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The hematopoietic stem cell (HSC) niche is a specialized microenvironment, where a complex and dynamic network of interactions across multiple cell types regulates HSC function. During the last years, it became progressively clearer that changes in the HSC niche are responsible for specific alterations of HSC behavior. The aging of the bone marrow (BM) microenvironment has been shown to critically contribute to the decline in HSC function over time. Interestingly, while upon aging some niche structures within the BM are degenerated and negatively affect HSC functionality, other niche cells and specific signals are preserved and essential to retaining HSC function and regenerative capacity. These new findings on the role of the aging BM niche critically depend on the implementation of new technical tools, developed thanks to transdisciplinary approaches, which bring together different scientific fields. For example, the development of specific mouse models in addition to coculture systems, new 3D-imaging tools, ossicles, and ex-vivo BM mimicking systems is highlighting the importance of new technologies to unravel the complexity of the BM niche on aging. Of note, an exponential impact in the understanding of this biological system has been recently brought by single-cell sequencing techniques, spatial transcriptomics, and implementation of artificial intelligence and deep learning approaches to data analysis and integration. This review focuses on how the aging of the BM niche affects HSCs and on the new tools to investigate the specific alterations occurring in the BM upon aging. All these new advances in the understanding of the BM niche and its regulatory function on HSCs have the potential to lead to novel therapeutical approaches to preserve HSC function upon aging and disease.

Despite their emerging relevance to fully understand disease pathogenesis, we have as yet a poor understanding as to how biomechanical signals are integrated with specific biochemical pathways to determine cell behaviour. Mesothelial-to-mesenchymal transition (MMT) markers colocalized with TGF-β1-dependent signaling and yes-associated protein (YAP) activation across biopsies from different pathologies exhibiting peritoneal fibrosis, supporting mechanotransduction as a central driving component of these class of fibrotic lesions and its crosstalk with specific signaling pathways. Transcriptome and proteome profiling of the response of mesothelial cells (MCs) to linear cyclic stretch revealed molecular changes compatible with bona fide MMT, which (i) overlapped with established YAP target gene subsets, and were largely dependent on endogenous TGF-β1 signaling. Importantly, TGF-β1 blockade blunts the transcriptional upregulation of these gene signatures, but not the mechanical activation and nuclear translocation of YAP per se. We studied the role therein of caveolin-1 (CAV1), a plasma membrane mechanotransducer. Exposure of CAV1-deficient MCs to cyclic stretch led to a robust upregulation of MMT-related gene programs, which was blunted upon TGF-β1 inhibition. Conversely, CAV1 depletion enhanced both TGF-β1 and TGFBRI expression, whereas its re-expression blunted mechanical stretching-induced MMT. CAV1 genetic deficiency exacerbated MMT and adhesion formation in an experimental murine model of peritoneal ischaemic buttons. Taken together, these results support that CAV1-YAP/TAZ fine-tune the fibrotic response through the modulation of MMT, onto which TGF-β1-dependent signaling coordinately converges. Our findings reveal a cooperation between biomechanical and biochemical signals in the triggering of MMT, representing a novel potential opportunity to intervene mechanically induced disorders coursing with peritoneal fibrosis, such as post-surgical adhesions.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell ( cis ) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis -inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta. Notch signaling is critical for HSC emergence. Here, the authors identify a sub-set of hemogenic endothelial cells with high Notch activity that it is gradually shut down through cis inhibition of NOTCH1 by JAG1, and report that this process sustains HSC.

Background The cell-cycle inhibitor p57 kip2 plays a critical role in mammalian development by coordinating cell proliferation and differentiation in many cell types. p57 kip2 expression is finely regulated by several epigenetic mechanisms, including paternal imprinting. Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the p57 Kip2 imprinting domain, is expressed exclusively from the paternal allele and participates in the cis -silencing of the neighboring imprinted genes through chromatin-level regulation. In light of our previous evidence of a functional interaction between myogenic factors and imprinting control elements in the regulation of the maternal p57 Kip2 allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of p57 Kip2 expression in muscle cells. Results We found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional p57 Kip2 allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel p57 Kip2 regulatory region, located intragenically and containing two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion demonstrated that the LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal p57 kip2 intragenic region. Finally, upon differentiation, the binding of MyoD to this region and its physical interaction with Kcnq1ot1, analyzed by ChIP and RNA immunoprecipitation assays, correlate with the loss of EZH2 and H3K27me3 from chromatin and with p57 Kip2 de-repression. Conclusions These findings highlight the existence of an imprinting-independent role of Kcnq1ot1, adding new insights into the biology of a still mysterious LncRNA. Moreover, they expand our knowledge about the molecular mechanisms underlying the tight and fine regulation of p57 Kip2 during differentiation and, possibly, its aberrant silencing observed in several pathologic conditions.

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.

One goal of regenerative medicine is to rejuvenate tissues and extend lifespan by restoring the function of endogenous aged stem cells. However, evidence that somatic stem cells can be targeted in vivo to extend lifespan is still lacking. Here, we demonstrate that after a short systemic treatment with a specific inhibitor of the small RhoGTPase Cdc42 (CASIN), transplanting aged hematopoietic stem cells (HSCs) from treated mice is sufficient to extend the healthspan and lifespan of aged immunocompromised mice without additional treatment. In detail, we show that systemic CASIN treatment improves strength and endurance of aged mice by increasing the myogenic regenerative potential of aged skeletal muscle stem cells. Further, we show that CASIN modifies niche localization and H4K16ac polarity of HSCs in vivo. Single-cell profiling reveals changes in HSC transcriptome, which underlie enhanced lymphoid and regenerative capacity in serial transplantation assays. Overall, we provide proof-of-concept evidence that a short systemic treatment to decrease Cdc42 activity improves the regenerative capacity of different endogenous aged stem cells in vivo, and that rejuvenated HSCs exert a broad systemic effect sufficient to extend murine health- and lifespan.

Bone marrow-derived mesenchymal stromal cell- (BM-MSC-) based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP-) 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P) and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers) and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia), S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs’ ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair.

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57 , myogenin , Mef2C and p21 , while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration.