Periodic Reporting for period 2 - RENOIR (REcreating the ideal Niche: environmental control Of cell Identity in Regenerating and diseased muscles)
Periodo di rendicontazione: 2022-05-01 al 2025-04-30
The project aims to address one of the major challenges in Europe that is to have an ageing population, which is healthy and active, and to improve the quality of life of people affected by chronic diseases. Skeletal and cardiac muscle ischemia are amongst the major causes of exacerbation of several diseases and morbidity in the population that represent an important social and economic burden for our society.
In this context, the overall objective of RENOIR research program is to unravel, the decline in skeletal and cardiac muscle mass and function during ageing and disease, using advanced biotechnological tools.
Specific scientific objectives of the project are to identify molecular signatures and interaction patterns of muscle niche components and to combine biomaterials and biopharma for improved ex vivo models and in vivo regeneration. The project innovations will provide a strong basis for the growth of the involved biotechnology company.
In this context, the overall objective of RENOIR research program is to unravel, the decline in skeletal and cardiac muscle mass and function during ageing and disease, using advanced biotechnological tools.
Specific scientific objectives of the project are to identify molecular signatures and interaction patterns of muscle niche components and to combine biomaterials and biopharma for improved ex vivo models and in vivo regeneration. The project innovations will provide a strong basis for the growth of the involved biotechnology company.
The Project activities are on track. WPs led to advances in their specific aims. Main results for each WPs are the following:
WP1 aims at investigating how the different stem and progenitor cells such as SCs, ECs and cSCs in the muscle acquire and maintain their fate during homeostasis, traumatic injury and chronic degeneration/regeneration. WP1 led to the advances in the characterization of EC progenitors’ fate during muscle injury and in an altered inflammatory environment. Key achievements include optimization of in vivo EndoMT induction protocols, and the study of Cripto’s role in EndoMT (ESR1, ESR2). Major results also include the preliminary characterization of single cell RNAseq and bulk RNAseq data of mouse cardiac/stem progenitors (CSCs).
Most of the studies have also required the set-up of specific protocols to work with small number of cells (ESR1, 2, 5, 13, 10), and have involved the use of functional approaches (knock out and knockdown; gain-of-function experiments), lineage tracing approaches (ESR1, 5) genomic and epigenomic profiling (ATACseq, RNA-seq: ESR7, 9).
A review (ESR1, 2) highlighted key roles of MuSCs, macrophages, FAPs, ECs, and pericytes in orchestrating regenerative processes and pathogenesis in dystrophic conditions. Lineage tracing studies (ESR5) revealed that TNAP+ pericytes contribute to both skeletal and smooth muscle development in fetal life through unipotent progenitors. STAT3 inhibition (ESR7) was shown to rejuvenate aged MuSCs by restoring autophagy at transcriptional and cytoplasmic levels, suggesting therapeutic strategies to counteract sarcopenia. Single-cell RNAseq analysis (ESR1) demonstrating that macrophage depletion leads to EC EndMT via SPP1-dependent signaling in injured muscle.
WP2 investigates how inflammation, myogenesis, and vasculature coordinate muscle regeneration and fibrosis. Major results advanced the study of macrophage roles in dystrophic models, highlighting Bach1’s function and HMGB1-CD47 interactions (ESR4, ESR6, ESR12).
Additional studies further characterized the multifaceted role of macrophages in regeneration and disease. A complementary study (ESR6) dissected regenerative inflammation, Other work (ESR6) optimized xenogeneic-free media for corneal endothelial cultures using PRGF, advancing regenerative applications. ESR12 also contributed to the elucidation of the CXCR4/CXCL12 axis and its role in anti-tumor immune mechanisms.
WP3 aims at developing new in vitro systems to mimic the muscle regenerating niche and comprising all cellular and extracellular components, using new bioactive materials/devices. Another objective is the developing of new and more efficient biomaterial delivery vehicle for temporal and spatial control of therapeutic proteins (Cripto and HMGB).
WP3 developed a microfluidic prototype to study flow effects and EndoMT using Flexdym (ESR10, 13). WP3 also defined bioactive materials mimicking muscle signals (ESR8), simulation and optimisation of the fabrication of high-volume microfluidic extracorporeal circuit (ESR13) and optimization of hydrogel microbeads manufacturing protocol for the large scale production of Cripto protein using bioreactors (ESR11).
Additional outputs include a review on how microfluidic design strategies (ESR10, 13) can model thrombogenicity, aiming to optimize shear flow and material architecture to reduce clot risk. These findings enhance tools for engineering biomimetic niches and therapies.
WP4 led to advances in training ESRs in cutting edge technologies in complementary research areas. All ESRs received multidisciplinary research training, improving both technical and soft skills. ESRs (all) are enrolled in structured PhD Programs and attended local and RENOIR Training Activities, Seminars and Conferences on different research areas and expertise.
WP5 led to advance in Dissemination and Outreach activities. ESRs have been involved in dissemination and outreach activities, including RENOIR promotion through the RENOIR website and through the other online platforms such as Twitter, Facebook or UNIMIB’s online research magazine. ESRs prepared short presentations in English to present RENOIR and their single projects (available on the website). Communications of RENOIR results have been achieved through active participation of the ESRs at national and international meetings/conferences, including the EMBO Conference in Myogenesis (ESR1, 2, 7, 8, 11), where ESR2 won the best Poster award.
Joint journal clubs and seminars fostered knowledge exchange. All these activities involved both ESRs working in public and Private Institutions and contribute to favor the exchanges between these two sectors.
WP1 aims at investigating how the different stem and progenitor cells such as SCs, ECs and cSCs in the muscle acquire and maintain their fate during homeostasis, traumatic injury and chronic degeneration/regeneration. WP1 led to the advances in the characterization of EC progenitors’ fate during muscle injury and in an altered inflammatory environment. Key achievements include optimization of in vivo EndoMT induction protocols, and the study of Cripto’s role in EndoMT (ESR1, ESR2). Major results also include the preliminary characterization of single cell RNAseq and bulk RNAseq data of mouse cardiac/stem progenitors (CSCs).
Most of the studies have also required the set-up of specific protocols to work with small number of cells (ESR1, 2, 5, 13, 10), and have involved the use of functional approaches (knock out and knockdown; gain-of-function experiments), lineage tracing approaches (ESR1, 5) genomic and epigenomic profiling (ATACseq, RNA-seq: ESR7, 9).
A review (ESR1, 2) highlighted key roles of MuSCs, macrophages, FAPs, ECs, and pericytes in orchestrating regenerative processes and pathogenesis in dystrophic conditions. Lineage tracing studies (ESR5) revealed that TNAP+ pericytes contribute to both skeletal and smooth muscle development in fetal life through unipotent progenitors. STAT3 inhibition (ESR7) was shown to rejuvenate aged MuSCs by restoring autophagy at transcriptional and cytoplasmic levels, suggesting therapeutic strategies to counteract sarcopenia. Single-cell RNAseq analysis (ESR1) demonstrating that macrophage depletion leads to EC EndMT via SPP1-dependent signaling in injured muscle.
WP2 investigates how inflammation, myogenesis, and vasculature coordinate muscle regeneration and fibrosis. Major results advanced the study of macrophage roles in dystrophic models, highlighting Bach1’s function and HMGB1-CD47 interactions (ESR4, ESR6, ESR12).
Additional studies further characterized the multifaceted role of macrophages in regeneration and disease. A complementary study (ESR6) dissected regenerative inflammation, Other work (ESR6) optimized xenogeneic-free media for corneal endothelial cultures using PRGF, advancing regenerative applications. ESR12 also contributed to the elucidation of the CXCR4/CXCL12 axis and its role in anti-tumor immune mechanisms.
