood via the dorsal caudate putamen to induce cerebral hemorrhage; （2） sham-surgery group （n = 5）, rats underwent surgery but did not receive blood injection; （3） blank control group （n = 5）, rats received no surgery and blood administration. At 2 hours after surgery, all rats were intraperitoneally administered BrdU. MAIN OUTCOME MEASURES： Distribution and proliferation of BrdU-positive cells were observed by immunohistochemical staining. BrdU-positive cell differentiation into neurons and glial cells in the peripheral hemorrhagic region was detected by double-label immunofluorescence. RESULTS： Immunohistochemistry results revealed that BrdU-positive cells existed not only in the peripheral hemorrhagic region, such as the subependymal layer and hippocampal dentate gyrus, but also in the lateral septal nucleus, diagonal band, habenular nucleus, and cerebral cortex. Following cerebral hemorrhage, BrdU-positive cells in the peripheral hemorrhagic region gradually increased （P 〈 0.05）, and peaked at 7 14 days. Double-label immunofluorescence showed that with time after cerebral hemorrhage, BrdU/nestin-positive cells decreased, but BrdU/GFAP- and BrdU/NSE-positive cells increased in the peripheral cerebral hemorrhagic region （P 〈 0.05）. CONCLUSION： Cerebral hemorrhage can induce the proliferation of endogenous NPCs, which peaks at 1-2 weeks after hemorrhage. NPCs can also migrate towards the regions distal to the hemorrhagic focus, such as a diagonal band or lateral septal nucleus. NPCs can gradually differentiate with increasing time after hemorrhage.

Inhibitory Effect of LPS on the Proliferation of Oligodendrocyte Precursor Cells through the Notch Signaling Pathway inIntrauterine Infection-induced Rats

Periventricular white matter injury (PWMI)is very common in survivors of premature birth,and the final outcomes are a reduction in myelinated neurons leading to white matter hypomyelination.How and (or) why the oligodendrocyte lineage develops abnormally and myelination is reduced is a hot topic in the field.This study focuses on the effect of intrauterine inflammation on the proliferation of oligodendrocyte lineage cells and the underlying mechanisms.Lipopolysaccharide (LPS)(300μg/kg)was intraperitoneally injected into pregnant Sprague-Dawley rats at embryonic days 19 and 20 to establish a rat model of intrauterine infection-induced white matter injury.Corpus callosum tissues were collected at postnatal day 14(P14)to quantify the number of oligodendrocytes,the number and proliferation of oligodendrocyte precursor cells (OPCs), and the expression of myelin proteins (MBP and PLP).Furthermore,the expression of Writ and Notch signaling-related proteins was analyzed.The results showed that the number of oligodendrocytes in the corpus callosum tissues of LPS-treated rats was reduced,and the expression levels of myelinating proteins were down-regulated.Further analysis showed that the Notch signaling pathway was down-regulated in the LPS-treated group.These results indicate that intrauterine LPS may inhibit the proliferation of OPCs by down-regulating the Notch rather than the Writ signaling pathway,leading to hypomyelination of white matter.

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUND The development of regenerative therapy for human spinal cord injury(SCI)is dramatically restricted by two main challenges:the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing.Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge.The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIM To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells(drNPCs).METHODS Seven non-human primates with verified complete thoracic SCI were divided into two groups:drNPC group(n=4)was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury,and lesion control(n=3)was injected identically with the equivalent volume of vehicle.RESULTS Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways.Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation.Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk,migrating to areas of axon growth cones.CONCLUSION Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI,based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation.The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs.Instead,directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support,thereby further supporting the regeneration processes.

CXCR4-Expressing Glial Precursor Cells Demonstrate Enhanced Migratory Tropism for Glioma

Malignant glioma remains one of the most intractable of human cancers principally due to the highly infiltrative nature of these neoplasms. The use of neural precursor cells (NPC) has received considerable attention based on their ability to selectively migrate towards disseminated areas of tumor in vivo and their described ability to deliver tumor-directed therapies specifically to infiltrating tumor cells. Fundamental to optimizing the use of these cells for potential clinical translation is the development of an understanding regarding the biologic cues that govern their ability to migrate towards infiltrative glioma foci. To this end, in this paper we detail that NPC selected for double-expression of the glial-precursor marker A2B5 and the cell-surface chemokine receptor, CXCR4, demonstrate enhanced in vitro gliomadirected tropism. These findings demonstrate the relevance of these markers for the phenotypic segregation of an optimally tumor-tropic NPC sub-population as a means of enhancing NPC-based therapeutic strategies for the treatment of glioma.

Development of neural precursor cells from mouse embryonic stem cells

Objective:To exploretheserum-freecultureconditionsfordifferentiatingmouseembryonicstemcells(ES cells)intoneuralprecursorcells(NPC)andcomparetheeffectsof humanembryonicfibroblasts(HEF)as thefeederlayer of ES withthatof mouseembryonicfibroblasts(MEF)in vitro.Methods:MouseES cellswereculturedin or notin feederlayer cellsmediumcontainingor notleukemiainhibitoryfactorto suppresstheirdifferentiation.Immunocytochemicalmethod was usedto identifyNPCby detectingnestinantigenandalkalinephosphatase.Results: TheES cellsculturedin HEF werepositiveto alkalinephosphatase.Serum-freemediumallowedthedifferentiationof ES cellsintoNPC.Conclusion:HEFcouldreplaceMEFandkeeptheundifferentiatedconditionof ES cellswithmorebenefits.NPCof highpuritycould be culturedfromEScellsby serum-freeculturemethod.

Human umbilical cord Wharton's jelly-derived oligodendrocyte precursor-like cells for axon and myelin sheath regeneration

Human umbilical mesenchymal stem cells from Wharton＇s jelly of the umbilical cord were induced to differentiate into oligodendrocyte precursor-like cells in vitro. Oligodendrocyte precursor cells were transplanted into contused rat spinal cords. Immunofluorescence double staining indicated that transplanted cells survived in injured spinal cord, and differentiated into mature and immature oligodendrocyte precursor cells. Biotinylated dextran amine tracing results showed that cell transplantation promoted a higher density of the corticospinal tract in the central and caudal parts of the injured spinal cord. Luxol fast blue and toluidine blue staining showed that the volume of residual myelin was significantly increased at 1 and 2 mm rostral and caudal to the lesion epicenter after cell transplantation. Furthermore, immunofluorescence staining verified that the newly regenerated myelin sheath was derived from the central nervous system. Basso, Beattie and Bresnahan testing showed an evident behavioral recovery. These results suggest that human umbilical mesenchymal stem cell-derived oligodendrocyte precursor cells promote the regeneration of spinal axons and myelin sheaths.

Propofol increases in vitro proliferation of cultured rat hippocampal precursor cells inhibited by corticosterone

BACKGROUND： Previous studies have shown that propofol enhances proliferation of cultured hippocampal precursor cells in vitro and increases proliferation of cultured hippocampal precursor cells inhibited by corticosterone. Because gamma-aminobutyric acid A （GABA-A） receptor is the functional target for propofol, the proliferative effects of propofol are thought to take place through GABA-A receptor. OBJECTIVE： To determine whether propofol enhances proliferation of rat hippocampal precursor cells inhibited by corticosterone by upregulating expression of GABA-A receptor. DESIGN, TIME AND SETTING： A comparative, observational, in vitro experiment was performed at the Beijing Institute of Pharmacology and Toxicology from April 2005 to April 2006. MATERIALS： Propofol was purchased from AstraZeneca, italy; corticosterone was purchased from Sigma, USA; bicuculline was purchased from Alexis, Switzerland. METHODS： Hippocampal precursor cells were isolated from newborn Wistar rats and cultured in vitro. The second passage of precursor cells was grouped according to the various drugs added to the culture medium： 0.5 μmol/L propofol; 2.5 pmol/L propofol; 100 μmol/L corticosterone; 10 μmol/L bicuculline; 100 μmol/L corticosterone and 0.5 μmol/L propofol; 100 μmol/L corticosterone and 2.5 μmol/L propofol; 100 μmol/L corticosterone, 10 μmol/L bicuculline, and 0.5 μmol/L propofol; 100 μmol/L corticosterone, 10 μmol/L bicuculline, and 2.5 μmol/L propofol; 100 μmol/L corticosterone and 10 pmol/L bicuculline. The cells were cultured for 24 hours with medium containing the respective concentration of drug. The control group consisted of precursor cells absent of drug treatment. MAIN OUTCOME MEASURES： The MTT and ^3H-TdR incorporation assays were used to detect proliferative effects of propofol and bicuculline on cultured rat hippocampal precursor cells inhibited by corticosterone. Immunocytochemistry was used to detect GABA-A receptor expression. Enzyme-linked irnmunosorbent assay was used to quantify GABA-A receptor expression. RESULTS： Propofol, at a concentration of 0.5 and 2.5 μmol/L, increased proliferation of cultured rat hippocampal precursor cells inhibited by corticosterone, while bicuculline antagonized the effects of propofol （P 〈 0.05 or P 〈 0.01 ）. Corticosterone （100μmol/L） decreased expression of GABA-A receptor in the hippocampal precursor cells （P〈 0.05）, and GABA-A receptor expression was upregulated when propofol （2.5μmol/L） was added to the culture medium （P〈 0.05）. CONCLUSION： Low concentrations of propofol increased expression of GABA-A receptor. These results suggest that GABA-A receptor is involved in increased proliferation of cortisone-inhibited rat hippocampal precursor cells in vitro.

STUDY ON DIFFERENTIATION OF RATS EMBRYONIC STEM CELLS CULTURED IN BRL-CM INTO NEURAL PRECURSOR CELLS

Objective To investigate whether buffalo rat liver cell conditioned medium (BRL CM) can be used as the culture medium of embryonic stem (ES) cells, and to get relatively pure neural precursor cells (NPCs) for treatment aim. Methods Mouse ES cells were cultured in BRL CM and medium contain leukemia inhibitory factor (LIF), respectively. NPCs were selectively cultured in serum free medium. Alkaline phosphatase activity was visualized with NBT/BCIP and nestin antigen was detected with immunocytochemical methods. Results BRL CM could be used as an efficiency culture condition instead of LIF in ES cells culture. About 86% of cells derived from ES cells in the serum free culture were NPCs. Conclusion BRL CM can replace LIF to use in ES cell culture. High purity of NPC can be induced from ES cells with serum free culture method.

Expression of the Capsid Precursor Protein gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector

We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.