Periodic Reporting for period 1 - REGENERATE-IT (Learning from animals how to regenerate: multidisciplinary training programme in regenerative biology)
Période du rapport: 2023-03-01 au 2025-02-28
Regenerative medicine holds transformative potential for treating injuries, degenerative diseases, and aging-related conditions. However, a critical gap remains in understanding the biological mechanisms that enable certain animals to regenerate complex tissues and organs-a capability humans largely lack. The REGENERATE-IT project, funded under the Horizon Europe Marie Skłodowska-Curie Actions, addresses this gap by studying regenerative processes in diverse animal models, including zebrafish, salamanders, and planarians. By integrating comparative biology, genomics, and bioengineering, the project aims to uncover universal principles of regeneration that could inform new therapeutic strategies for human health.
The project’s objectives are twofold:
• Scientific: Decipher the molecular, cellular, and evolutionary underpinnings of regeneration to identify pathways that could be harnessed for medical applications.
• Training: Develop a multidisciplinary cohort of researchers skilled in cutting-edge techniques, from single-cell sequencing to advanced imaging, fostering innovation in regenerative biology.
By bridging fundamental research and translational applications, REGENERATE-IT aligns with the EU’s strategic priorities in health innovation and sustainable biotechnology, contributing to the European Health Union and Green Deal agendas.
The project’s objectives are twofold:
• Scientific: Decipher the molecular, cellular, and evolutionary underpinnings of regeneration to identify pathways that could be harnessed for medical applications.
• Training: Develop a multidisciplinary cohort of researchers skilled in cutting-edge techniques, from single-cell sequencing to advanced imaging, fostering innovation in regenerative biology.
By bridging fundamental research and translational applications, REGENERATE-IT aligns with the EU’s strategic priorities in health innovation and sustainable biotechnology, contributing to the European Health Union and Green Deal agendas.
Since its inception, REGENERATE-IT has structured 12 individual research projects across 9 European institutions, each focusing on distinct aspects of regeneration (e.g. limb regrowth, stem cell dynamics, and immune system interactions) and trained 12 doctoral candidates through hands-on workshops, secondments, annual consortium meetings and a bioinformatics summer school.
During the project, substantial progress was achieved across diverse research areas. One early milestone was the development of OneSABER, an improved RNA in situ hybridization method offering greater flexibility and lower costs, making gene expression studies more accessible.
A regeneration sufficiency assay was established to systematically characterize the regenerative potential of specific genes, providing insights into tissue permissibility, spatial and temporal aspects of regenerative responses, and identifying genes capable of inducing regeneration—knowledge applicable to mammalian systems.
By selecting and cloning regeneration-specific genes based on transcriptomic activity pre- and post-injury, researchers began mapping their spatial expression, with several showing localized activation at amputation sites. An ATACseq dataset covering the entire regeneration timeline was generated to identify enhancer elements that drive gene expression specifically during regeneration, helping minimize side effects.
In Parhyale, new tools were created to study microbial influences on wound healing and regeneration.
In planarians, glutathione-responsive nanocarriers enabled real-time imaging of redox dynamics, revealing spatial antioxidant regulation when combined with antibody staining and FISH. Enhanced immobilization techniques using linalool improved live imaging of these processes.
A reproducible 3D spheroid culture system for axolotl fibroblasts was developed, offering a robust platform to screen for factors that promote multipotency and limb regeneration. The discovery of novel species-specific microRNAs in regenerating limbs provided new insight into regenerative mechanisms.
In mammalian systems, spatial transcriptomics and single-cell RNA sequencing identified a unique macrophage subtype in regenerating spiny mouse tissue that may promote regeneration. Tools to study gene and protein expression in these cells were developed, and protocols were established for differentiating mouse, spiny mouse, and human macrophages into pro-regenerative types. Efforts are underway to derive pro-regenerative macrophages from human iPSCs.
Spiny mouse satellite cells showed enhanced differentiation potential over those from standard lab mice, highlighting new therapeutic strategies for muscle regeneration and muscular dystrophy. In studies on colonic epithelial regeneration, canonical Wnt signaling was shown to play a dynamic, essential role, with critical activation windows identified using reporter mice and ongoing single-cell RNA-seq to dissect pathway control.
