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Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity
Published on: February 24, 2017
Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro
Glyn D Palmer1, Andre Steinert, Arnulf Pascher
1Center for Molecular Orthopaedics, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Adult mesenchymal stem cells (MSCs) have the capacity to differentiate into various connective tissues such as cartilage and bone following stimulation with certain growth factors. However, less is known about the capacity of these cells to undergo chondrogenesis when these proteins are delivered via gene transfer. In this study, we investigated chondrogenesis of primary, bone marrow-derived MSCs in aggregate cultures following genetic modification with adenoviral vectors encoding chondrogenic growth factors. We found that adenoviral-mediated expression of TGF-beta1 and BMP-2, but not IGF-1, induced chondrogenesis of MSCs as evidenced by toluidine blue metachromasia and immunohistochemical detection of type II collagen. Chondrogenesis correlated with the level and duration of expressed protein and was strongest in aggregates expressing 10-100 ng/ml transgene product. Transgene expression in all aggregates was highly transient, showing a marked decrease after 7 days. Chondrogenesis was inhibited in aggregates modified to express >100 ng/ml TGF-beta1 or BMP-2; however, this was found to be partly due to the inhibitory effect of exposure to high adenoviral loads. Our findings indicate that parameters such as these are important functional considerations for adapting gene transfer technologies to induce chondrogenesis of MSCs.
Insights
Gene transfer of TGF-beta1 and BMP-2 effectively induced chondrogenesis in mesenchymal stem cells (MSCs). Optimal results were achieved with moderate protein levels, while high levels or viral loads inhibited the process.
Area of Science:
- Regenerative Medicine
- Molecular Biology
- Biotechnology
Background:
- Adult mesenchymal stem cells (MSCs) can differentiate into cartilage and bone.
- The role of gene transfer in inducing MSC chondrogenesis is not well understood.
Purpose of the Study:
- To investigate chondrogenesis of MSCs using adenoviral gene transfer of growth factors.
- To determine the impact of growth factor expression levels and duration on chondrogenesis.
Main Methods:
- Primary bone marrow-derived MSCs were cultured in aggregates.
- MSCs were genetically modified using adenoviral vectors encoding TGF-beta1, BMP-2, or IGF-1.
- Chondrogenesis was assessed via toluidine blue staining and type II collagen detection.
Main Results:
- Adenoviral expression of TGF-beta1 and BMP-2 induced MSC chondrogenesis.
- Chondrogenesis correlated with protein expression levels (10-100 ng/ml) and duration.
- High expression levels (>100 ng/ml) and high adenoviral loads inhibited chondrogenesis.
Conclusions:
- Gene transfer of specific growth factors can induce MSC chondrogenesis.
- Expression levels, duration, and viral load are critical parameters for successful gene therapy-guided chondrogenesis.
- These findings are important for developing gene transfer strategies for cartilage regeneration.
