BACKGROUND:Islet beta cell replacement therapy is one of the most promising approaches for treating type 1 diabetes mellitus. However, its large scale application is hampered by a shortage of islet beta cells for transplantation. Pluripotent stem cells are one of ideal seed cells for islet beta cell replacement therapy, but pancreatic beta-cell differentiation is time-consuming and labor-intensive. OBJECTIVE:To construct a high efficient pancreatic and duodenal homeobox 1 (Pdx1)/insulin dual-reporter vector and to monitor the key genes expression during pancreatic beta-cell differentiation from pluripotent stem cells. METHODS:In order to construct a high efficient Pdx1/insulin dual-reporter vector, puromycin resistance gene was firstly introduced into pTiger vector, and then the original 410 bp mouse Ins1 promoter of the vector was replaced by 646 bp mouse Ins1 promoter. Finally, the dual-reporter vector was transduced into INS-1 and human induced pluripotent stem cells to testify its function. RESULTS AND CONCLUSION:The high efficient Pdx1/insulin dual-reporter vector was constructed successfully. The vector successfully acquired puromycin resistance gene and high gene expression efficacy of insulin in INS-1 cells. The specific gene expression pattern of Pdx1/insulin was first found in INS-1 cells. To conclude, the real-time monitoring function of Pdx1/insulin expression is preliminarily confirmed during pancreatic beta-cell differentiation.

Objective:To construct the lentiviral expression vector pLVX-EF1α-CD19t-IRES-Puro for truncated CD19(CD19t) in cytoplasmic region,and examine its expression in K562 cells.Methods:CD19t gene was synthesized and cloned into pLVX-EF1α-IRES-Puro vector followed by virus packaging in 293FT cells.Human erythroleukemia K562 cells were infected with recombinant lentivirus containing CD19t to construct K562/CD19t cells.The expression of CD19t was examined by Western blot and immunofluorescence staining.ELISA was used to analyze the secreted IL-2 in co-culture supernatant of K562/CD19t with CAR-Jurkat.Results:Lentiviral expression vector pLVX-EF1α-CD19t-IRES-Puro was confirmed by double enzymatic digestion and sequencing.The expression of CD19t in K562 cells was detected by Western blot and immunofluorescence staining.ELISA showed that CAR-Jurkat secreted IL-2,which was stimulated by CD19t expression.Conclusion:The lentiviral vector for CD19t expression has been constructed and CD19t expression can be detected in K562/CD19t cells,which would be helpful to make animal model for B cell leukemia and test CAR-T performance.

Objective:To construct the retroviral expression vector for RRAGD-EGFP fusion protein,and examine its subcellular localization in MDA-MB-436 cells.Methods:Total RNA was extracted from HEK293 cells and reverse transcribed into cDNA.RRAGD(Ras-related GTP binding protein D) gene was amplified by PCR and cloned into the retroviral expression vector pQCXIP-EGFP-N1 to construct pQCXIP-RRAGD-EGFP,followed by retrovirus was made and infected MDA-MB-436,a cell line of breast cancer.Afterwards,time lapse imaging was performed.Results:The retroviral expression vector for RRAGD-EGFP fusion protein was successfully constructed.RRAGD-EGFP fusion protein was found in cytoplasmic vesicles and nucleus.Conclusion:Localization of RRAGD-EGFP fusion protein in cytoplasmic vesicles is consistent with its role in lysosomal biogenesis,which has laid foundation for the further study on the role of RRAGD in cell-in-cell.

目的:构建原钙黏蛋白7b(PCDH7b)与绿色荧光蛋白(GFP)的融合基因表达载体pQCXIP-PCDH7b-EGFP,并检测PCDH7b-GFP融合蛋白在细胞中的表达定位.方法:以MDA-MB-436细胞基因组DNA为模板,PCR扩增人PCDH7b基因,克隆入逆转录病毒载体pQCXIP-EGFP-N1构建pQCXIP-PCDH7b-EGFP,病毒包装后感染人乳腺上皮细胞系MCF10A,免疫荧光染色检测其与膜蛋白E钙黏蛋白的相对表达定位.结果:构建获得逆转录病毒载体pQCXIP-PCDH7b-EGFP,融合蛋白PCDH7b-GFP定位于细胞浆和细胞膜,与E钙黏蛋白有共定位.结论:PCDH7b-GFP融合蛋白的表达,为后续活细胞成像分析PCDH7b在细胞内的动态分布奠定了基础.

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as “EML method” based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.

Although cell-in-cell structures (CICs) could be detected in a wide range of human tumors, homotypic CICs formed between tumor cells occur at low rate for most of them. We recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin (α-catenin), a molecular linker between cadherin-mediated adherens junctions and F-actin. Expression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation in a ROCK kinase-dependent way. Thus, our work identified α-catenin as another molecule in addition to E- and P-cadherin that were targeted to inactivate homotypic CICs formation in human tumor cells.

To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.

Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.

