Disc-Derived Induced Pluripotent Stem Cells and Environmental Cues for Nucleus Pulposus Regeneration

Notochordal cells (NCs) are linked to a healthy state of the nucleus pulposus (NP) and are therefore considered a promising candidate for cell-based regenerative 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 generation of notochordal-like cells (NLCs) from human induced pluripotent stem cells (hiPSCs) is considered a viable alternative. Therefore, this study aimed to build on the instructive capacity of decellularized notochordal cell-derived matrix (dNCM) and the epigenetic memory of tissue-specific hiPSCs derived from TIE2+ nucleus pulposus-progenitor cells (NPPCs) to improve hiPSC differentiation towards mature, healthy matrix-producing NLCs. As a comparison, donor-matched minimally invasive peripheral blood mononuclear cell (PBMC)-derived hiPSCs were generated. The results showed that the use of tissue-specific derived hiPSCs instructed by a mix of natural cues provided by dNCM allowed for an improved differentiation capacity, indicated by the increased expression of key phenotypic and functional NC/NPC markers. Additionally, within this in vitro environment, the cell-source influenced off-target differentiation resulting in increased neural- and immune-cell fate for the PBMC-derived hiPSC lines. In the end, Understanding how in vivo microenvironmental cues will direct differentiation and maturation of the hMEPCs is imperative to refine the differentiation protocol also taking into account the hiPSC-derived NLC functionality and survival within the challenging environment of the intervertebral disc. Overall design: Donor-matched intervertebral disc biopsies and blood samples of three patients (no 190, 191, 192) were collected at Inselspital in Bern after informed consent and ethical approval (no. b2019-00097). Both cell types were reprogrammed using Sendai virus vectors carrying the Oct4, Klf4, Sox, Myc (OSKM) reprogramming factors. The iPSC lines were differentiated into mesendoderm progenitor cells (MEPCs) according to the publish protocol by Warin et al. (2024) (https://doi.org/10.1016/j.isci.2024.109018). At this timepoint the cells were transfected with NOTO mRNA and simulteneously plated into low-adherent culture plates to obtain 3D cell pellets of 35,000 cells in NP-genic medium (hgDMEM (11965, Thermo Fisher) supplemented with 1% ITS+, 40 µg/ml L-proline (P5607, Sigma-Aldrich), 1% NEAA, 50 µg/mL Ascorbic acid-2-Phosphate (ASAP; A8960, Sigma-Aldrich), and 10 ng/mL TGF-ß1 (240-B-010, R&D Systems, Minneapolis, USA)). On day 7, half of the samples were switched to NP-genic medium in which the TGF-ß1 was replaced by decellularized notochordal cell-derived matrix, the other half was continued in TGF-ß1. The sample were cultured until day 28 with daily medium refreshment whereafter they were collected for histology, biochemical analysis, and gene expression profiling.