Multilineage Differentiation of Dental Pulp Stem Cells from Green Fluorescent Protein Transgenic Mice

Aim The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells （DPSCs） from green fluorescent protein （GFP） transgenic mice. The expression of GFP in DPSCs was also observed during differentiation. Methodology DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR. Results The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation. Conclusion As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have success- fully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

Repair of spinal cord injury by neural stem cells transfected with brain-derived neurotrophic factor-green fluorescent protein in rats A double effect of stem cells and growth factors?

Brain-derived neurotrophic factor（BDNF）can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein（GFP）gene persists for a long time after transfection into neural stem cells.Nevertheless,little is known about the biological characteristics of BDNF-GFP modified nerve stem cells in vivo and their ability to induce BDNF expression or repair spinal cord injury.In the present study,we transplanted BDNF-GFP transgenic neural stem cells into a hemisection model of rats.Rats with BDNF-GFP stem cells exhibited significantly increased BDNF expression and better locomotor function compared with stem cells alone.Cellular therapy with BDNF-GFP transgenic stem cells can improve outcomes better than stem cells alone and may have therapeutic potential for spinal cord injury.

Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

Objective:To study the role of bone marrow mesenchymal stem cells(BMSCs)in construction of vascularized engineered tissue.Methods:hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein(GFP)-CMV.Then the recombinant shuttle plasmid was transfected into BMSCs with Lipofectamine^(TM)2000 for packaging and amplifying.hVTGF165 mRNA expression in BMSCs cells was tested.Results:The sequence of hVEGFI65 in pShutlle-GFP-hVFGF165 plasmid was confirimed by double-enzyme cleavage method and sequencing.hVECF165 was highly expressed in BMSCs.Conclusions:The GFP/hVECF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells,which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT EGFP was constructed The linearized vector was introduced into ES cells by electroporation The G418 r6TG r cell clones were obtained after selection with G418 and 6TG media The integration patterns of these resistant cell clones were identified with Southern blotting Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated They have normal properties, such as karyotype, viability and di fferentiation ability The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells Cultured in suspension, the 'green' ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges The 'green' embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence Conclusions This generation of 'green' targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell replacement therapy as well as for other biomedical a nd research purposes

The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the 'green' ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The 'green' embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

Construction of Ad-EGFP-BDNF vector and its expression in neural stem cells

BACKGROUND： Brain-derived neurotrophic factor （BDNF） provides nourishment to injured neurons. Neural stem cells can differentiate into neurons to repair neuronal injury in vivo. It has been hypothesized that continuous secretion of BDNF from neural stem cells could benefit brain injury repair. OBJECTIVE： To transfect BDNF and enhanced green fluorescent protein （EGFP） into neural stem cells with adenovirus vector and to observe expression of BDNF and EGFP in transfected neural stem cells. DESIGN, TIME AND SETTING： Observational, cellular, molecular study was performed at the Biochemistry Laboratory, Tongji University School of Medicine, China from July 2004 to September 2006. MATERIALS： Neural stem cells were provided by the Anatomy and Histoembryology Laboratory of Fudan University Medical School, China. METHODS： BDNF cDNA was extracted by reverse transcription polymerase chain reaction from the rat hippocampus. Following gene cloning and packaging by HEK293.BDNF, the EGFP gene was transfected into cultured neural stem cells with the Ad-EGFP-BDNF vector. BDNF-expressing neural stem cell clones were selected by G418 selection. MAIN OUTCOME MEASURES： EGFP expression and cell morphology were observed by fluorescent microscopy; neural stem cell expressing BDNF mRNA was examined by reverse transcription polymerase chain reaction; BDNF expression was detected by enzyme-linked immunosorbent assay from supematant of infected neural stem cells. RESULTS： High transfection efficiency was obtained using 5×10^8 virus titers to transfect neural stem cells. G418-resistant neural stem cell clones integrated BDNF mRNA fragments. Enzyme-linked immunosorbent assay results showed that BDNF expression in the supernatant increased with increasing culture time and peaked at 72 hours. CONCLUSION： Adenovirus-mediated BDNF and EGFP genes were successfully transfected into neural stem cells and were expressed in neural stem cells for a long period of time.

Can muscle-derived stem cells serve as seed cells to repair spinal cord injury?

Muscle-derived stem cells （MDSCs） can come from a number of different sources, which are easy to isolate and culture, and are also useful in the transformation and expression of exogenous genes. Therefore, MDSCs could possibly be used for gene therapy in the treatment of neurological diseases. However, research on MDSCs has focused on identifying phenotypes and induced differentiation, with few in vivo animal experiments conducted. In this study, MDSCs were selected as seed cells and implanted into the rat spinal cord injury area. Results demonstrated that the MDSCs survived, migrated, and were distributed along the spinal nerves. Moreover, the motor function of rat lower limbs improved significantly, suggesting that MDSCs could be used as seed cells to repair spinal cord injury.

Neurogenic Differentiation of Murine Adipose Derived Stem Cells Transfected with EGFP in vitro

Some studies indicate that adipose derived stem cells(ADSCs)can differentiate into adipogenic,chondrogenic,myogenic,and osteogenic cells in vitro.However,whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated.In this study,the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene,and then the cells were induced for neural differentiation.The morphology of those ADSCs began to change within two days which developed i...

Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP- C1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluoresce- nce microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection. Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

The chimeric mice derived from umbilical cord blood stem cells of EGFP-transgenic mouse

Objective:The chimeric mice were prepared by microinjection of blastocyst cavity using umbilical cord blood stem cells(UCBSCs)of Enhanced Green Fluorescent Protein(EGFP)-transgenic mouse,which was expected to provide a theoretical and experimental basis for the study of in-vivo differentiation of adult stem cells.Methods:Mouse UCBSCs expressing green fluorescence was microinjected into blastocyst cavity and several blastocysts were transferred into uterus of pseudo pregnant mouse.First of all,new-born candidate chimeric mice were observed through feather color.Secondly,the genomic DNA and total RNA were extracted to analyze chimeric rate in several tissues.Finally,flow cytometry was used to detect percentage of green fluorescent cells mice in several tissues.Results:The UCBSCs expressing green fluorescent protein were successfully isolated.After flow cytometry analysis,the proportion of cells expressing green fluorescence was 80.25%.Through microinjection and embryo transfer,we got five white new-born mice and no chimeric feather color was observed.The analyses of PCR and RT-PCR were carried out to detect EGFP gene using six tissues including heart muscle,liver,lung,skin,leg muscle and adipose tissue.The results showed that the leg muscle and adipose tissue of two mice were positive and the other tissues and six tissues of the other 3 mice were all negative.The leg muscle and adipose tissue of two positive mice were digested into single-cells suspension and were carried out flow cytometry analysis.The results showed that the average chimeric rates of leg muscle and adipose tissue of two positive mice were 9.87% and 5.78%,respectively.Conclusions:The results demonstrated that adult UCBSCs could differentiate into leg muscle and adipose tissue in vivo.

A versatile tool for tracking the differentiation of human embryonic stem cells

The ability of human embryonic stem cells(hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are power-ful tools for tracking cell types of interest in differentiated cell populations.Here,we describe the construction of modular lentivectors containing different tissue-specific promoters(Tα1 ofα-tubulin;aP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla greenfluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to be traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.

小鼠pLVX-Wnt3a-IRES-ZsGreen1慢病毒载体的构建及神经干细胞转染

目的构建共表达小鼠Wnt3a(mWnt3a)与绿色荧光蛋白(GFP)慢病毒载体,感染神经干细胞(NSCs),观察mWnt3a在NSCs中的表达。方法利用同源重组技术将mWnt3a基因插入慢病毒载体pLVX-IRES-ZsGreen1,构建pLVX-Wnt3a-IRES-Zs-Green1慢病毒重组质粒,通过瞬时转染法包装出病毒上清,感染NSCs,设为Wnt3a-NSCs组;同时设GFP感染NSCs组(GFP-NSCs组)和未感染NSCs组(NSCs组)作为对照。免疫荧光染色法对Wnt3a-NSCs组NSCs进行nestin鉴定;Real-Time PCR检测各组细胞mWnt3a mRNA的表达;Western blotting检测各组细胞mWnt3a、β-catenin蛋白的表达。结果经限制性内切酶检测、基因测序和绿色荧光观察证实成功构建了携带mWnt3a基因的重组慢病毒,且慢病毒滴度达3×108TU/mL。Wnt3a-NSCs组NSCs在荧光显微镜下证实有绿色荧光,且nestin表达阳性。Real-Time PCR和Western blotting结果显示感染后7 d,Wnt3a-NSCs组mWnt3a mRNA和蛋白以及β-catenin蛋白均明显高于GFP-NSCs组和NSCs组(P<0.01)。结论成功构建了表达mWnt3a基因的慢病毒载体,在体外培养条件下可以成功转染NSCs。

