Wen-Song Tan

Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34+ cells

Hypoxia maintained biological characteristics of CD34(+) cells through keeping lower intracellular reactive oxygen specials (ROS) levels. The effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state were compared in this study. Hypoxia decreased the mRNA expression of both catalase (CAT) and glutathione peroxidase (GPX), but not affected mRNAs expression of superoxide dismutase (SOD). While the cellular GPX activities under hypoxia were apparently less than those under normoxia, neither SOD activities nor CAT activities were affected by hypoxia. The analysis of glutathione redox status and ROS products showed the lower oxidized glutathione (GSSG) levels, the higher reduced glutathione (GSH) levels, the higher GSH/GSSG ratios, and the less O(2)- and H(2)O(2) generation under hypoxia (versus normoxia). Meanwhile more primary CD34(+)CD38(-) cells were obtained when cultivation was performed under hypoxia or with N-acetyl cysteine (the precursor of GSH) under normoxia. These results demonstrated the different responses of anti-oxidative mechanism between normoxia and hypoxia. Additionally, the present study suggested that the GSH-GPX antioxidant system played an important role in HSPCs preservation by reducing peroxidation.

The efficacy of combination therapy using adeno-associated virus-TRAIL targeting to telomerase activity and cisplatin in a mice model of hepatocellular carcinoma.

PurposeTNF-related apoptosis-inducing ligand (TRAIL) functions as a soluble cytokine and has been demonstrated significant antitumor activity against a variety of cancer cell lines without toxicity to most normal cells. Cisplatin is a potent anticancer agent and is widely used in the clinical for treatment of human cancers. Adeno-associated virus (AAV2) is a promising gene delivery vehicle for its advantage of low pathogenicity and long-term gene expression. However, lack of tissue specificity caused low efficiency of AAV transfer to target cells. The promoter of human telomerase reverse transcriptase (hTERT) is a good candidate to enhance targeting efficiency of AAV in cancer cells. Although AAV-mediated TRAIL controlled by hTERT promoter (AAV-hTERT-TRAIL) has obvious antitumor activity, the tumor cannot be completely eradicated. In this study, we first examined the effectiveness of combination therapy of cisplatin and AAV-hTERT-TRAIL on human hepatocellular carcinoma (HCC) in vitro and in vivo.MethodsFor in vitro experiments, tumor cell lines were treated with cisplatin, virus, or both. The transgene TRAIL expression controlled by hTERT promoter was evaluated in BEL7404 HCC cell line. Cytotoxicity was performed by MTT analysis. Cell apoptosis was detected by flow cytometry analysis. The in vivo antitumor efficacy of combination treatment with cisplatin and AAV-hTERT-TRAIL was assessed in human hepatocellular carcinoma xenografts mouse model.ResultsThe enhanced TRAIL expression was observed in BEL7404 cells treated with AAV-hTERT-TRAIL plus cisplatin. Treatment with both AAV-hTERT-TRAIL and cisplatin exhibited stronger cytotoxicity and induced more significant apoptosis in cancer cells compared with AAV-hTERT-TRAIL or cisplatin alone, respectively. Moreover, in animal experiments, the combined treatment greatly suppressed tumor growth and resulted in tumor cell death.ConclusionsAAV-mediated therapeutic gene expression in combination with chemotherapy provides a promising therapeutic strategy for human cancers. These data suggest that combined use of AAV-hTERT-TRAIL and cisplatin may have potential clinical application.

A comparative gene-expression analysis of CD34+ hematopoietic stem and progenitor cells grown in static and stirred culture systems

[This corrects the article DOI: 10.2478/s11658-006-0039-x.].

Hematopoietic reconstitution of CD34+ cells grown in static and stirred culture systems in NOD/SCID mice.

The hematopoietic reconstitution of cord blood (CB) CD34+ cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.

Effect of culture temperature on TNFR-Fc productivity in recombinant glutamine synthetase-chinese hamster ovary cells

Lowering the culture temperature is often useful to improve the production of many recombinant proteins in Chinese hamster ovary (CHO) cells. Batch cultivation of GS-CHO cells expressing TNFR-Fc antibody was therefore carried out at 30, 33.5 and 37°C. TNFR-Fc productivity, qTNFR-Fc, increased as culture temperature decreased; and the maximum qTNFR-Fc was 20xa0mg/(109 cells·day) at 30°C which was three times that at 37°C. Increasing the viable cell density (VCD) to above 2.2xa0×xa0106 cells/ml, however, decreased the qTNFR-Fc at 30°C, which was due to a reduction in transcription of the TNFR-Fc gene. Taken together, lowering temperature can improve qTNFR-Fc but the negative effect of increasing VCD compromises this effect. Further process development addressing the issue of cell density-dependent TNFR-Fc productivity is therefore needed.

Differential gene expression of human CD34+ hematopoietic stem and progenitor cells before and after culture.

Ex vivo expanded CD34+ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34+ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34+ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing.

Effects of telomerase activity and apoptosis on ex vivo expansion of cord blood CD34+ cells

Ex vivo expansion of CD34+ cells has become critically important in order to obtain sufficient haematopoietic stem cells for clinical application. Among major regulators involved in ex vivo expansion, telomerase activity and apoptosis have been revealed to be closely linked to cell cycle progression. However, all exact roles remain to be elucidated. Here, change in telomerase activity and level of apoptosis in cord blood (CB) CD34+ cells were evaluated together with specific cell population growth rate during ex vivo culture.

The improvement of adenovirus vector production by increased expression of coxsackie adenovirus receptor

Adenoviruses are promising vectors for gene therapy. The production of adenoviral vectors (AdV), however, is limited by the cell density effect, namely when cell infection is performed at above 106 cells/ml, a drop in cell-specific adenovirus productivity occurs. Our results also show that the coxsackie adenovirus receptor (CAR) plays an important role in AdV production. CAR expression of infected cells varied with culture time and the cell-specific AdV productivity dropped rapidly along with decreased CAR expression. Furthermore, CAR expression of cells was maintained at a high level by replacing the medium or supplementing it with trichostatin A, which could improve the cell-specific productivity. Thus, a higher CAR expression level at infection time could improve cell-specific AdV productivity at high cell densities.