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ABSTRACT: 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.
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.
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.
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.
The Journal of heart and lung transplantation: the official publication of the International Society for Heart Transplantation 02/2012; 31(2):204-12. · 3.54 Impact Factor
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ABSTRACT: 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.
Biological & Pharmaceutical Bulletin 01/2012; 35(3):308-16. · 1.66 Impact Factor
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ABSTRACT: 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.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2011; 27(8):1198-205.
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ABSTRACT: 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.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2011; 27(8):1225-31.
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ABSTRACT: Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2011; 27(2):240-6.
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ABSTRACT: With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2010; 26(8):1116-22.
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ABSTRACT: By using the cell density, cell viability, Pro-UK activity, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)), yield of lactate to glucose (Y(lac/glc)) and as the evaluation indexes, the growth and metabolism characteristics of pro-urokinase (Pro-UK) expressing CHO cells in serum-free suspension batch culture were examined and compared to those in serum-containing suspension batch culture. We observed hardly differences in growth and metabolism characteristics between the CHO cell populations grown in serum-free suspension batch culture and serum-containing suspension batch culture. The optimal mathematical model parameters for the CHO cells grown in suspension batch culture were obtained by non-linear programming of data representing the growth, substrate consumption and product formation of the CHO cells during logarithmic growth phase using MATLAB software, and the kinetic model of the cell growth and metabolism in serum-free culture were established.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 01/2010; 26(1):85-92.
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ABSTRACT: We constructed and identified cardiac-specific a-myosin heavy chain (alpha-MHC) promoter-driven expression vector. alpha-MHC promoter was amplified by PCR by using mouse genomic DNA as template and inserted into pGEM-T Easy vector. The inserted fragment was released by enzyme digestion, and then the cytomegalovirus (CMV) promoter in pcDNA3.1(+)-EGFP-hygro vector was replaced by the alpha-MHC promoter to construct alpha-MHC-EGFP expression vector. After identification with enzyme digestion, alpha-MHC-EGFP was transfected into mouse primary cardiomyocytes by electroporation. Green fluorescence could be observed in transfected cardiomyocytes, but not in transfected non-cardiomyocytes. Alpha-MHC-EGFP expression vector was specifically expressed in cardiomyocytes, and could be used to purify embryonic stem cell-derived cardiomyocytes.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 10/2009; 25(10):1546-51.
