Julie Warin

Intervertebral disc (IVD) degeneration, a major cause of low back pain, is driven by the disruption of nucleus pulposus (NP) tissue homeostasis. The NP arises from embryonic notochord cells that differentiate and adapt to the hypoxic, matrix-rich environment of the developing disc. Recapitulating the developmental trajectory from notochordal progenitors to NP-like cells in vitro remains challenging, as most protocols bypass early specification steps and fail to capture this understudied lineage. Here, using a three-dimensional (3D) system, we reconstruct this developmental path from human pluripotent stem cells into mesendoderm progenitor cells (MEPCs), notochord-like cells (NLCs), and NP-like cells, guided by single-cell transcriptomics and spatial profiling. We identify MEPCs as a distinct progenitor population with a unique gene signature and conserved regulatory networks ( GSC , KLF8 , LHX1 , MIXL1 ) shared with early organizer and mesendodermal cells. Self-organization in MEPCs-derived aggregates induced spontaneous expression of NOTO , a key transcription factor orchestrating notochordal identity, and enhanced commitment to notochordal fate. Long-term culture in a medium containing TGF-β1 and GDF-5 promoted maturation into SOX9 ⁺ , KRT8 ⁺ , CD109 ⁺ NP-like cells exhibiting robust matrix deposition and prominent LAMP1-positive intracytoplasmic vacuoles. This scalable, physiologically relevant model enables fundamental studies of human disc biology and early degenerative processes, while advancing regenerative strategies.

The notochord is a defining feature of chordates. It acts as mechanical support and a source of signals to surrounding tissues during development. In mammals, notochord-derived cells persist within intervertebral discs, where they form the nucleus pulposus, the cartilage in between vertebrae units that provides the spine with flexibility. Here, we synthesise developmental knowledge with recent advances in notochord biology and insights from single-cell molecular approaches. We discuss the developmental processes from notochord initiation during gastrulation through to disc formation, highlighting signalling pathways that govern axial mesoderm specification and notochordal lineage commitment. Knowledge gained from in vivo studies has guided the development of pluripotent stem cell-based models in mice and humans, including monolayer and micropatterned systems and 3D organoids. These models recapitulate key developmental aspects of notochord formation and pave the way for disease modelling and regenerative applications. We discuss their relevance to the study of developmental disorders arising from notochord dysfunction and notochordal cell roles in disc homeostasis. Finally, we outline remaining questions and examine how developmental insights and stem cell innovations can advance our understanding of tissue formation, function and homeostasis while fostering the integration of basic mechanistic insights with translational applications.

In this issue of Genes & Development , Unger and colleagues (doi:10.1101/gad.353153.125) combined human pluripotent stem cell-derived in vitro models with targeted in vivo mouse models to reveal multiple developmental defects triggered by HNF1A mutations causing maturity-onset diabetes of the young. This work paints the picture of a disorder that starts well before diabetes manifests, highlighting its complexity arising from the diverse roles of HNF1A across distinct cell types, each potentially differentially impacted by different mutations.

Background: The notochord is a midline structure essential for vertebrate embryogenesis, contributing to the development of the nervous system, digestive tract, and vertebral column. In particular, notochord signaling is indispensable for proper patterning and coordinated development of alternating vertebrae and intervertebral discs (IVDs). Later, notochordal cells (NCs) mature and adopt a characteristic vacuolated morphology before giving rise to the core of the forming IVD, the nucleus pulposus (NP). Postnatally, NCs play pivotal role in maintaining disc integrity through the secretion of specific factors and extracellular matrix (ECM). Despite its importance in disc formation and homeostasis, the morphogenetic mechanisms underlying the notochord’s transformation into the NP are insufficiently characterized. Results: We conducted a comprehensive histological and immunohistochemical analysis to investigate the cellular events governing NP formation in the mouse developing spine. Temporal analysis of intracytoplasmic vacuole formation using Lamp1 marker revealed their contribution to NP growth, while cell density progressively decreased. In addition, quantitative analyses demonstrated a notable proliferative capacity within notochordal cells coupled with region-specific apoptotic activity in the sclerotome, at future disc sites. Conclusions: This study highlights the intricate balance of cellular proliferation, programmed cell death, matrix remodeling, and vacuolation dynamics as key determinants in shaping the NP along the rostro-caudal axis. Key Findings: - Spatiotemporal cellular changes drive the transition from notochord to nucleus pulposus - Future disc regions show selective notochord proliferation and sclerotome cell death - Notochord vacuolization and matrix deposition contribute to nucleus pulposus morphogenesis