重组慢病毒转染兔骨髓间充质干细胞与脱钙骨基质构建转基因组织工程材料

背景:缺损组织的修复重建受限于自体或异体可替代移植材料的来源问题而导致临床应用受限,转基因干细胞和组织工程材料研究开辟了新的治疗思路。目的:探究增强型绿色荧光蛋白重组慢病毒转染兔骨髓间充质干细胞在体外的生物学特性以及与脱钙骨基质体外构建转基因组织工程材料的生物矿化特性。方法:细胞贴壁及密度离心法获得兔骨髓间充质干细胞,增强型绿色荧光蛋白重组慢病毒以感染复数为100转染第5代兔骨髓间充质干细胞,体外观察转染细胞与未转染细胞的增殖能力、细胞表型、细胞周期以及成骨诱导后碱性磷酸酶、Runx2、骨钙素表达的差异;增强型绿色荧光蛋白重组慢病毒转染骨髓间充质干细胞与脱钙骨基质在体外构建转基因组织工程材料,对其进行扫描电镜观察及元素能谱分析。结果与结论:增强型绿色荧光蛋白重组慢病毒成功转染骨髓间充质干细胞后,在转染24,48 h细胞增殖较未转染细胞缓慢(P <0.05);在转染72 h后,细胞表型未发生变异,细胞周期、细胞增殖能力以及成骨诱导后碱性磷酸酶、Runx2、骨钙素表达量与未转染细胞无明显差异(P> 0.05);增强型绿色荧光蛋白标记的骨髓间充质干细胞在脱钙骨基质支架上有较好的生物相容性,根据荧光表达强度推测目的基因在2周左右发挥最大生物学功能,且出现了钙磷矿化物沉积,体现出优越的生物矿化特性。

携带Fluc和ZsGreen双报告基因小鼠iPS细胞系的建立

背景与目的:诱导性多潜能干细胞(induced pluripotent stem cells,iPS细胞)在修复受损组织器官和治疗人类疾病方面已展示了良好的应用前景,但iPS细胞具有形成畸胎瘤的作用,这是其安全应用于临床的巨大障碍之一。为此本研究拟利用慢病毒体外感染的方法,建立携带萤火虫荧光素酶(firefly luciferase,Fluc)和绿色荧光蛋白(green fluorescent protein,ZsGreen)双报告基因的小鼠iPS细胞系,为活体动态监测畸胎瘤形成、定植和形成畸胎瘤所需最小细胞剂量等提供实验基础。方法:构建含Fluc和ZsGreen双报告基因的慢病毒载体。以pLH2BmRFP质粒为模板,PCR扩增获得Fluc基因片段,将其克隆至利用BamHⅠ酶切的pHAGE-fullEF1a-MCS-IZsGreen质粒中,挑选一个阳性克隆质粒进行测序,最终获得携带双报告基因的慢病毒载体pEF1a-Fluc-IRES-ZsGreen(pELZG)。采用脂质体介导的瞬时转染生产携带Fluc和ZsGreen基因的病毒,收集病毒上清,并感染iPS细胞,数天后荧光显微镜下观察是否有绿色荧光的iPS细胞克隆,不断挑取ZsGreen阳性的iPS细胞克隆以进一步纯化。将标记好的iPS细胞移植入裸鼠皮下,观察成瘤情况。结果:pELZG载体分别经XbaⅠ、PvuⅡ、SacⅠ单酶切,得到的片段大小分别为10.005、3.282和6.723 kb,2.441、3.401和6.604 kb,预示成功构建了Fluc和ZsGreen双报告基因的慢病毒载体。利用其生产的病毒上清成功感染iPS细胞,经过不断的纯化,最终获得含稳定表达Fluc和ZsGreen双报告基因的小鼠iPS细胞系。将标记好的iPS细胞植入裸鼠皮下后,观察到畸胎瘤的形成。结论:成功建立了携带Fluc和ZsGreen双报告基因的小鼠iPS细胞系,为相关后续研究打下了良好的基础。标记后的iPS细胞对畸胎瘤的形成和发展具有很好的示踪作用。

