Fabio Rossi

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Fibrosis causes nearly 45% of all deaths in industrialized nations. Currently, no efficient antifibrotic therapy exists. Detailed understanding of the mechanisms governing efficient tissue regeneration and of how they go awry in fibrotic pathologies will likely inform therapeutic approaches. The remarkable healing capacity of skeletal muscle and development of rodent models for muscular dystrophies, in which fibrosis is a common outcome, makes skeletal muscle an excellent candidate to study tissue regeneration and fibrosis. In contrast to infiltrating macrophages, the role of tissue resident macrophages (RMs) during regeneration or fibrosis is not well-understood, mainly due to the lack of a specific marker for their identification. In the first part of this study, using a combination of parabiosis, lineage tracing, and single-cell transcriptomics we identified a population of muscle resident LYVE1+TIM4+ macrophages that locally self-renew (self-renewing resident macrophages, SRRMs). Using a Colony Stimulating Factor 1 Receptor (CSF1R) inhibition/withdrawal approach to specifically deplete SRRMs, we showed that SRRMs provide a non-redundant function in clearing damage-induced apoptotic cells. We further found that depletion of RMs through CSF1R inhibition changed muscle fiber composition from damage-sensitive glycolytic fibers towards damage-resistant oxidative fibers, protecting dystrophic muscle against contraction induced injury. This finding has therapeutic potential in light of the ongoing clinical testing of CSF1R inhibitors.As a well-known pro-inflammatory cytokine with diverse roles in antimicrobial and antitumor immunity, a small amount of conflicting evidence exists regarding the source and function of IFNγ during the regeneration process following a sterile injury. In the second part of this study, we showed that a population of natural killer cells is the main source of IFNγ in regenerating muscle. We found that the absence of IFNγ per se has no effect on regeneration. However, the role of IFNγ gets unmasked when TNFα is neutralized. Using three different transgenic mice strains to conditionally deplete the IFNγ receptor 1 on the surface of satellite cells, fibro-adipogenic progenitors (FAPs), or macrophages, we found that IFNγ acts through macrophages and FAPs to support muscle regeneration. In contrast to the common belief, we found that IFNγ peaks when pro-inflammatory macrophages are switching to an anti-inflammatory phenotype.

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Hematopoietic stem cells (HSCs) are believed to reside and are protected in a special-perivascular microenvironment, which is called the hematopoietic stem cell niche. Within the niche, the fate of HSCs such as homing, self-renewal, quiescence, and differentiation is determined based on an array of intra and extracellular signals. For many decades, the identity and function of major cellular components of niches for HSCs has been a long-standing debate. One key candidate is a population of mesenchymal stromal cells (MSCs). Due to the complexity of the bone marrow matrix, it has been very challenging to identify a signature marker that specifically labels these MSCs. Recently, CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells or leptin receptor-expressing (LepR⁺) cells have been found to play an important role in hematopoiesis. They are not only a major cellular component of HSC niches but also can give rise to osteoblasts and adipocytes in bone marrow. However, it remains unclear how osteogenesis and adipogenesis are prevented in most CAR/LepR⁺ cells to maintain HSC niches and marrow cavities. Moreover, how the number of niche is controlled is not yet fully understood, as only about 0.01% of whole bone marrow cells are found to be HSCs being present at any time under homeostasis. Our preliminary data indicated that the transcription factor Hic1 is preferentially expressed in MSCs across all tissues in the body, including bone marrow. Hic1-expressing cells have been described as quiescent mesenchymal stromal cells that have the full capacity to become adipocytes and/or osteoblasts. Single cell RNA sequencing analysis revealed that Hic1⁺ MSCs isolated from bone marrow are heterogeneous but with the majority of such cells expressing high levels of CXCL12 and LepR. When Hic1 is deleted, MSCs numbers expand dramatically, and in mice there is an associated increase in HSC activity and HSC frequency in bone marrow compared to wild type individuals. Thus, we propose that Hic1 acts as a quiescence regulator of MSCs in bone marrow and is responsible for controlling the size of the hematopoietic stem cell niche.

