摘要
AIM: To investigate adenoviral transduction in mesenchymal stem cells(MSCs) and effects on stemness in vitro and function as a cell therapy in vivo.METHODS: Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine(PEI)-mediated transfection of pc DNA3-e GFP or adenoviral transduction of green fluorescent protein(GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness(i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2(BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology.RESULTS: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs(81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs(78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs(7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression(0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs(1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitrowere not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult(P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells(P < 0.05). AdBMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs(83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone(1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05).CONCLUSION: Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.
AIM To investigate adenoviral transduction inmesenchymal stem cells （MSCs） and effects onstemness in vitro and function as a cell therapy in vivo .METHODS： Bone marrow-derived adult and fetal MSCwere isolated from an equine source and expandedin monolayer tissue culture. Polyethylenimine （PEI）-mediated transfection of pcDNA3-eGFP or adenoviraltransduction of green fluorescent protein （GFP）was evaluated in fetal MSCs. Adenoviral-mediatedtransduction was chosen for subsequent experiments. Allexperiments were carried out at least in triplicate unlessotherwise noted. Outcome assessment was obtained byflow cytometry or immunohystochemistry and includedtransduction efficiency, cell viability, stemness （i.e. ,cell proliferation, osteogenic and chondrogenic celldifferentiation）, and quantification of GFP expression.Fetal and adult MSCs were then transduced with anadenoviral vector containing the gene for the bonemorphogenic protein 2 （BMP2）. In vitro BMP2 expressionwas assessed by enzyme linked immunosorbent assay.In addition, MSC-mediated gene delivery of BMP2 wasevaluated in vivo in an osteoinduction nude mousequadriceps model. New bone formation was evaluated bymicroradiography and histology.RESULTS： PEI provided greater transfection andviability in fetal MSCs than other commercial chemicalreagents. Adenoviral transduction efficiency wassuperior to PEI-mediated transfection of GFP in fetalMSCs （81.3% ± 1.3% vs 35.0% ± 1.6%, P 〈 0.05） andwas similar in adult MSCs （78.1% ± 1.9%）. Adenoviraltransduction provided significantly greater expressionof GFP in fetal than adult MSCs （7.4 ± 0.1 vs 4.4 ± 0.3millions of mean fluorescence intensity units, P 〈 0.01）as well as significantly greater in vitro BMP2 expression（0.16 pg/cell-day vs 0.10 pg/cell-day, P 〈 0.01）. Fractionof fetal MSC GFP positive cells decreased significantlyfaster than adult MSCs （1.15% ± 0.05% vs 11.4% ±2.1% GFP positive at 2 wk post-transduction, P 〈 0.05）.Cell proliferation and osteogenic differentiation in vitro were not affected by Ad transduction in both fetal andadult MSCs, but fetal MSCs had reduced chondrogenicdifferentiation in vitro when compared to adult （P 〈0.01）. Chondrogenic differentiation was also significantlyreduced in Ad-GFP transduced cells （P 〈 0.05）. Ad-BMP2 transduced adult MSCs induced new boneformation in more thighs than Ad-BMP2 transduced fetalMSCs （83% vs 17% of the six treated thighs per group,P 〈 0.05） and resulted in increased femur midshaftdiameter due to greater extent of periosteal new bone（1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P 〈 0.05）.CONCLUSION： Fetal MSCs may be genetically manipulatedex vivo with adenoviral vectors. Nonetheless, theabbreviated expression of the exogenous gene may limittheir applications in vivo .
基金
The Ohio State University College of Medicine Roessler Research Scholarship(In part)
in part by National Cancer Institution of the United States grant No.P30 CA016058(Our histological examination was performed at The OSU Comparative Pathology and Mouse Phenotyping Shared Resource)
