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Yu-Chen Hu

Publications and source records attributed to Yu-Chen Hu.

At least 19 recordsLinked to original sources
PubMed2007

Variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs) and MSCs-derived adipogenic, chondrogenic, and osteogenic progenitors without compromising the differentiation capacity. Remarkably, the transgene expression level and duration varied widely with the differentiation states at which the progenitors were transduced. However, whether the variation was a general phenomenon and what caused the variation were unclear. Here we demonstrated that transduction of the MSCs and MSC-derived progenitors using baculoviruses carrying egfp driven by CMV, EF-1alpha or CAG promoter resulted in a general trend of varied expression, that is, the chondrogenic progenitors allowed for the poorest expression while the adipogenic progenitors conferred the best expression. Quantification of the nuclear and cytoplasmic egfp gene copy numbers by quantitative real-time PCR revealed that the varied expression did not arise from the discrepancies in gene delivery efficiency nor was it due to the disparities in nuclear transport efficiency. In contrast, the transcription levels paralleled the overall expression levels. These data suggested that although the egfp genes could be efficiently delivered into the nuclei of chondrogenic progenitors, they were not transcribed as well as they were in the adipogenic progenitors. In conclusion, the rapidly altering cellular transcription machinery in the course of differentiation progression predominantly led to the varied expression levels.

Baculoviridae↗
PubMed2007

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle.

BACKGROUND: We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene-based cartilage tissue engineering. However, baculovirus-mediated gene expression in the chondrocytes is transient. METHODS: In this study, we attempted to prolong the expression by supertransduction, but uncovered that after long-term culture the chondrocytes became more refractory to baculovirus transduction. Therefore, the correlation between baculovirus-mediated enhanced green fluorescent protein (EGFP) expression and cell cycle was investigated by comparing the cycling chondrocytes and chondrocytes rich in quiescent cells, in terms of EGFP expression, virus uptake, cell cycle distribution, nuclear import and methylation of viral DNA. RESULTS: We demonstrated, for the first time, that baculovirus-mediated transduction of chondrocytes is correlated with the cell cycle. The chondrocytes predominantly in G2/M phase were approximately twice as efficient in EGFP expression as the cycling cells, while the cells in S and G1 phases expressed EGFP as efficiently as the cycling cells. Notably, the chondrocyte populations rich in quiescent cells resulted in efficient virus uptake, but less effective nuclear transport of baculoviral DNA and higher degree of methylation, and hence poorer transgene expression. CONCLUSIONS: These findings unravel the practical limitations when employing baculovirus in cartilage tissue engineering. The implications and possible solutions are discussed.

Active Transport, Cell Nucleus↗
PubMed2006

Bioreactors for tissue engineering.

Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.

Animals↗
PubMed2006

Accelerated induction of apoptosis in insect cells by baculovirus-expressed SARS-CoV membrane protein.

It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.

Animals↗
PubMed2006

Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells.

AIM: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg). METHODS: A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) was constructed to transduce baby hamster kidney (BHK) cells by a simplified transduction protocol. RESULTS: The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles. CONCLUSION: The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive post-translational modifications.

Animals↗
PubMed2006

Determination of the baculovirus transducing titer in mammalian cells.

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.

Baculoviridae↗
PubMed2006

Expression, purification and characterization of enterovirus-71 virus-like particles.

AIM: Enterovirus 71 (EV71) has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS: The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses: Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS: Both single infection by Bac-P1-3CD and co-infection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins self-assembled into clusters of virus-like particles (VLP) resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION: Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.

Animals↗
PubMed2006

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering.

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.

Aggrecans↗
PubMed2006

Baculovirus vectors for gene therapy.

Since the discovery that baculoviruses can efficiently transduce mammalian cells, baculoviruses have been extensively studied as potential vectors for both in vitro and in vivo gene therapy. This chapter reviews the history of this research area, cells permissive to baculovirus transduction, factors influencing transduction and transgene expression, efforts to improve transduction, mechanisms of virus entry and intracellular trafficking, applications for in vivo and ex vivo gene therapy, as well as advantages, limitations, and safety issues concerning use of baculoviruses as gene therapy vectors. Recent progress and efforts directed toward overcoming existing bottlenecks are emphasized.

Animals↗
PubMed2005

Rational development of GAG-augmented chitosan membranes by fractional factorial design methodology.

To develop a novel biomaterial for chondrocyte culture, 8 glycosaminoglycan (GAG)/chitosan membranes (groups N1-N8) were prepared, with the aid of a 2-level 2(4-1) fractional factorial design, by co-immobilizing chondroitin-4-sulfate (CSA), chondroitin-6-sulfate (CSC), dermatan sulfate (DS), and heparin to chitosan membranes. The fractional factorial design allowed us to partly interpret the effects of individual GAGs and two-way interactions between GAGs. Within the level range of -1 and +1, low CSA level (2.6 mg) is favorable for collagen synthesis but not for cell proliferation. High CSC level (1.3 mg) is favorable for GAG production but not for cell proliferation. Conversely, high heparin (0.33 mg) and DS (0.13 mg) levels are desired for cell proliferation but not for the production of collagen and GAG. Moreover, the two-way interactions between GAGs influence the cell behavior. Among the 8 GAG/chitosan membranes, N1 and N4 (containing low CSA and heparin levels) lead to the maintenance of proper chondrocyte phenotype, as judged by the chondrocyte-like morphology, modest cell expansion, higher GAG and collagen production and proper cartilage marker gene expression. In conclusion, this approach provides a means of rationally predicting and evaluating the proper formulation of GAG/chitosan membranes and may facilitate the rational design of other tissue engineering scaffolds.

Animals↗
PubMed2005

Baculovirus-mediated production of HDV-like particles in BHK cells using a novel oscillating bioreactor.

We have recently demonstrated the assembly of hepatitis delta virus-like particles (HDV VLP) by co-transducing hepatoma cells using two recombinant baculoviruses, one encoding hepatitis B surface antigen (HBsAg), and one encoding large delta antigen (L-HDAg). In this study, we further demonstrated the assembly and secretion of VLP in other mammalian cells. The assembly efficiency varied depending on cell lines, the baculovirus constructs and the relative dosage of both recombinant viruses. The co-transduction of BHK cells led to the formation of VLPs resembling authentic virions in size and appearance. The production process was transferred to a novel oscillating packed bed bioreactor, BelloCell, in which the transduction efficiency was up to approximately 90% for a high cell density of 1.5 x 10(7) cells/cm(3) bed and a total yield of 427 microg based on HBsAg in the VLP (harvested from 940 ml medium) was obtained. The particle yield corresponded to an average volumetric yield of 454 ngml(-1) and a specific yield of 285 microg/10(9) cells, and is significantly superior to that can be obtained by the commonly employed transfection method. The combination of baculovirus transduction and BelloCell reactor, thus, may represent a simple and efficient approach for the production of HDV VLP and viral vectors.

Animals↗
PubMed2005

Baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells.

Baculovirus has been employed for a wide variety of applications. In this study, we further expanded the application to the high-level expression of hepatitis delta virus (HDV) antigens and the formation of virus-like particles (VLP) in transduced mammalian cells. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen (L-HDAg) and hepatitis B surface antigen (HBsAg) under mammalian promoters. With a simplified transduction protocol using unconcentrated virus, high transduction efficiencies were achieved in hepatoma cells, in which L-HDAg and HBsAg were expressed abundantly, allowing for easy colorimetric detection in Western blots. L-HDAg alone was nucleus-bound and HBsAg alone was secreted; formation and secretion of HDV-like particles were readily detected upon coexpression, indicating that the baculovirus-expressed proteins were processed correctly as the authentic proteins. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus. Furthermore, superinfection introduced more baculovirus DNA into cells in the long-term culture as revealed by Q-PCR, thereby enhancing and prolonging the expression. In summary, baculovirus transduction can be an attractive method as an alternative to the plasmid transfection commonly employed for HDV research thanks to the significantly higher gene delivery efficiencies as well as the abundant expression and proper processing. Baculovirus can also be envisaged as a useful tool for investigating protein-cell interactions and virus assembly.

Animals↗
PubMed2005

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS: A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS: In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS: These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.

Adipocytes↗
PubMed2005

Expression and purification of N and E proteins from severe acute respiratory syndrome (SARS)-associated coronavirus: a comparative study.

Histidine-tagged N (rNH) and E (rEH) proteins of Severe Acute Respiratory Syndrome (SARS)-coronovirus were expressed in the baculovirus/insect cell system and purified by immobilized metal affinity chromatography. rNH and rEH proteins differed markedly with respect to expression levels, cell death kinetics and subcellular localizations that led to different extraction and purification schemes. The features of both proteins are compared and the potential applications of purified rNH and rEH are discussed.

Animals↗
PubMed2005

A novel oscillating bioreactor BelloCell: implications for insect cell culture and recombinant protein production.

BelloCell is a novel packed bed bioreactor that allows alternating nutrient and gas transfer to a culture. Spodoptera frugiperda Sf-9 grown in the BelloCell (300 ml culture) reached 1.3-1.5x10(7) cells ml-1 in 7-8 days and the total baculovirus-expressed protein yield was 2.3-times that in a stirred tank bioreactor (600 ml culture). The superior cell and protein yields underline the potential of BelloCell for cell culture and recombinant protein production.

Animals↗
PubMed2004

Highly efficient baculovirus-mediated gene transfer into rat chondrocytes.

To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.

Animals↗
PubMed2004

Investigation of optimal transduction conditions for baculovirus-mediated gene delivery into mammalian cells.

Although baculovirus-mediated gene delivery into mammalian cells has been documented in a wealth of the literature, systematic investigation of the optimal transduction conditions remains unavailable. In this work, a transduction protocol using unconcentrated baculovirus is proposed for simple and efficient gene delivery into HeLa cells. We found that approximately 75-85% of the cells could be readily transduced and express the reporter protein when virus transduction occurred for 4 h at 25 degrees C using Dulbecco's phosphate-buffered saline (D-PBS) as the surrounding solution. This method contrasts with previous protocols in which transduction occurs for 1 h at 37 degrees C using growth medium (e.g., DMEM) as the surrounding solution. Investigation of the physical parameters led to the findings that: 1) baculovirus uptake by HeLa cells continued for at least 4 h in the event of high virus dosage, which led to higher gene expression; 2) the half-life of baculovirus dramatically decreased at 37 degrees C; 3) EGTA pretreatment did not apparently facilitate the gene delivery when the cells grew to multilayers; and 4) lower transduction efficiency and gene expression were obtained when DMEM was used (in comparison with D-PBS and TNM-FH), suggesting that DMEM contains certain inhibitory factors for baculovirus transduction. Our data uncovered several aspects that were not investigated before and the optimized transduction conditions allowed for gene delivery as efficient as that by the protocols commonly employed by others, but eliminated the need for virus ultracentrifugation. The protocol not only represented a simpler approach, but also considerably reduced possible virus inactivation during ultracentrifugation, thus making it easier to convert the baculovirus/mammalian cell system to a tool for eukaryotic protein production on a larger scale.

Animals↗