Periodic Reporting for period 4 - EpiCrest2Reg (From Epigenetics of Cranial Neural Crest Plasticity to Intervertebral Disc Regeneration)
Período documentado: 2023-09-01 hasta 2025-02-28
A general purpose of this ERC Synergy collaborative project is to overcome the typical boundaries segregating the research domains of developmental, stem cell, and chromatin biology and human regenerative medicine, opening a new scientific dimension. This general paradigm shift will be exemplified in a specific context. Namely, the Rijli laboratory has a long-standing interest in the dissection of the transcriptional mechanisms of craniofacial development and morphogenesis in the vertebrate embryo, mostly focusing on the mouse as a model system, and in particular in the epigenetic control of cranial neural crest cell fate and plasticity. His contribution has led to breakthrough novel understanding in the morphogenetic potential of cranial neural crest cells. However, such knowledge is missing the opportunity to be extended to a human setting and to result in a possible clinical benefit. The Martin laboratory has developed new strategies in the generation of cell-based grafts, for the treatment of musculoskeletal diseases. His contribution has led to different clinical studies, including the first-in-man assessment of cranial neural crest-derived nasal septum chondrocytes (NCs) as a cell source for articular cartilage repair. However, along with the discovery of NC regeneration capacity and environmental plasticity, such translational work requires a deeper understanding of mechanisms of action, in order to gain robustness in efficacy. Indeed, one of the general bottlenecks of the field of regenerative medicine is the limited knowledge on the associated biological processes, which often leads to ‘trial-and-error’ approaches and reduces the perspectives of improvement of promising proof-of-principle studies.
Lower back pain, mostly caused by intervertebral disc (IVD) degeneration, is a leading cause of disabilities and therefore a significant socio-economic burden to the society. Most surgical treatments cannot cure the pathology of this degenerative disc disease (DDD) and while cell therapy treatments for DDD have been conducted, no ideal cell source that could survive and contribute to the regeneration of the degenerated tissue could be identified so far. While trials could validate safety of cellular injection therapies, the desired outcome expectations, such as restoration and repair of the destroyed disc tissue, could not be met. To date, the assumption for the lack of success in clinical trials is that injected cells do not adapt or even survive for a reasonable time in the exceptional environment of a progressively degenerating disc. Our previous work showed that NCs can maintain their ability to form a cartilage tissue even under inflammatory conditions, such as present in the IVD, suggesting their possible feasibility for the treatment of DDD.
General understanding and controlling of the molecular and cellular mechanisms regulating the capacity of human NCs to adapt to a new environment would ideally enhance the quality of repair tissue in the targeted degenerated IVD, and could further be envisioned to be translated also to other tissue types and diseases.
Lower back pain, mostly caused by intervertebral disc (IVD) degeneration, is a leading cause of disabilities and therefore a significant socio-economic burden to the society. Most surgical treatments cannot cure the pathology of this degenerative disc disease (DDD) and while cell therapy treatments for DDD have been conducted, no ideal cell source that could survive and contribute to the regeneration of the degenerated tissue could be identified so far. While trials could validate safety of cellular injection therapies, the desired outcome expectations, such as restoration and repair of the destroyed disc tissue, could not be met. To date, the assumption for the lack of success in clinical trials is that injected cells do not adapt or even survive for a reasonable time in the exceptional environment of a progressively degenerating disc. Our previous work showed that NCs can maintain their ability to form a cartilage tissue even under inflammatory conditions, such as present in the IVD, suggesting their possible feasibility for the treatment of DDD.
General understanding and controlling of the molecular and cellular mechanisms regulating the capacity of human NCs to adapt to a new environment would ideally enhance the quality of repair tissue in the targeted degenerated IVD, and could further be envisioned to be translated also to other tissue types and diseases.
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
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
The practical benefits of the synergistic cooperation, beyond a cultural shift and the generation of unprecedented and valuable insights into the mechanisms of transcriptional regulation of mammalian craniofacial development and into the regenerative potential and plasticity of adult neural crest-derived cartilage, will include the identification of cellular markers and cell culture requirements for human nasal derived chondrocytes to be used for replacement strategies. These will be crucial towards definition of quality settings for repeatable clinical outcome in the use of NCs for IVD regeneration.