Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-β1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. Stem Cells 2016;34:653–667

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.

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.

Understanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-β inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro. Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research.

The development of human induced pluripotent stem cell (iPSC)–based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN’s prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter’s versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.