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REcreating the ideal Niche: environmental control Of cell Identity in Regenerating and diseased muscles

Periodic Reporting for period 1 - RENOIR (REcreating the ideal Niche: environmental control Of cell Identity in Regenerating and diseased muscles)

Période du rapport: 2020-05-01 au 2022-04-30

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, using novel and integrated biotechnological tools, the progressive decline in skeletal and cardiac muscle mass and functionality, that represents the most critical and complex features associated with human ageing and disease.
Specific scientific objectives of the project are to identify the distinctive molecular fingerprint of the different cell components of the muscle regenerating niche, to define their pattern of the interactions and to combine the use of advanced biomaterials and biopharma to optimize ex vivo modelling of the niche and to achieve in vivo delivery of compounds for efficient tissue maintenance and 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. Main results include the optimization of new protocols to induce EndoMT in vivo (ESR1), the characterization of the role of Cripto in restricting EndoMT in vitro and in vivo (ESR2), and -the optimization of cell culture conditions to be used in the developing of a microfluidic device to study EndoMT (ESR1, 13). 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).
WP2 focuses on investigating how inflammatory, myogenic and vascular components integrate to coordinate muscle regeneration and how fibrosis develops in pathological conditions. Major results led to advances in the characterization of macrophages in two murine models of muscle dystrophy, mdx, and D2-mdx (ESR4), elucidating the role of the transcription factor Bach1 in monocyte/macrophages, showing a faster conversion from proinflammatory macrophages to resolution phase in Bach1 loss of function mice (ESR6), and in determining the interactions between HMGB1 and its ligand and CD47 (ESR12)
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 led to advances in designing and fabricating a microfluidic prototype for i) investigating flow effects on cell attachment and survival, as well as coating/matrix deposition, integrity and longevity; ii) and EndoMT, using Flexdym (ESR10, 13). Main results also include definition of the ideal bioactive materials that can recapitulate in a simplified but consistent form the role of biophysical/biochemical signals in muscle (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).
WP4 led to advances in training ESRs in cutting edge technologies in complementary research areas. All the ESRs received multidisciplinary and intense research training improving their research skills such as inter-discipline thinking, formulation of hypotheses, design of experiments, and complementary 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.
Joined Journal clubs and seminars helped to create a network of shared knowledge and expertise. 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 network to apply this method in the field and thanks to new resources and to the multidisciplinary nature of the consortium, this project will pave the way to the development of i) innovative alternative methods to animal experimentations, in line with the EEC priorities, and ii) novel therapeutic approaches to muscle diseases characterized by fibrotic accumulation and/or inefficient healing (e.g. in MD).

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
Finally, we expect RENOIR to provide ESRs with new and better career prospective in the field of stem cell biology and tissue regeneration, inflammation, fibrosis and biomaterials.
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