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La fibrodisplasia ossificante progressiva (FOP) è una rara malattia congenita che provoca l'ossificazione eterotopica (HO) nei muscoli scheletrici. Deriva da una mutazione con guadagno di funzione (R206H) nel gene Acvr1 che codifica per il recettore dell'activina di tipo I, che porta all'attivazione aberrante delle proteine morfogenetiche ossee e delle vie di segnalazione dell'activina A. I pazienti sperimentano riacutizzazioni infiammatorie episodiche nei muscoli scheletrici che innescano l'HO. Il ruolo preciso dei macrofagi nei tessuti affetti da HO rimane poco chiaro e richiede ulteriori chiarimenti. Per modellare FOP abbiamo utilizzato il ceppo murino transgenico condizionale Acvr1R206Hlox/lox; Gt(ROSA26)SorCreERT2. La tomografia computerizzata (TC) ha rivelato che dopo l'espressione del recettore mutato, i topi FOP sviluppano osso ectopico dopo aver ricevuto danno muscolare a 14 e 21 giorni. Al fine di esplorare il coinvolgimento del sistema immunitario innato nell'inizio e nella progressione dell'HO, abbiamo diminuito selettivamente i monociti circolanti tramite quattro iniezioni endovenose di liposomi contenenti clodronato in topi FOP. Le scansioni TC hanno rivelato una formazione ossea ectopica significativamente ridotta nei topi FOP impoveriti di macrofagi rispetto ai controlli a 14 e 21 giorni dopo il danno. Per ottenere ulteriori informazioni sulla segnalazione precoce che porta all'HO, il sequenziamento dell'RNA a singola cellula è stato eseguito sui muscoli di topi FOP a 5 e 7 giorni dopo il danno. L'analisi bioinformatica ha rivelato che i progenitori fibro-adipogenici (FAP) sono arricchiti in vie di segnalazione correlate alla condro/osteogenesi e all'ipossia nei topi FOP. Inoltre, i macrofagi FOP hanno mostrato una maggiore espressione dei marcatori di differenziazione degli osteoclasti e hanno mostrato un profilo pro-infiammatorio sovraregolato. In sintesi, questi risultati affermano che i topi FOP replicano fedelmente le caratteristiche osservate, Fibrodysplasia Ossificans Progressiva (FOP) is a rare congenital disease that results in heterotopic ossification (HO) in skeletal muscles. It arises from a gain-of-function mutation (R206H) in the Acvr1 gene encoding for the activin type I receptor, which leads to the aberrant activation of the bone morphogenetic proteins and activin A signalling pathways. Patients experience episodic inflammatory flare-ups in skeletal muscles that trigger HO. The precise role of macrophages in the tissues affected by HO remains unclear and requires further elucidation. To model FOP we used the Acvr1R206Hlox/lox;Gt(ROSA26)SorCreERT2 conditional transgenic mouse strain. Computerized tomography (CT) revealed that upon expression of the mutated receptor, FOP mice develop ectopic bone after receiving muscle injury at 14 and 21 days. In order to explore the involvement of the innate immune system in the initiation and progression of HO, we selectively depleted circulating monocytes via four intravenous injections of clodronate liposomes in FOP mice. CT scans revealed significantly reduced ectopic bone formation in macrophage-depleted FOP mice compared to controls at 14 and 21 days post-injury. To gain further insights into the early signalling leading to HO, single-cell RNA sequencing was performed on muscles of FOP mice 5 and 7 days after pinch injury. Bioinformatic analysis revealed that fibro-adipogenic progenitors (FAPs) were enriched in pathways related to chondro/osteogenesis and hypoxia in FOP mice. Furthermore, FOP macrophages exhibited heightened expression of osteoclast differentiation markers and displayed an upregulated pro-inflammatory profile. In summary, these findings affirm that FOP mice faithfully replicate the features observed in patients and underscore the critical role of macrophages in HO. Importantly, single-cell transcriptomics suggests that macrophages and FAPs collaborate to form a cellular environment that promotes and sustains bone formation, even in the ear

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Our knowledge of the complexity of the developing hematopoietic system has dramatically expanded over the course of the last few decades. We now know that, while hematopoietic stem cells (HSCs) firmly reside at the top of the adult hematopoietic hierarchy, multiple HSC-independent progenitor populations play variegated and fundamental roles during fetal life, which reflect on adult physiology and can lead to disease if subject to perturbations. The importance of obtaining a high-resolution picture of the mechanisms by which the developing embryo establishes a functional hematopoietic system is demonstrated by many recent indications showing that ontogeny is a primary determinant of function of multiple critical cell types. This review will specifically focus on exploring the diversity of hematopoietic stem and progenitor cells unique to embryonic and fetal life. We will initially examine the evidence demonstrating heterogeneity within the hemogenic endothelium, precursor to all definitive hematopoietic cells. Next, we will summarize the dynamics and characteristics of the so-called “hematopoietic waves” taking place during vertebrate development. For each of these waves, we will define the cellular identities of their components, the extent and relevance of their respective contributions as well as potential drivers of heterogeneity.