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Despite its rigid structure, bone is a highly dynamic tissue that is constantly remodeled throughout life to maintain its structure and function. Remodeling is mediated by bone resorbing osteoclasts and bone depositing osteoblasts. While the hematopoietic origins of osteoclasts are well understood, the identity of the skeletal mesenchymal progenitors that give rise to osteoblasts remains elusive. Understanding the identity and biology of these skeletal mesenchymal progenitors is critical to the development of therapeutics for the treatment of bone-related disorders from trauma and surgical-induced heterotopic ossification to osteoporosis-related fractures. Previous studies have attempted to identify skeletal stem and progenitor cell populations, however, though the periosteum is known to be a source of progenitors crucial to bone regeneration, none of the identified populations were periosteal. While PDGFRα expression has been identified in many putative skeletal progenitors, we show that it is expressed ubiquitously throughout skeletal tissues and therefore does not constitute a unique marker for skeletal progenitors. We identified HIC1 as a marker of a novel injury-inducible periosteal skeletal progenitor that contributes to bone homeostasis and regeneration, an important advancement in the understanding of skeletal biology.Contrary to skeletal tissues, in skeletal muscle, expression of PDGFRα is restricted to fibro-adipogenic progenitors (FAPs) which have been suggested as the cellular source of heterotopic ossification. Using a new inducible PDGFRαCreERT2 transgenic model, we confirmed that tissue resident PDGFRα+ FAPs give rise to HO in skeletal muscle and that they have inherent osteogenic potential that is induced by an altered inflammatory environment following muscle damage. This presents novel cellular and therapeutic targets for the prevention of acquired HO. The TGFβ and BMP pathways are well known for their effects on FAPs, inducing ectopic bone formation and fibrosis. Using a PDGFRαCreERT2/Smad4Flox transgenic model, we examined the role of mesenchymal TGFβ and BMP signaling in adult tissues. We found that impairment of SMAD-mediated TGFβ/BMP signaling in PDGFRα+ cells induced increased proliferation and uncoupling of bone formation and resorption resulting in high turnover bone loss. This suggests constitutive SMAD-mediated TGFβ/BMP signaling is required for adult murine skeletal homeostasis and deepens our scientific understanding of adult skeletal biology.

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The euchromatic histone methyltransferase 2 (Ehmt2), aka. G9a, is responsible for methylating histone H3 at lysine 9. However, it is a multifaceted gene whose functions sometimes exceed histone-tail modifications, and the nature and importance of its regulatory effects differ vastly between different tissue systems. A number of lines of evidence hint at its critical role in the developmental biology and tissue biology of mesenchymal tissues. In this work I assessed the role of Ehmt2 in skeletal muscle, adipose tissues, and craniofacial development. Previous publications employing immortalised cell lines have proposed that Ehmt2 is an important inhibitor of myogenic differentiation. In addition to the well-known repressive effects of H3K9 dimethylation, for which Ehmt2 is largely responsible, it was postulated that it can directly methylate MYOD, a master regulator of myogenesis, and lead to repression of myogenic cell differentiation. In a mouse model to validate this, I found conditional knockout muscle stem cells activated, proliferated, and differentiated normally without Ehmt2. Knockout mice under the control of Myod-Cre developed and regenerated normally after acute injury to leg muscle, refuting the previous theory that Ehmt2 is required for myogenesis. The global loss of H3K9 dimethylation in normal myogenesis also signalled that this histone tail modification is largely irrelevant in skeletal muscle.Despite the gene’s dispensability in a number of developmental tissues, Ehmt2-/- mice die early during embryogenesis. In order to uncover the role of Ehmt2 in other mesenchymal tissues, I generated a transgenic mouse line to conditionally delete Ehmt2 during mesoderm and neural crest development. I found that the loss of Ehmt2 in the Pdgfra developmental lineage resulted in striking yet highly reproducible craniofacial malformations. Adipose tissue is also an important topic in the understanding of Ehmt2, especially since previous publications have found its importance in white adipose tissue, and its homologue, Ehmt1/GLP, to be required for brown adipose tissue specification and activation. The PdgfraCre Ehmt2floxed/null mouse model revealed novel in vivo insight for the role in which Ehmt2 limits lipid accumulation and is required for normal brown adipose development.

