Assembly of large biochemical networks can be achieved by confronting new cell-specific experimental data with an interaction subspace constrained by prior literature evidence. The SIGnaling Network Open Resource, SIGNOR (available on line at http://signor.uniroma2.it), was developed to support such a strategy by providing a scaffold of prior experimental evidence of causal relationships between biological entities. The core of SIGNOR is a collection of approximately 12 000 manually-annotated causal relationships between over 2800 human proteins participating in signal transduction. Other entities annotated in SIGNOR are complexes, chemicals, phenotypes and stimuli. The information captured in SIGNOR can be represented as a signed directed graph illustrating the activation/inactivation relationships between signalling entities. Each entry is associated to the post-translational modifications that cause the activation/inactivation of the target proteins. More than 4900 modified residues causing a change in protein concentration or activity have been curated and linked to the modifying enzymes (about 351 human kinases and 94 phosphatases). Additional modifications such as ubiquitinations, sumoylations, acetylations and their effect on the modified target proteins are also annotated. This wealth of structured information can support experimental approaches based on multi-parametric analysis of cell systems after physiological or pathological perturbations and to assemble large logic models.

Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. An unbiased mass spectrometry–based proteomic analysis of FAPs from muscles of wild-type and mdx mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in mdx FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.

Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product—a cell‐free collagen‐glycosaminoglycan (GAG) scaffold—demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic‐like state. Recent findings describe how iPSCs‐derived fibroblasts (“post‐iPSF”) are proangiogenic, produce more ECM than their somatic precursors (“pre‐iPSF”), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar‐free). ECM production is 45% higher from post‐iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze‐dried scaffold using ECM grown by post‐iPSF cells (Post‐iPSF Coll) is developed and tested vs precursors ECM‐activated scaffolds (Pre‐iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM‐derived scaffolds have more diverse ECM and more robust immune responses to cues. Post‐iPSF‐Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin‐1‐receptor antagonist (IL‐1ra) vs. pre‐iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds.

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The regeneration of the muscle tissue relies on the capacity of the satellite stem cell (SC) population to exit quiescence, divide asymmetrically, proliferate, and differentiate. In age-related muscle atrophy (sarcopenia) and several dystrophies, regeneration cannot compensate for the loss of muscle tissue. These disorders are associated with the depletion of the satellite cell pool or with the loss of satellite cell functionality. Recently, the establishment and maintenance of quiescence in satellite cells have been linked to their metabolic state. In this work, we aimed to modulate metabolism in order to preserve the satellite cell pool. We made use of metformin, a calorie restriction mimicking drug, to ask whether metformin has an effect on quiescence, proliferation, and differentiation of satellite cells. We report that satellite cells, when treated with metformin in vitro, ex vivo, or in vivo, delay activation, Pax7 downregulation, and terminal myogenic differentiation. We correlate the metformin-induced delay in satellite cell activation with the inhibition of the ribosome protein RPS6, one of the downstream effectors of the mTOR pathway. Moreover, in vivo administration of metformin induces a belated regeneration of cardiotoxin- (CTX-) damaged skeletal muscle. Interestingly, satellite cells treated with metformin immediately after isolation are smaller in size and exhibit reduced pyronin Y levels, which suggests that metformin-treated satellite cells are transcriptionally less active. Thus, our study suggests that metformin delays satellite cell activation and differentiation by favoring a quiescent, low metabolic state.