Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

Abstract

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 pcDNA3-eGFP 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 vitro Clinical Trials Studywere 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). Ad-BMP2 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 .

Human fetal mesenchymal stem cell secretome enhances bone consolidation in distraction osteogenesis

“…However, the stem cells have poor differentiation and a poor survival rate following their transplantation in vivo that has limited their regenerative potential [ 26 ]. Although the proliferative and differentiation capacities of hFMSCs can be retained and their osteogenic potentials may be enhanced through gene modulation [ 25 , 27 ], the genetic modification procedure is rather complicated and further studies are still needed before their clinical application. In contrast, cell-free secretome harvested from the hFMSC conditioned medium centrifugation is an easy and cost-effective procedure.…”

Dental-Pulp Stem Cells as a Therapeutic Strategy for Ischemic Stroke

“…Lentiviral and retroviral vectors can be used to insert genes into MSCs/DPSCs with high transduction efficiency, although these vectors may induce off-target effects and activate oncogenes owing to insertional mutagenesis [60,69]. The adenovirus vector is effective when transient expression is required in MSCs/DPSCs [60,70]. Adenovirus vectors demonstrate low genotoxicity; however, there is a risk for cytotoxicity at high titers.…”

<p>Growth Factor Gene-Modified Mesenchymal Stem Cells in Tissue Regeneration</p>

“…[31][32][33][34][35][36][37][38][39] Using viral vectors to insert genes into MSCs is a high transduction efficiency approach that has the potential to induce off-target effects owing to insertional mutagenesis. 32,35,40,41 Viral systems are also limited by relatively small transgene cargo capacity, high production cost, difficulties in production and scale-up, and adverse immune reactions. There are advantages and disadvantages to all known viral vectors, with the selection of an appropriate vector being dependent upon transduction rates and the desired duration of treatment and target gene expression.…”