Ching-Kuang Chuang

Baculovirus transduction of mesenchymal stem cells triggers the toll-like receptor 3 pathway.

ABSTRACT Human mesenchymal stem cells (hMSCs) can be genetically modified with viral vectors and hold promise as a cell source for regenerative medicine, yet how hMSCs respond to viral vector transduction remains poorly understood, leaving the safety concerns unaddressed. Here, we explored the responses of hMSCs against an emerging DNA viral vector, baculovirus (BV), and discovered that BV transduction perturbed the transcription of 816 genes associated with five signaling pathways. Surprisingly, Toll-like receptor-3 (TLR3), a receptor that generally recognizes double-stranded RNA, was apparently upregulated by BV transduction, as confirmed by microarray, PCR array, flow cytometry, and confocal microscopy. Cytokine array data showed that BV transduction triggered robust secretion of interleukin-6 (IL-6) and IL-8 but not of other inflammatory cytokines and beta interferon (IFN-β). BV transduction activated the signaling molecules (e.g., Toll/interleukin-1 receptor domain-containing adaptor-inducing IFN-β, NF-κB, and IFN regulatory factor 3) downstream of TLR3, while silencing the TLR3 gene with small interfering RNA considerably abolished cytokine expression and promoted cell migration. These data demonstrate, for the first time, that a DNA viral vector can activate the TLR3 pathway in hMSCs and lead to a cytokine expression profile distinct from that in immune cells. These findings underscore the importance of evaluating whether the TLR3 signaling cascade plays roles in the immune response provoked by other DNA vectors (e.g., adenovirus). Nonetheless, BV transduction barely disturbed surface marker expression and induced only transient and mild cytokine responses, thereby easing the safety concerns of using BV for hMSCs engineering.

The repair of osteochondral defects using baculovirus-mediated gene transfer with de-differentiated chondrocytes in bioreactor culture

Baculovirus has emerged as a promising gene delivery vector. Hereby de-differentiated rabbit chondrocytes were transduced ex vivo with a recombinant baculovirus expressing BMP-2 (Bac-CB), seeded to scaffolds and cultured statically for 1 day (Bac-w0 group) or in a rotating-shaft bioreactor (RSB) for 1 week (Bac-w1 group) or 3 weeks (Bac-w3 group). Mock-transduced constructs were cultured statically for 1 day to serve as the control (Mock-w0 group). We unraveled that Bac-CB transduction and increasing culture time in the RSB yielded more mature cartilaginous constructs in vitro. Eight weeks after implanting into the rabbit osteochondral defects, Mock-w0 constructs failed to repair the lesion while Bac-w0 constructs resulted in augmented, yet incomplete, repair. Bac-w1 constructs yielded neocartilage layers rich in glycosaminoglycans and collagen II, but the integration between the graft and host cartilages was not complete. In contrast, Bac-w3 constructs led to the regeneration of hyaline cartilages as characterized by cartilage-like appearance, improved integration, chondrocytes clustered in lacunae, smooth and homogeneous matrix rich in collagen II and glycosaminoglycans but deficient in collagen I. In conclusion, combining baculovirus-modified de-differentiated chondrocytes and RSB culture creates constructs that repair osteochondral defects, and in vitro culture time dictates the construct maturation and subsequent in vivo repair.

Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression

Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximately 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.

Baculovirus Transduction of Mesenchymal Stem Cells: In Vitro Responses and In Vivo Immune Responses After Cell Transplantation

Baculovirus holds great promise for the genetic modification of mesenchymal stem cells (MSCs). However, whether baculovirus transduction provokes undesired MSCs responses that might compromise their in vivo applications has yet to be examined. Hereby, we unraveled that baculovirus transduction of human MSCs upregulated the transcription of interleukin (IL)-1beta, interferon (IFN)-alpha and IL-6, but not tumor necrosis factor (TNF)-alpha and IFN-gamma. However, only IL-6 secretion was detectable by enzyme-linked immunosorbent assay (ELISA). Baculovirus transduction also stimulated transient, low level upregulation of human leukocyte antigen I (HLA-I) on the human MSCs surface, yet it did not either altered the HLA-II expression or impaired the MSCs ability to inhibit lymphocyte proliferation. After transplantation into allogeneic rats, the transduced rat MSCs elicited transient, mild macrophage responses, but the cells remained tolerant as judged by the persistence of transplanted cells and absence of CD8(+) T cells infiltration. Besides, transplantation of the transduced MSCs did not provoke systemic induction of monocytes and CD8(+) T cells. This study, for the first time, explores the responses of MSCs to virus transduction and confirms the safety of transplanting baculovirus-engineered MSCs into immunocompetent animals for cell-based gene therapy.