The blastoderm cells isolated from the stage X embryos of chicken were cultured on feeder layer of mouse embryonic fibroblasts(MEF)with high glucose DMEM supplemented with 10 ng/mL bFGF,20 ng/mL hIGF-1,2 ng/mL mSCF,2 ng/mL hIL-11 and 1 000 U/mL LIF.Typical chicken embryonic stem cells(cESCs)colonies with high endogenous AKP activity were observed in the culture system.The cESCs derived from blastoderm cells expressed pluripotent markers SSEA-1 and Oct-4 as determined by immunocytological analysis.RT-PCR analysis further confirmed the expression of cENS-1,a gene specifically expressed in cESCs and early embryo.These results demonstrate the feasibility for using the culture system in isolating cESCs from the stage X chicken embryos and supporting the growth of cESCs with undifferentiating state in vitro.

The efficient expression vector for recombinant human pro-urokinase(rhPro-UK) was constructed with the combination of chicken β-globin MAR sequences,human elongation factor alpha1(hEF-1α) housekeeping gene regulatory sequences,and RNA stability and the output sequence.The rhPro-UK expression level reached 1 299 IU/cells6/d in CHO(Chinese hamster ovary) cell lines.The rhPro-UK containing supernatant purified with cation exchange chromatography,hydrophobic interaction chromatography,gel exclusion chromatography process resulted in rhPro-UK purity over 95% with recovery rate of 60%-70%.

BACKGROUND: The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue constructed by a combination of cells and scaffolds in vitro. However, this strategy has been hampered by the lack of cardiomyocytes and the significant cell death after transplantation in vivo.METHODS: In this study we explored the feasibility of in vitro construction of vascularized cardiac muscle using genetically modified mouse embryonic stem cells (ESCs) transfected by pMHC-neo/SV40-hygro. A stirred bioreactor was used to facilitate the formation of a large number of ESC-derived cardiomyocytes, which were then mixed with human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) in a liquid collagen scaffold to construct highly vascularized cardiac tissue in vitro.RESULTS: The resulting tissue constructs were transplanted into dorsal subcutaneous sites of nude mice. Tumor formation was not detected in all samples and vascularized cardiac tissue could survive after transplantation. Vascularization of the implanted cardiac muscle was significantly enhanced by the addition of HUVECs and MEFs, which resulted in a thicker myocardium. The combination of genetically modified ESCs and stirred bioreactor cultivation not only benefited the large-scale production of pure ESC-derived cardiomyocytes, but also effectively controlled the potential risk of undifferentiated ESCs.CONCLUSIONS: Using liquid collagen as scaffold, the enriched cardiomyocytes derived from genetically modified ESCs mixed with HUVECs and MEFs in 3-dimensional culture resulted in highly vascularized cardiac tissues. J Heart Lung Transplant 2012;31:204-12 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.

Embryonic stem cells (ESCs) can propagate unlimitedly in vitro and differentiate into cardiomyocytes, which have been proposed as unlimited cell sources for cardiac cell therapy. This was limited by difficulties in large-scale generation of pure cardiomyocytes. In this study, we used stirred bioreactors to optimize the differentiation condition for mass production of embryoid bodies (EBs) derived from genetically modified mouse ESCs. Stirred suspension culture could more efficiently produce EBs and have a more uniform EB population without large necrotic centers, compared with the conventional static culture. Importantly, the cardiac-specific gene expressions (GATA binding protein 4, α-cardiac myosin heavy chain and myosin light chain-2v) were increased within EBs cultured in stirred bioreactor. Stirred suspension culture significantly increased the proportion of spontaneously contracting EBs, yielded a greater percentage of α-sarcomeric actinin-positive cells detected via flow cytometry, and harvested relatively more cardiomyocytes after G418 selection. Stirred suspension culture provided a more ideal culture condition facilitating the growth of EBs and enhancing the cardiogenic differentiation of genetically modified ESCs, which may be valuable in large-scale generation of pure cardiomyocytes.

Objective:To investigate the growth and metabolism of Vero cells immobilized cultured on various microcarriers.Methods:Cell viability and cell morphology of the Vero cells cultured in 1%(v/v) newborn bovine serum DMEM/F12,the growth of the Vero cells cultured on 2D MicroHex,Biosilon,Cytodex 1 and Cytopore 1 was evaluated on the viable cell density;The specific consumption rate of glucose(qglc),the specific production rate of lactate(qlac),the specific consumption rate of glutamine(qgln),and the specific consumption rate of glutamate(qglu) were used as the evaluation indexes,the metabolism of the Vero cells cultured on various microcarriers was determined.Results:After 7 d in culture the viable cell density of the Vero cells cultured on 2D MicroHex,Biosilon,Cytodex 1 and Cytopore 1 was 18.4×105 cells/ml,21.9×105 cells/ml,23.9×105 cells/ml and 16.2×105 cells/ml,respectively.And,the Vero cells growth on Cytodex 1 formed compact cell layers with distinct cell morphology;Metabolic indexes of the Vero cells cultured on various microcarriers were basically the same.Conclusion:Among these microcarriers,Cytodex 1 is comparatively good for the growth of Vero cells,and could be used as the favor choice of microcarrier for the large-scale cultivation of Vero cells for viral vaccine production.