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Fibrosis is a response to injury that involves cell proliferation and deposition of extracellular matrix; pathological fibrosis can be considered to be imperfect wound healing that impairs organ function. Our understanding of the role and source of cardiac fibroblasts (CFs) in the development of pathological fibrosis is severely hampered by a lack of robust in vivo markers and understanding of the heterogeneity within this vaguely defined population. Here we demonstrate the existence of cardiac-resident mesenchymal progenitor cells (cMSCs, identified as CD31-:CD45-:PDGFRα+:Sca-1+) and show that their in vivo pharmacological modulation with the receptor tyrosine kinase inhibitors nilotinib or imatinib reduces fibrosis and improves cardiac function following acute or chronic cardiac injury. We observed in vivo the initial expansion of Sca-1+ cMSCs following acute cardiac injury contributes to mature collagen-producing PDGFRα+:Sca-1- CFs. In two models of acute injury, isoproterenol treatment and ligation of the left anterior descending artery (LAD), treatment with nilotinib led to reduced proliferation of both cMSCs and CFs but an increased relative prevalence of cMSCs, suggesting a blockade in the fibrogenic differentiation of these cells. Extending these observations into mdx mice, a model of chronic myocardial dystrophy displaying cardiac fibrosis after one year of age, we investigated the effects of long-term treatment with imatinib as a potential pharmacologic therapy. mdx mice treated with imatinib for 15 months displayed improved cardiac function and reduced cardiac fibrosis. These improvements were matched by a reduced quantity of cMSCs within treated myocardial tissue. Finally, given the well-known role of PDGFRα+ Sca-1+ mesenchymal progenitors in the development of fibrofatty infiltration in skeletal muscle, we postulated a role for phenotypically similar cMSCs in the fibrofatty infiltrate observed inarrhythmogenic cardiomyopathy. Using a genetic model that dysregulates quiescence in mesenchymal populations, we observed hyperproliferation of cMSCs and spontaneous generation of adipose deposits within the myocardium, supporting our hypothesis that cMSCs are the cellular source of the fibrofatty infiltrate. In their entirety, our findings demonstrate the importance of differing subsets of mesenchymal cell populations within the heart and potential therapeutic benefit of targeting them pharmacologically following acute ischemic damage or during chronic dystrophic injury.

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Expression of the cell surface sialomucin CD34 is common to many adult stem cell types, including muscle satellite cells. However, no clear stem cell or regeneration-related phenotype has ever been reported in mice lacking CD34, and its function on these cells remains poorly understood. Here, we assess the functional role of CD34 on satellite cell-mediated muscle regeneration. Using an optimized flow cytometry-based method to analyze myogenic progenitors, we show that CD34’s expression is tightly regulated early during the muscle regeneration process. Following this, we show that Cd34⁻/⁻ mice, which have no obvious developmental phenotype, display a defect in muscle regeneration when challenged with either acute or chronic muscle injury, resulting in impaired myofibre hypertrophy. In vivo engraftment efficiency and BrdU proliferation assays comparing WT and Cd34⁻/⁻ myogenic progenitors attribute this defect to impaired myogenic progenitor cell function in Cd34⁻/⁻ animals. Lastly, the culture of isolated single myofibres demonstrate that this overall muscle regenerative defect is caused by a delay in the activation of satellite cells lacking CD34 as well as impaired proliferation following activation. Consistent with the reported anti-adhesive function of CD34, Cd34⁻/⁻ satellite cells also show decreased motility along their host myofibre. Altogether, our results identify a role for CD34 in the poorly understood early steps of satellite cell activation, and provide the first evidence that beyond being a stem cell marker, CD34 may play an important function in modulating satellite cell activity.

