Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System
Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration. 1. Introduction Nonviral gene delivery is of great interest to stimulate cells for a direct potential clinical application for a wide range of musculoskeletal diseases. For repair and regeneration of the spinal intervertebral disc, cells would be required to match the native population in the intervertebral disc (IVD) niche. The nucleus pulposus cells, at the centre of the disc, and the annulus fibrosus cells, which populate the IVD “niches,” are difficult to reproduce in the laboratory since the unique markers to identify these cells are not known yet [1–4]. The IVD niche is defined as a low pH, very dense extracellular matrix consisting of collagen and glycosaminoglycan such as aggrecan and relative low cellularity [5, 6]; therefore the disc environment causes a major challenge for implanted cells such as MSCs. One strategy if cells are to be transplanted into a complex niche such as the IVD, which is populated by highly specialised and perfectly adapted native cell population, is to precondition the cells with some specialised growth factor (GF) cocktail (which is not known yet) or by additional mechanical stimuli [7]. Without doubt bone marrow derived mesenchymal stem cells (MSCs) have been proposed in many fields of musculoskeletal research because they can be isolated relatively easily, show a fast proliferation, and hold the potency to differentiate into different
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