Beta-cell transplantation is considered to be the most effective approach to cure type 1 diabetes (T1D). Unfortunately, the scarce availability of donor tissue limits the applicability of this therapy. Recent stem cell research progress shows stem cell therapy may be a potential means to solve this problem. Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewable and multipotent adult stem cells which can differentiate into the three germ layers. Here we aimed to investigate whether MSCs could be reprogrammed into insulin-producing cells (IPCs). We isolated and characterized MSCs obtained from rat bone marrow. Then MSCs were induced to transdifferentiate into IPCs under specific conditions containing high concentrations of glucose, activin A, all-trans retinoic acid, and other maturation factors. The induced cells expressed multiple genes related to pancreatic beta-cell development and function, such as insulin1, glucagon, Pdx1, Pax6, and Glut-2. Insulin1 and C-peptide production were identified by immunocytochemistry. In vitro glucose challenge studies showed the induced cells secreted insulin in a glucose-dependent manner, as do normal pancreatic beta-cells. Transplantation of these MSC-derived insulin-positive cells could reverse the hyperglycemia of streptozotcin (STZ)-induced diabetic rats. These results demonstrated that MSCs could be reprogrammed into IPCs and might be a potential autologous cell source for transplantation therapy of T1D.

Apoptosis plays an important role in limiting viable cell density and production performance in large-scale animal cell culture.Overexpression of anti-apoptotic genes is one of the most common strategies used to improve apoptosis resistance of industrial cells.HEK293 cells with adenoviral anti-apoptotic E1B-19K gene were transfected and picked out several representative clones.These clones were further investigated in the apoptotic percentages under various inducing culture conditions and cell growth and metabolism under normal culture condition.The results showed that the overexpression of E1B-19K in HEK293 cells,endowed cells anti-apoptotic ability by decreasing cell death by 60%~80% under inducing culture conditions with reduced glucose,low serum or without glutamine,delayed cells' entry into decay phase by 2 d under normal culture conditions;and had no significant affect on cell metabolisms of glucose,lactate and glutamine.The above results indicate that the overexpression of E1B-19K is a promising strategy to reduce cell death in mammalian cell culture.

Objective: To investigate the effect of p27 inducible-expression on the growth and metabolism of HEK293 cells.Methods: The vector pTet-on and Dox-responsive p27-expressing vector were co-transfected into HEK293 cells and individual clones were isolated randomly.Cell growth and metabolism were assessed by cell-cycle distribution and viable cell density,Qglc,Qlac and Qgln as indexes,respectively.Results: The expression of p27 increased percentage of G1 cells,reduced consumption of glucose and production of lactate of HEK293 cells.Conclusions: The inducible-expression of p27 is an effective strategy for G1-phase arrest of HEK293 cells.

Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.

Pluripotent embryonic stem cells (ESC) have the ability to differentiate into a variety of cell lineages in vitro , including cardiomyocytes. Successful applications of ESC-derived cardiomyocytes in cell therapy and tissue engineering were limited by difficulties in selecting the desired cells from the heterogeneous cell population. We describe a simple method to generate relatively pure cardiomyocytes from mouse ESCs. A construct comprising mouse cardiac α-myosin heavy chain (MHC) promoter driving the neomycin resistance gene and SV40 promoter driving the hygromycin resistant gene designated pMHCneo/ SV40-hygro, was stably transfected into mouse ESCs. The transgenic ESC line, designated MN6 retained the undifferentiated state and the potential of cardiogenic differentiation. After G418 selection, more than 99% of cells expressed α-sarcomeric actin. Immunocytological and ultrastructural analysis demonstrated that, the selected cardiomyocytes were highly differentiated. Our results represent a simple genetic manipulation used to product essentially pure cardiomyocytes from differentiating ESCs. It may facilitate the development of cell therapy in heart diseases. Key words: Embryonic stem cells, α-myosin heavy chain promoter, cardiomyocytes, differentiation, genetic enrichment.

Objective: To evaluate the influences of constitutively overexpressing human telomerase reverse transcriptase(hTERT) on the growth and metabolism of Vero cells cultured in vitro.Methods: Using the viable cell density,cell viability as the major evaluation indexes,in combination with cell morphology and stretching dynamics,the growth of both the hTERT-overexpressing Vero cell line T1 and wild-type Vero cells cultured in static attachment and suspended microcarriers was evaluated;Using the specific consumption rate of glucose(qglc),specific consumption rate of glutamine(qgln),specific production rate of lactate(qlac) and yield of lactate to glucose(Ylac/glc) as the evaluation indexes,the metabolism of both T1 cells and wild-type Vero cells cultured in static attachment and suspended microcarriers was determined.Results: Constitutively hTERT overexpression in T1 cells resulted in poorer capability of adhesion and extension,and reduced serum dependence,followed by the ability of adhesion independent growth in vitro to some extent.And no significant differences in metabolism between T1 cells and wild-type Vero cells cultured in static attachment and suspended microcarriers were observed.Conclusion: Constitutive overexpression of hTERT could reduce the dependency on serum and adhesion of Vero cells cultured in vitro,which indicated that constitutive overexpression of hTERT is a potential technical approach to optimize the characteristics of animal cells cultured in vitro.

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.