Co-Conjugating Chondroitin-6-Sulfate/Dermatan Sulfate to Chitosan Scaffold Alters Chondrocyte Gene Expression and Signaling Profiles

Co‐conjugating chondroitin‐6‐sulfate (CSC) and dermatan sulfate (DS) to chitosan scaffolds improves chondrocyte differentiation and extracellular matrix (ECM) production. To further elucidate the cellular responses to CSC/DS conjugation, gene expression profiles for the rat chondrocytes cultured on the CSC/DS/chitosan and chitosan‐only scaffolds were compared by reverse‐transcription PCR (RT‐PCR) and quantitative real‐time RT‐PCR (qRT‐PCR). Our data unraveled that the CSC/DS/chitosan scaffold resulted in low‐level expression of collagen I, IIA and X and potentiated the aggrecan, collagen II (including collagen IIB) and TIMP3 expression, but downregulated the decorin expression. Therefore CSC/DS/chitosan scaffold maintained the chondrocyte differentiation while minimized de‐differentiation and hypertrophy. Furthermore, CSC/DS conjugation affected the expression of 11 genes implicated in 9 signaling pathways (as unveiled by cDNA microarray) and upregulated the expression of TGF‐β1, Sox9, BMP2, PTHrP and Ihh (as confirmed by qRT‐PCR). These data suggested that the CSC/DS/chitosan scaffold potentiated the TGF‐β and Hedgehog pathways, which activated the expression of PTHrP and its downstream Sox9. The signals were transduced to elevate the expression of aggrecan, collagen II and TIMP3, and contributed to the well‐differentiated chondrocyte phenotype. Altogether, this study for the first time elucidated the roles of GAGs‐conjugated biomaterials in matrix production and breakdown, cellular differentiation and signal transduction at the molecular levels. Biotechnol. Bioeng. 2008;101: 821–830.

Baculovirus-mediated growth factor expression in dedifferentiated chondrocytes accelerates redifferentiation: effects of combinational transduction.

Transduction of partially dedifferentiated rabbit chondrocytes with a baculovirus (Bac-CB) expressing bone morphogenetic protein-2 (BMP-2) reverses dedifferentiation and enhances matrix production. Hereby we examined whether transduction with Bac-CB in combination with another baculovirus expressing transforming growth factor-beta1 (TGF-beta1) or insulin-like growth factor-1 (IGF-1) synergistically augmented chondrogenic differentiation. Passage 3 rabbit articular chondrocytes were transduced by different baculovirus combinations: single transduction with Bac-CB, cotransduction with Bac-CB and Bac-CT (expressing TGF-beta1), cotransduction with Bac-CB and Bac-CI (expressing IGF-1), and transduction with Bac-CB followed by repeated transduction with Bac-CT, Bac-CI, or Bac-CB 5 days later. Transduced cells were encapsulated into alginate beads for culture. Among these strategies, only cotransduction with Bac-CB and Bac-CT led to improved redifferentiation when compared with Bac-CB single transduction, as evidenced by the enhanced expression of aggrecan and collagen IIB (Col IIB), suppressed expression of Col I and Col X, emergence of chondrocyte-specific lacunae, and elevated deposition of matrix molecules. The cotransduction also accelerated the expression of Sox9, Col IIB, and aggrecan. In summary, baculovirus-mediated coexpression of TGF-beta1 and BMP-2 synergistically accelerates the chondrocyte redifferentiation process and improves the maintenance of chondrocyte phenotype and accumulation of cartilage-specific matrix molecules.

Electron emitters synthesized by selected area deposition of carbon nanotubes on silicon substrates

Abstract We have successfully attained patterned carbon nanotubes grown on two-dimensional Ni arrays of square blocks of various sizes on Si by a microwave-heated chemical vapor deposition process. The Ni blocks are either freestanding or isolated by the silicon dioxide. For patterned carbon nanotube emitters, grown on freestanding Ni blocks, the emission current–density increases with the size of the Ni blocks. To the contrary, patterned carbon nanotube emitters grown on 2 μm by 2 μm Ni blocks isolated by the silicon diode have exhibited an emission behavior as excellent as the un-patterned emitters; both emitters have very low turn-on and threshold fields being at ∼0.1 V/μm and ∼1.50 V/μm, respectively and can emit current density exceeding 120 mA/cm2.

Baculovirus transduction of chondrocytes elicits interferon-α/β and suppresses transgene expression

Baculovirus is an effective vector for gene delivery into primary chondrocytes and repeated baculovirus transduction (i.e. supertransduction) appears to be promising for prolonging transgene expression, but how supertransduction may influence baculovirus‐mediated gene delivery is unknown.

Effect of nickel thickness and microwave power on the growth of carbon nanotubes by microwave-heated chemical vapor deposition

The effect of Ni thickness and microwave power on the growth of carbon nanotubes (CNTs) by microwaveheated chemical vapor deposition is reported. A 5-100-nm-thick nickel layer was deposited with an e-gun in a vacuum of 10-6 Torr. It was found that the diameter and length of CNTs increase with Ni layer thickness. The emission I-V curves clearly show two groups of characteristics marked off at the Ni thickness of 50 nm. The low field emission for those films grown on nickel thickness below 50 nm is consistent with the carbonaceous particles and carbon overlayers observed using SEM. The microwave power that determines the substrate temperature also affects the morphology and emission property of CNT films. A CNT film grown on 80-nm-thick Ni layer at 900 W for 18 min has shown excellent emission characteristics with very low turn-on field of 0.056 V/µm and a high current density of 160 mA/cm2 at 4.5 V/µm, which is comparable to the best field emission samples ever reported.

Baculovirus-Mediated Gene Transfer into Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- and gene-based therapies. Additionally, baculovirus has emerged as a novel gene therapy vector because of its large cloning capacity for insertion of multiple genes, its minimal cytotoxic effects, and its inability to replicate in mammalian cells. These features have prompted efforts to employ baculovirus vectors carrying mammalian expression cassettes for gene delivery into MSCs. This chapter demonstrates the use of GFP expression to monitor baculovirus-mediated gene transfer into MSCs.