For skin regeneration, a group of gene markers was identified to assess stem cell content in skin grafts, offering a molecular tool for evaluating graft quality. A reference transcriptomic model of human epidermal stem cells was created, while a CRISPRa-based platform now enables high-throughput screening of genes for reprogramming progenitors into durable epidermal stem cells.
A new theoretical model describes how mitochondrial electron transport chain conformation affects metabolism and regenerative ability in the zebrafish heart. Standardized methods were introduced to evaluate caudal fin regeneration in adult zebrafish, with preliminary evidence that specific tyrosine phosphatases downstream of ROS may influence the process, pending further validation.
During the project, substantial progress was achieved across diverse research areas. One early milestone was the development of OneSABER, an improved RNA in situ hybridization method offering greater flexibility and lower costs, making gene expression studies more accessible.
A regeneration sufficiency assay was established to systematically characterize the regenerative potential of specific genes, providing insights into tissue permissibility, spatial and temporal aspects of regenerative responses, and identifying genes capable of inducing regeneration—knowledge applicable to mammalian systems.
By selecting and cloning regeneration-specific genes based on transcriptomic activity pre- and post-injury, researchers began mapping their spatial expression, with several showing localized activation at amputation sites. An ATACseq dataset covering the entire regeneration timeline was generated to identify enhancer elements that drive gene expression specifically during regeneration, helping minimize side effects.
In Parhyale, new tools were created to study microbial influences on wound healing and regeneration.
In planarians, glutathione-responsive nanocarriers enabled real-time imaging of redox dynamics, revealing spatial antioxidant regulation when combined with antibody staining and FISH. Enhanced immobilization techniques using linalool improved live imaging of these processes.
A reproducible 3D spheroid culture system for axolotl fibroblasts was developed, offering a robust platform to screen for factors that promote multipotency and limb regeneration. The discovery of novel species-specific microRNAs in regenerating limbs provided new insight into regenerative mechanisms.
In mammalian systems, spatial transcriptomics and single-cell RNA sequencing identified a unique macrophage subtype in regenerating spiny mouse tissue that may promote regeneration. Tools to study gene and protein expression in these cells were developed, and protocols were established for differentiating mouse, spiny mouse, and human macrophages into pro-regenerative types. Efforts are underway to derive pro-regenerative macrophages from human iPSCs.
Spiny mouse satellite cells showed enhanced differentiation potential over those from standard lab mice, highlighting new therapeutic strategies for muscle regeneration and muscular dystrophy. In studies on colonic epithelial regeneration, canonical Wnt signaling was shown to play a dynamic, essential role, with critical activation windows identified using reporter mice and ongoing single-cell RNA-seq to dissect pathway control.
For skin regeneration, a group of gene markers was identified to assess stem cell content in skin grafts, offering a molecular tool for evaluating graft quality. A reference transcriptomic model of human epidermal stem cells was created, while a CRISPRa-based platform now enables high-throughput screening of genes for reprogramming progenitors into durable epidermal stem cells.
A new theoretical model describes how mitochondrial electron transport chain conformation affects metabolism and regenerative ability in the zebrafish heart. Standardized methods were introduced to evaluate caudal fin regeneration in adult zebrafish, with preliminary evidence that specific tyrosine phosphatases downstream of ROS may influence the process, pending further validation.
The REGENERATE-IT project has delivered a suite of innovative tools, models, and insights that set new standards in regenerative biology. The OneSABER method and standardized regeneration assays enhance the efficiency and reproducibility of gene expression and regeneration studies, benefiting the wider research community. The identification of regeneration-specific genes, novel cell types, and regulatory networks paves the way for the development of targeted regenerative therapies. Diagnostic gene markers and advanced cell screening platforms have direct translational potential, improving the selection and success of cell and tissue therapies. The multidisciplinary approach, integrating molecular biology, imaging, and bioengineering, exemplifies the collaborative spirit needed to tackle complex biomedical challenges. Continued research, validation, and eventual clinical translation will be essential to fully realize the therapeutic potential of these discoveries.