Employing epigenetic memory and native instructive stimuli to stimulate iPS-NLC differentiation

Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and are considered a promising candidate for cell-based therapies. However, NCs are scarcely available as they are lost early in life, and attempts at in vitro expansion have failed because NCs lose their specific phenotype. The production of notochordal-like cells (NLCs) from human induced pluripotent stem cells (hiPSCs) is a viable alternative. Therefore, this study aimed to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD-progenitor cells (TIE2+-cells) and the instructive capacity of decellularized notochordal cell-derived matrix (dNCM)2 to improve hiPSC differentiation towards mature, healthy matrix-producing NLCs. hiPSCs were generated from TIE2+-IVD cells of three adult donors. As a comparison donor-matched minimally invasive peripheral blood mononuclear (PBM)-derived iPSCs were used. Firstly, hiPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)1 for 2 days. Thereafter, the cells were further driven towards the NC-lineage by transfection with synthetic NOTO mRNA1 and matured by switching to a 3D-cell pellet culture in discogenic medium containing 10ng/mL TGF-β1 or 3mg/mL dNCM until day 28. Read-outs included cell morphology, gene and protein expression and matrix deposition. Both TIE2+- and PBM-cell derived hiPSC showed successful differentiation towards mesendodermal progenitors 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 and Nodal signaling (LEF1, NODAL) and mesendodermal markers (FOXA2, TBXT) was detected, compared to mTeSR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. On day 3, we detected a significant increase in NOTO mRNA levels in all donor lines after transfection compared to untransfected cell pellets. 3D-pellets of all donor lines showed glycosaminoglycan (GAG)- and collagen type II-rich areas after dNCM- but not TGF-β1-treatment on day 28. This was confirmed with the DMMB-assay, showing a significantly increased GAG content in the 3D-pellets treated with dNCM compared to TGF-β1. Next to that, TIE2+-cell derived iPSC pellets contained a significantly higher GAG content after dNCM-treatment compared to the PBM-cell derived hiPSC pellets. Immunohistochemical evaluation showed a heterogeneous cell population including cells positive for chondrogenic- (ACAN, SOX9), NPC/NC- (panKRT, T), and IVD progenitor- markers (CD24, TIE2). In conclusion, using tissue-specific TIE2+-cell derived hiPSCs combined with dNCM-treatment may allow for an improved differentiation capacity indicated by the increased deposition of GAG and collagen type II-rich matrix. However, the obtained cell population is still very heterogeneous and further transcriptome analysis could unravel whether the 3D-pellets contain cells which were successfully driven towards the notochordal-lineage and how these can be enriched based on unique NC-specific markers. Next to that, delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the observed differences in their differentiation capacity. These insights could be used for further optimization of iPS-NLC differentiation and allow for a more purified population of mature, healthy matrix-producing NLCs. This work was funded by Horizon 2020 (no. 825925) and the Dutch Arthritis Society (LLP22). References 1Colombier, P. et al. (2020). NOTO transcription factor directs human induced pluripotent stem cell-derived mesendoderm progenitors to a notochordal fate. Cells, 9(2), 509. 2Bach, Frances C., et al. "Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside." Oncotarget 9.41 (2018): 26507

Recommendations for intervertebral disc notochordal cell investigation: from isolation to characterization

Background Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called “the notochord”. However, changes in the cells derived from the notochord which form the NP (i.e., notochordal cells [NCs]), in terms of their phenotype and functional identity from early developmental stages to skeletal maturation are less understood. These key issues require further investigation to better comprehend the role of NCs in homeostasis and degeneration as well as their potential for regeneration. Progress in utilizing NCs is currently hampered due to poor consistency and lack of consensus methodology for in vitro NC extraction, manipulation, and characterization. Methods Here, an international group has come together to provide key recommendations and methodologies for NC isolation within key species, numeration, in vitro manipulation and culture, and characterization. Results Recommeded protocols are provided for isolation and culture of NCs. Experimental testing provided recommended methodology for numeration of NCs. The issues of cryopreservation are demonstrated, and a pannel of immunohistochemical markers are provided to inform NC characterization. Conclusions Together we hope this article provides a road map for in vitro studies of NCs to support advances in research into NC physiology and their potential in regenerative therapies