WP3 aims at developing new in vitro systems to mimic the muscle regenerating niche and comprising all cellular and extracellular components, using new bioactive materials/devices. Another objective is the developing of new and more efficient biomaterial delivery vehicle for temporal and spatial control of therapeutic proteins (Cripto and HMGB).
WP3 developed a microfluidic prototype to study flow effects and EndoMT using Flexdym (ESR10, 13). WP3 also defined bioactive materials mimicking muscle signals (ESR8), simulation and optimisation of the fabrication of high-volume microfluidic extracorporeal circuit (ESR13) and optimization of hydrogel microbeads manufacturing protocol for the large scale production of Cripto protein using bioreactors (ESR11).
Additional outputs include a review on how microfluidic design strategies (ESR10, 13) can model thrombogenicity, aiming to optimize shear flow and material architecture to reduce clot risk. These findings enhance tools for engineering biomimetic niches and therapies.
WP4 led to advances in training ESRs in cutting edge technologies in complementary research areas. All ESRs received multidisciplinary research training, improving both technical and soft skills. ESRs (all) are enrolled in structured PhD Programs and attended local and RENOIR Training Activities, Seminars and Conferences on different research areas and expertise.
WP5 led to advance in Dissemination and Outreach activities. ESRs have been involved in dissemination and outreach activities, including RENOIR promotion through the RENOIR website and through the other online platforms such as Twitter, Facebook or UNIMIB’s online research magazine. ESRs prepared short presentations in English to present RENOIR and their single projects (available on the website). Communications of RENOIR results have been achieved through active participation of the ESRs at national and international meetings/conferences, including the EMBO Conference in Myogenesis (ESR1, 2, 7, 8, 11), where ESR2 won the best Poster award.
Joint journal clubs and seminars fostered knowledge exchange. All these activities involved both ESRs working in public and Private Institutions and contribute to favor the exchanges between these two sectors.
The scientific innovative aspect of RENOIR resides in considering the whole network of cellular interactions rather than focusing on the single components of the remodelling process, with the final aim of diverting the fate of the different cell types from inducing fibrosis to orchestrating muscle remodelling and tissue regeneration. RENOIR is the first to apply this holistic approach, paving the way for alternatives to animal models and new therapies for fibrotic muscle diseases.
The use of LOF and GOF mouse models are widening our knowledge on Cripto genetic/epigenetic regulation of myogenic precursors and macrophages. This will also allow to optimize administration of soluble Cripto therapeutic intervention.
Deep sequencing technologies and analysis is providing novel and specific markers of different progenitor cells and macrophages in both cardiac and skeletal muscles in physiological and pathological condition, to be exploited as diagnostic/prognostic tools and target of therapeutic intervention.
3D culture mimicking niche is being optimized, by exploiting advanced biomaterials in combination with different cell types and signaling. This will lead to a deeper knowledge of the cell-cell interactions and possibly to an approach that could also replace animal experimentation in several contexts.
Advanced biomaterials are also being testing for stem cells and biopharma delivery: this will allow optimization of muscle regeneration in models of acute and chronic muscle
RENOIR also offers ESRs strong career prospects in stem cell biology, tissue regeneration, and biomaterials.
The use of LOF and GOF mouse models are widening our knowledge on Cripto genetic/epigenetic regulation of myogenic precursors and macrophages. This will also allow to optimize administration of soluble Cripto therapeutic intervention.
Deep sequencing technologies and analysis is providing novel and specific markers of different progenitor cells and macrophages in both cardiac and skeletal muscles in physiological and pathological condition, to be exploited as diagnostic/prognostic tools and target of therapeutic intervention.
3D culture mimicking niche is being optimized, by exploiting advanced biomaterials in combination with different cell types and signaling. This will lead to a deeper knowledge of the cell-cell interactions and possibly to an approach that could also replace animal experimentation in several contexts.
Advanced biomaterials are also being testing for stem cells and biopharma delivery: this will allow optimization of muscle regeneration in models of acute and chronic muscle
RENOIR also offers ESRs strong career prospects in stem cell biology, tissue regeneration, and biomaterials.