Advancing stem cell-based technologies for intervertebral disc regeneration: Exploring biology-driven cues to improve iPSCs differentiation toward notochordal-like cells

Intervertebral disc (IVD) degeneration is a primary cause of low back pain (LBP), creating a significant global burden. Stem cell-based regenerative strategies, especially those using induced pluripotent stem cells (iPSCs), are drawing attention for their potential to repair or even reverse disc degeneration. This thesis focuses on generating iPSC-derived notochordal-like cells (NLCs), inspired by the anabolic and anti-inflammatory functions of natural notochordal cells (NCs) in healthy IVDs. However, clinical translation of iPSC-derived NLCs is currently limited by challenges in achieving optimal differentiation and cell purification. To overcome these hurdles, in this thesis, a multidisciplinary approach has been employed, including the development of an iPSC reporter line to monitor differentiation from a functional perspective, the application of CRISPR activation (CRISPRa) in iPS-NLC differentiation to achieve multigene activation mimicking notochord development, and the exploration of genetic and epigenetic signatures of dog NCs. Single-cell RNA sequencing (scRNA-seq) and single-cell transcriptome + Chromatin ImmunoCleavage sequencing (T-ChIC-seq) where the chromatin state together with transcription profiles are captured with the sort-assisted single-cell ChIC (sortChIC) and vast transcriptome analysis of single cells by dA-tailing’ (VASA-seq) were used to identify cellular instructions in the native dog disc that can be harnessed for regenerative therapy. In Chapter 2, two human ACAN-2A-mScarlet reporter iPSC lines were created and validated for aggrecan (ACAN) expression. These lines provide a robust platform for refining iPSC protocols and studying cell fate decisions. Building on these findings, Chapter 3 presents a stepwise differentiation protocol using dCas9-SAM CRISPRa to activate transcription factors (NOTO, TBXT, FOXA2, SOX9, SOX5, and SOX6). Single-cell transcriptomics showed that co-expression of SOX9, SOX5, and SOX6 is vital for generating a larger population of NLCs. The ACAN-2A-mScarlet reporter also confirmed that 3D pellet culture enhances the maturation of CRISPRa-treated iPSCs into functional, matrix-producing cells. Chapter 4 extends these insights by providing the first single-cell atlas of NC-rich IVDs in non-chondrodystrophic dogs. It identifies NC clusters involved in disc maturation and NC-to-nucleus pulposus cell (NPC) transitions, along with fibrocyte and inflammatory clusters typical of advanced degeneration. Further T-ChIC analysis highlights the role of H3K27me3-mediated repression in regulating NC and NPC fates, and shows that removing this barrier in human NPCs can reverse gene silencing and potentially restore healthier phenotypes. Finally, Chapter 5 discusses the possible reasons leading to off-target differentiation during iPS-NLC differentiation, such as overlapping signaling pathways (e.g., WNT/β-catenin, NODAL/SMAD, and BMP) and culture conditions that do not fully simulate the native disc environment. To address these challenges, CRISPR-based technologies offer a more promising means of precisely regulating iPSC differentiation, and conducting experiments under physiologically relevant conditions like 3D culture with biomechanical loading, may improve iPSC differentiation efficiency. These strategies and insights have the potential to improve the efficiency of iPS-NLC differentiation and lay the foundation for the development of future stem cell-based regenerative treatments for intervertebral disc degeneration.