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A key regulator of central nervous system (CNS) inflammatory responses is a highly specialized subset of tissue macrophages that reside in the CNS parenchymal and perivascular spaces known as “microglia”. Microgliosis is a common response to multiple types of damage within the CNS and is commonly characterized by an increase in microglial cells. What remains elusive, however, is the origin of cells involved in this phenomenon and whether the increase in the number of cells is due to local expansion or recruitment of myeloid progenitors from the bloodstream. Here, we investigated the origin of microglia using chimeric animals obtained by parabiosis. We found no evidence of circulating myeloid cells recruitment under healthy conditions and in denervation or CNS neurodegenerative disease suggesting that microglia can respond to CNS trauma and degeneration by expanding in situ independently of the contribution of blood-derived myeloid precursors. Furthermore, I investigated the extent to which blood-derived myeloid cells contribute to the microglia population in conditions where other circulating blood-borne cells have access to the CNS, such as in multiple sclerosis, an autoimmune disease of CNS and its murine model experimental autoimmune encephalitis (EAE).Using a novel approach to specifically replace circulating progenitors without affecting CNS-resident microglia, we found a strong correlation between monocyte infiltration and progression to the paralytic stage of EAE. Inhibition of chemokine receptor-dependent recruitment of monocytes to the CNS blocked EAE progression suggesting that these infiltrating cells are essential for pathogenesis. Finally, we found that although microglia can enter the cell cycle and return to quiescence following remission, recruited monocytes vanish, thus not ultimately contributing to the resident microglial pool. These findings collectively demonstrate that microglia constitute a unique myeloid cell population that are capable of long-term self-renewal within the CNS, and can respond to CNS trauma and degeneration by expanding in situ independently of the contribution of blood-derived myeloid precursors. Furthermore, two distinct subsets of myelomonocytic cells with unique roles in neuroinflammation and disease progression were identified under conditions where the blood-brain barrier is damaged and blood-derived leukocytes have access to the CNS parenchyma.

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White adipose tissue, or fat, is a complex endocrine tissue important for energy storage and metabolism, and has significant effects on various physiological phenomena, including growth, behaviour, reproduction and immune-modulation. It has been proposed that fat cells, or adipocytes, arise from connective tissue cells that fill with lipid; however, mounting evidence suggests that adipocytes represent a distinct lineage with its own cellular origins. Yet very little is known about the cells that give rise to new adipocytes. Here, my colleagues and I developed a strategy to isolate purified populations of adipogenic progenitor (AP) cells from subcutaneous fat, visceral fat and skeletal muscle, using fluorescence-activated cell sorting. These cells are capable of robust adipogenic differentiation, even at the single cell level. We confirmed their commitment to the adipogenic lineage using a variety of assays, and reveal that they are lineage-restricted cells, incapable of osteogenic, chondrogenic or myogenic differentiation. Thus, we have developed an enabling technology to allow interrogation of the adipocyte lineage among different tissues and fat depots, during different physiological, pathological or developmental stages. Recent evidence suggests that fat depots with a greater ability to generate new adipocytes are associated with lower metabolic risk. Using our isolation strategy, we confirmed that metabolically healthier depots are associated with greater AP abundance and activity, uncovering a link between stem cell biology and metabolic disease. However, adipocyte production in non-adipose tissues, such as skeletal muscle and bone marrow, is associated with chronic disease and aging. To explore possible reasons for this dichotomy, we examined the role of APs in a model of skeletal muscle injury. Our results suggest that APs expand after damage to assist in muscle regeneration by establishing a pro-myogenic niche, ascribing to them a novel function that is independent of adipogenesis. Together, our strategy to interrogate the adipogenic lineage has allowed us to formulate new hypotheses to explain adipose and skeletal muscle physiology. This technology forms the basis for future work that will to allow us to understand how new adipocytes are formed, and perhaps permit the manipulation of adipogenic progenitors for therapeutic benefit.