基于CRISPR/Cas9n技术建立携带mT-F2A-EGFP报告系统的小鼠胚胎干细胞系

目的·通过CRISPR/Cas9n(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nickase)介导的同源定向修复(homologous-directed repair,HDR)技术在小鼠胚胎干细胞(mouse embryonic stem cell,mESC)的中内胚层关键调控分子T-box转录因子Brachyury(即T基因)末端依次敲入手足口病毒2A(foot-and-mouth disease virus 2A,F2A)和增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)以建立T荧光报告细胞系(mTF2A-EGFP)。方法·首先,针对T基因构建特异性单链导向RNA(single guide RNA,sgRNA)质粒和包含F2A-EGFP的供体质粒。利用电穿孔将这2种质粒递送到mESC E14Tg2a(E14)内,通过HDR在T基因末端插入F2A-EGFP。然后,通过药物筛选和基因测序验证所获得的单克隆细胞,并将其诱导形成类胚体(embryonic body,EB)进行分化。通过荧光显微镜和流式细胞技术分别监测mT-F2A-EGFP细胞克隆在分化前后的荧光信号变化,并进行实时定量聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction,RT-qPCR)来检测多能性标志基因、中内胚层以及外胚层标志基因的转录水平变化。同时,检测克隆细胞的周期、生长曲线,并利用碱性磷酸酶(alkaline phosphatase,AP)染色检测候选克隆干细胞特性。最后,挑选克隆细胞系T1进行了EB分化。利用流式细胞技术分选出分化细胞群中EGFP荧光表达细胞(EGFP+)和无荧光表达的细胞(EGFP-),并检测各谱系基因的表达情况。结果·EGFP被正确插入到E14细胞的T基因,其荧光强度能正确反映T基因表达水平且未产生明显的不良反应。当T1报告克隆分化时,通过流式细胞技术分选出的包含mT-F2A-EGFP的荧光细胞主要高表达中内胚层标志基因。结论·成功构建携带mT-F2A-EGFP的mESC,可实现对T基因调控程度的快速监测,并实时追踪分化过程中表达T基因标记EGFP的中内胚层细胞。

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.

Construction of an allogenic chimeric mouse model for the study of the behaviors of donor stem cells in vivo

It is essential to establish an animal model for the elucidation of the biological behaviors of stem cells in vivo. We constructed a chimeric animal model by in utero transplantation for investigation of stem cell transplantation.

Mesenchymal stem cells transduced by PLEGFP-N1 retroviral vector maintain their biological features and differentiation

Background Enhanced green fluorescent protein （EGFP） has been an important reporter gene for gene therapy. Human mesenchymal stem cells （hMSCs） are ideal target cells in cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated by PLEGFP-N1 retroviral transduction.Methods hMSCs were isolated from human bone marrow by density gradient fractionation and adherence to plastic flasks. Individual colonies were selected and cultured in tissue dishes. Packaging cells PT67 were transfected by PLEGFP-N1 retroviral vector , and hMSCs were transduced by viral supernatant infection. Meanwhile, hMSCs-EGFP were identified by immune phenotypes and whether it could differentiate into osteoblasts or adipocytes under conditioned media was investigated.Results The rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morphological features. Flow cytometric analyses showed that hMSCs-EGFP were positive for CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced into osteocytes or adipocytes under conditioned media. These biological features of hMSCs-EGFP were consistent with those of hMSCs.Conclusions hMSCs transduced by PLEGFP-N1 retroviral vector can be used in vivo securely because they can maintain their biological characteristics and differentiation. It is a simple and reliable way to trace the changes of hMSCs in vivo by EGFP during cell transplantation and gene therapy.

The experimental study of genetic engineering human neural stem cells mediated by lentivirus to express multigene

Objective: To explore the feasibility to construct genetic engineering human neural stem cells (hNSCs) mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI). Methods: Human neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3 (NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot. Results: Genetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot. Conclusions: Genetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.