Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6.

Current medical treatments for IVD degeneration rely on conservative therapies or surgery. Surgical treatments (e.g. spinal fusion,) have shown satisfactory results in alleviating pain, but long-term clinical outcomes remain poor. Thus, there is an urgent need for alternative cell based regenerative therapies focussed on correcting the underlying pathogenesis of IVD degeneration. However, for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix must be identified. Additionally, extensive in vitro studies are needed to establish and support further pre-clinical and potential commercial development. We have demonstrated that stimulation of both BM-MSCs and AD-MSCs with GDF6 results in improved differentiation to a nucleus pulposus (NP)-like phenotype and synthesis of proteoglycan rich matrix with micromechanical properties akin to the healthy IVD. Significantly, these studies have highlighted that AD-MSCs are the more appropriate cell source. Furthermore, our studies have shown hat GDF6 has anabolic effects on degenerate human NP cells, stimulating adoption of a more normal NP phenotype and increasing appropriate atrix synthesis. This suggests that delivery of GDF6 as part of an MSC-based therapy may be beneficial both in directing lineage-specific MSC differentiation, but also in restoring a more anabolic phenotype in native NP cells, thereby having a dual regenerative effect.

Clinical trials are underway to elucidate a successful MSC-based therapy for the repair and regeneration of intervertebral disc (IVD) tissue. Currently, there is a lack of knowledge surrounding the relationship between naïve MSCs and the inflammatory microenvironment of the degenerate disc. To inform a phase II clinical trial, this study tests the hypothesis that cytokines, IL-1ß and TNFα regulate the expression of neuropeptides and neurotrophic factors from MSCs, thus exacerbating pain in those patients that have the presence of sensory nerve fibres within the IVD. Patient-matched MSCs derived from bone marrow (BM) or adipose (AD) tissue were stimulated with IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours in either 21% or 5% O2. qRT-PCR was performed to assess expression of trophic factors involved in the survival or nerves (NGF & BDNF), blood vessels (VEGF) as well as pain related peptides (SP & CGRP) and inflammatory factors. Conditioned culture medium was analysed using ELISAs to identify secretion of soluble factors. IL-1β did not regulate neurotrophic factor expression from BM-MSCs under normoxic or hypoxic conditions. However, TNFα increased NGF, BDNF, SP and CGRP under normoxic conditions. In ADMSCs, VEGF was increased following IL-1β and TNFα stimulation; with TNFα also increasing NGF and CGRP under normoxic conditions. When exposed to hypoxia, the trophic effect of TNFα on human BM-MSCs was reduced. Overall this data suggests a role for priming or pre-stimulation of naïve MSCs prior to implantation to prevent exacerbation of pain from sensory nerve fibres.