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The ability of bone marrow derived cells to contribute to skeletal muscle repair may represent a novel means of cell therapy for myopathies. However, this phenomenon takes place at exceedingly low frequencies and has failed to yield any measurable functional improvement in disease models. In an effort to increase the efficiency of this process, we designed Cre/loxP-based tracing strategies to identify the lineages and mechanisms involved. However, these experiments were complicated by a previously unknown limitation of common Cre-reporter strains. We have studied the Z/AP, Z/EG and R26R-EYFP reporter strains and have demonstrated that although each reporter can be reliably activated by Cre during early development, exposure to Cre in adult hematopoietic cells results in a much lower frequency of reporter-positive cells in the Z/AP or Z/EG strains than in the R26R-EYFP strain. In reporter-negative cells derived from the Z/AP and Z/EG strains, the transgenic promoter is methylated and Cre-mediated recombination of the locus is inhibited. These findings suggest that the Z/AP and Z/EG strains may not be suitable for the investigation of developmental plasticity in adult models.As bone marrow derived cells are now believed to contribute to skeletal muscle repair primarily via fusion, we also constructed a chimeric measles hemagglutinin, Hα7, which efficiently mediates the fusion of diverse cell types with skeletal muscle. When compared directly to polyethylene glycol in vitro, Hα7 consistently generated a ten to fifteen fold increase in heterokaryon yield and induced insignificant levels of toxicity. More importantly, Hα7 was also capable of increasing the contribution of mouse and human bone marrow derived cells to skeletal muscle repair in vivo.

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Increasing evidence suggests that site-specific lysine methylation of histone and non-histone proteins is fundamentally involved in epigenetic regulation of gene expression during cellular differentiation and tumorigenesis. To study the in vivo relevance of the lysine methyltransferases Set7/9 and G9a, I have generated and characterized two conditional knockout mouse strains. Despite its widely proposed role in transcriptional activation through the methylation of histone H3 lysine 4 (H3K4) and a number of transcription factors, I found that Set7/9 knockout mice develop normally and do not display any overt phenotypic alteration. Since Set7/9 was shown to methylate the tumor suppressor protein p53 and was suggested to be important for its activity, I mainly focused my characterization of the Set7/9 knockout strain towards the proposed impairment of p53 function in these mice. Contrary to all reports, I found that in the absence of Set7/9, the p53 target genes p21WAF¹/CIP¹, Mdm2, Puma and Bax are normally expressed under basal and stressed conditions in different cell types. As a consequence, no functional p53 impairment was detectable upon DNA damage or in response to ectopic oncogene expression in Set7/9⁻/⁻ cells. Hence, my data demonstrates that Set7/9-mediated methylation of p53 represents, if at all, only a minor event in its regulation and does not appreciably control p53 activity in vivo. In the generated conditional G9a knockout strain, I primarily focused my efforts towards describing its role in the hematopoietic system. Mice that conditionally lack G9a expression in the blood, develop normally and can sustain the development of all hematopoietic cell types under homeostatic conditions. Interestingly however, when performing competitive bone marrow transplantation assays, I detected a marked impairment in G9a knockout bone marrow cells in the reconstitution of the hematopoietic system. Consistently, G9a-deficient myeloid and erythroid progenitors are dramatically reduced in their proliferation capacity. My experiments indicate for the first time, that G9a is specifically important for the biology of hematopoietic stem and progenitor cells under stress conditions and its inactivation might represent a promising way to interfere with blood development in pathological and regenerative settings.

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