3 research outputs found
Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses
Background/Aims: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. Methods: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. Results: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. Conclusion: The RAPA cells are highly efficient for adenovirus production and amplification
Gelatin-Derived GrapheneāSilicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells
Graphene-based
materials are used in many fields but have found only limited applications
in biomedicine, including bone tissue engineering. Here, we demonstrate
that novel hybrid materials consisting of gelatin-derived graphene
and silicate nanosheets of Laponite (GL) are biocompatible and promote
osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous
cell attachment, long-term proliferation, and osteogenic differentiation
of MSCs on a GL-scaffold were confirmed using optical microscopy and
scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold
were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic
differentiation. GL-powders increased the alkaline phosphatase (ALP)
activity in immortalized mouse embryonic fibroblasts but decreased
the ALP activity in more-differentiated immortalized mouse adipose-derived
cells. Note, however, that GL-powders promoted BMP9-induced calcium
mineral deposits in both MSC lines, as assessed using qualitative
and quantitative alizarin red assays. Furthermore, the expression
of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARĪ³ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms