Characterising GDF6-mediated differentiation and GMP-compliant expansion of adipose stem cells for intervertebral disc regeneration

Abstract

A growth differentiation factor 6 (GDF6) and adipose stem cell (ASC) combined therapy for intervertebral disc degeneration (IVDD) has previously been proposed based on the efficiency of producing nucleus pulposus-like cells. Evidence suggests that bone morphogenetic receptor type 2 (BMPR2) and Smad/ERK intracellular signalling pathways are important in the differentiation process. In order to progress this therapy towards clinic, a more comprehensive understanding surrounding the transcriptional changes involved in the early stages of differentiation are required; therefore supplementing the more thoroughly understood late stage differentiation. Secondly, a reliable and effective good manufacturing practice (GMP)-compliant expansion protocol for ASCs is necessary to provide sufficient cell numbers for clinical application. ASCs stimulated with GDF6 for 2h, 6h and 12h were investigated using RNA sequencing to analyse the transcriptomic changes in the early stages of differentiation. A number of significantly expressed genes were found that are involved in trilineage differentiation, as well as the immediate early response gene EGR1, which were validated by RT-qPCR. This included genes encoding proteins involved in promotion of chondrogenesis (SOX8, SOX9, FGFR3, FGF18, SCX, GDF10, FOXF1 and PRG4) as well as inhibition of osteogenesis (RUNX2) and adipogenesis (CEBPA, CEBPD and NR4A1). Inhibiting pSmad1/5/8 and pERK1/2 using dorsomorphin and U0126, respectively, revealed a reliance on both signalling mechanisms for the gene expression changes. Also, translation inhibition using cyclohexamide distinguished the primary (EGR1, FGF18 and FOXF1) from the secondary (SOX8, SOX9, SCX, FGFR3, GDF10 and PRG4) response genes. Furthermore, PRG4 was shown to be significantly upregulated in the late stage of differentiation (14day). For establishing the ASC expansion protocol, GMP-compliant growth medium was used to expand the cells from distinct demographic cohorts (middle-aged/old, male/female) from passage 1 to passage 3 where clinically-relevant cell numbers were achieved. Whilst stem cell CD marker expression was maintained, BMPR2 expression and response to GDF6 increased in each cohort at passage 3 and homogeneity across cohorts improved. To conclude, the results from this thesis reveal a multifactorial early gene response that contributes to lineage commitment with the identification of a number of potentially useful early markers of differentiation. Furthermore, a reliable and effective GMP-complaint expansion protocol shows that ASCs improve in phenotype, response to GDF6 and homogeneity when expanded to clinically-relevant numbers.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Judith Hoyland (Supervisor) & Stephen Richardson (Supervisor)