Résumé
INTRODUCTION: Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and they are considered an exciting target for cell-based therapy. However, NCs are scarcely available as they are lost early in life, and attempts at in vivoexpansion have failed because NCs lose their specific phenotype. The production of Notochordal-like cells (NLCs) from human induced pluripotent stem cells (iPSCs) is a viable alternative. However, current attempts have been challenged by the low differentiation efficiency into the NC lineage. Therefore, the aim of this study was to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD progenitor cells (TIE2+-cells) to improve hiPSC differentiation towards mature, matrix-producing NLCs.
METHODS: hiPSCs were generated from TIE2⁺ cells of three adult donors. As a comparison, donormatched minimally invasive peripheral blood mononuclear (PBM) cell-derived iPSCs were used. Firstly, the iPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)¹. Thereafter, the cells were further driven towards the NClineage by transfection with synthetic NOTO mRNA¹ and further matured using a 3D pellet culture in discogenic medium containing 10ng/mL TGF-β1. Read-out parameters included cell morphology, gene and protein expression and matrix deposition.
RESULTS: Both TIE2⁺ and PBM cell-derived hiPSC showed successful differentiation towards mesendodermal progenitor cells following Wnt activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt-target genes (LEF1, NODAL) and mesendodermal markers (TBXT, FOXA2, TBX6) was observed compared to mTESR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. At day 3, we confirmed a 9-fold increase in NOTO mRNA levels after transfection in all donor lines. At day 28, the appearance of vacuolated NLCs was observed in both TIE2⁺ and PBM cell-derived pellet cultures confirming successful commitment towards the NC-lineage. Interestingly, while DMMB-assay detected GAG deposition in both lines, a significant increase in GAG content was seen in the TIE2⁺ cell-derived pellets. DISCUSSION & CONCLUSIONS: Tissue-specific TIE2⁺ cell-derived iPSCs may allow for an improved iPSNLC differentiation efficiency, indicated by the increased potency for deposition of GAG-rich matrix. Detailed analysis of the phenotypic markers and matrix deposited at the end of the 28 day maturation is ongoing to further document the phenotype of these iPS-NLCs. Delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the differences in their differentiation capacity.
REFERENCES: ¹Colombier et al., 2020