Novel regenerative therapies have the potential to restore function and relieve pain in patients with low back pain (LBP) caused by intervertebral disc (IVD) degeneration. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation into nucleus pulposus (NP) cells of the IVD, which have potential for IVD regeneration. We have also shown that GDF6 stimulation activates the Smad1/5/8 and ERK1/2 signalling cascades. The aim of this study was to progress our understanding of the immediate/early response mechanisms in ASCs (N=3) which may direct GDF6-induced differentiation. RNAseq was used to perform transcriptome-wide analysis across a 12-hour time course, post-stimulation. Gene ontology analysis revealed greater transcription factor and biological processes activity at 2hrs than at the 6hr and 12hr time points, where molecular and cellular activities appeared to stabilise. Interestingly, a number of lineage determining genes were identified as differentially expressed and work is ongoing to investigate whether the early response genes are maintained throughout differentiation, or whether they are responsible for early NP lineage commitment.Study purpose and background
Methods and results
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
