The overall objective of the proposal is to identify primary chondroprogenitor cell type(s) from adult native and/or de-differentiated nasal cartilage suitable for regenerative strategies. We aim to identify a subset of adult human NCs that share transcriptional and chromatin profiles with developing neural crest-derived chondroprogenitors and may be suited for cartilage reprogramming and repair of degenerated IVD. Despite some degree of delay due to the unpredicted Covid-19 pandemic, implementation of the work program has been progressing at a strong pace.
In the previous reporting periods, we set up the conditions to establish, in addition to human NCs, murine models of adult nasal septum and articular cartilage dissociation and de-differentiation. We then started to investigate the transcriptional composition of adult native human and mouse nasal septum cartilage at the single cell level. We have significantly progressed both with additional experiments and computational analyses of the results. In addition, we carried out extensive bulk and single cell transcriptional analyses of adult nasal chondrocytes through de-differentiation, both in mouse and human. In the previous reporting periods, we begun comparisons with mouse embryonic neural crest-derived chondroprogenitors and nasal cells, and with embryonic articular cartilage cells. During this reporting period, we have progressed with the computational comparisons of adult and embryonic mouse transcriptome datasets. Furthermore, we aimed to assess whether the regenerative potential and plasticity of differentiated adult NCs might be explained and maintained by conserved epigenetic chromatin signatures, similar to those we found in embryonic cranial neural crest and neuronal cells (PMID: 28360266, PMID: 33603234, PMID: 37095271). During this and previous reporting periods we pursued extensive chromatin epigenomic analyses of nasal embryonic, native, early dissociated, and de-differentiated chondrocytes, both in mouse and human. To assess whether the regenerative potential of these cells might also be partly encoded in their 3D chromatin architecture of promoter-promoter and promoter-enhancer contacts and how chromatin is re-wired during the de-differentiation and re-differentiation processes, we set out the conditions to carry out comparative promoter capture-HiC experiments on embryonic, de-differentiated, and adult neural crest-derived nasal chondrocytes. For instance, we have identified and published large clusters of enhancers in developing mouse cranial neural crest cells and established their role in making long-range contacts, regulating the expression of key craniofacial target genes (PMID: 37277355). During this reporting period, we have further increased our efforts both experimentally and computationally. We have specifically been focusing, on the one hand, on Polycomb-dependent chromatin signatures potentially underlying cell plasticity during de-differentiation. On the other hand, we started to compare 3D chromatin wiring diagrams of embryonic cranial neural crest cells, native, and de-differentiated adult nasal cartilage cells.
With the final aim to therapeutically apply NCs in patients with degenerated disc diseases, we have established and validated a method to generate and apply NCs in form of chondrospheres into the nucleus pulposus tissue of IVD (published PMID: 35269672, PMID: 36865027, PMID: 34339870). To assess the interactions between therapeutic cells (human NCs) and host cells (human nucleus pulposus cells (NPs) in the IVD), we have established several in vitro co-culture systems. Our most recently developed ex vivo co-culture model, where NC chondrospheres were injected into human IVD received from pathology, is the closest model to resemble the clinical scenario of therapeutic cell application in the patient. After ex vivo co-culture, NCs and NPs were sorted for single-cell RNA sequencing and are currently prepared for bioinformatical analyses. All the previously performed work led to the identification of NC chondrospheres as an envisioned therapeutical product, which is currently tested for its safety and feasibility in a large animal sheep model of induced IVD degeneration. In parallel, also protocols and procedures are currently being adapted and validated to comply with GMP standards, laying out the groundwork towards clinical translation of degenerated IVD with NC chondrospheres