Several lines of evidence suggest that childhood leukemia, the most common cancer in young age, originates during in utero development. However, our knowledge of the cellular origin of this large and heterogeneous group of malignancies is still incomplete. The identification and characterization of their cell of origin is of crucial importance in order to define the processes that initiate and sustain disease progression, to refine faithful animal models and to identify novel therapeutic approaches. During embryogenesis, hematopoiesis takes place at different anatomical sites in sequential waves, and occurs in both a hematopoietic stem cell (HSC)-dependent and a HSC-independent fashion. Despite the recently described relevance and complexity of HSC-independent hematopoiesis, few studies have so far investigated its potential involvement in leukemogenesis. Here, we review the current knowledge on prenatal origin of leukemias in the context of recent insights in developmental hematopoiesis.

Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs. The oxidation of HMGB1 cysteines switches its extracellular activities from the orchestration of tissue regeneration to the exacerbation of inflammation. Extracellular HMGB1 is present at high amount and undergoes oxidation in patients with MDs and in mouse models of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy 3 (LGMDR3) compared to controls. Genetic ablation of HMGB1 in muscles of DMD mice leads to an amelioration of the dystrophic phenotype as evidenced by the reduced inflammation and muscle degeneration, indicating that HMGB1 oxidation is a detrimental process in MDs. Pharmacological treatment with an engineered nonoxidizable variant of HMGB1, called 3S, improves functional performance, muscle regeneration, and satellite cell engraftment in dystrophic mice while reducing inflammation and fibrosis. Overall, our data demonstrate that the balance between HMGB1 redox isoforms dictates whether skeletal muscle is in an inflamed or regenerating state, and that the nonoxidizable form of HMGB1 is a possible therapeutic approach to counteract the progression of the dystrophic phenotype. Rebalancing the HMGB1 redox isoforms may also be a therapeutic strategy for other disorders characterized by chronic oxidative stress and inflammation.

Heterotopic ossification is defined as an aberrant formation of bone in extraskeletal soft tissue, for which both genetic and acquired conditions are known. This pathologic process may occur in many dierent sites such as the skin, subcutaneous tissue, skeletal muscle and fibrous tissue adjacent to joints, ligaments, walls of blood vessels, mesentery and other. The clinical spectrum of this disorder is wide: lesions may range from small foci of ossification to massive deposits of bone throughout the body, typical of the progressive genetically determined conditions such as fibrodysplasia ossificans progressiva, to mention one of the most severe and disabling forms. The ectopic bone formation may be regarded as a failed tissue repair process in response to a variety of triggers and evolving towards bone formation through a multistage dierentiation program, with several steps common to dierent clinical presentations and distinctive features. In this review, we aim at providing a comprehensive view of the genetic and acquired heterotopic ossification disorders by detailing the clinical and molecular features underlying the dierent human conditions in comparison with the corresponding, currently available mouse models.

The signals that control endothelial plasticity in inflamed tissues have only been partially characterized. For example, it has been shown that inadequate vasculogenesis in systemic sclerosis (SSc) has been associated with an endothelial defect. We used a genetic lineage tracing model to investigate whether endothelial cells die or change phenotypically after fibrosis induction and whether signals released by cells of the innate immune system and in the blood of patients influence their commitment. We observed that in the lineage-tracing transgenic mice Cdh5-CreERT2::R26R-EYFP, endothelial-derived cells (EdCs) underwent fibrosis after treatment with bleomycin, and EdCs retrieved from the lung showed expression of endothelial-to-mesenchymal transition (EndoMT) markers. Liposome-encapsulated clodronate was used to assess macrophage impact on EdCs. Clodronate treatment affected the number of alternatively activated macrophages in the lung, with upregulated expression of EndoMT markers in lung EdCs. Endothelial fate and function were investigated in vitro upon challenge with serum signals from SSc patients or released by activated macrophages. Sera of SSc patients with anti-Scl70 Abs, at higher risk of visceral organ fibrosis, induced EndoMT and jeopardized endothelial function. In conclusion, EdCs in SSc might be defective because of commitment to a mesenchymal fate, which is sustained by soluble signals in the patient's blood. Macrophages contribute to preserve the endothelial identity of precursor cells. Altered macrophage-dependent plasticity of EdCs could contribute to link vasculopathy with fibrosis.