Multi-layered spatial transcriptomics identify secretory factors promoting human hematopoietic stem cell development

Hematopoietic stem cells (HSCs) first emerge in the embryonic aorta-gonad-mesonephros (AGM) region. Studies of model organisms defined intersecting signaling pathways that converge to promote HSC emergence predominantly in the ventral domain of the dorsal aorta. Much less is known about mechanisms driving HSC development in humans. Here, to identify secreted signals underlying human HSC development, we combined spatial transcriptomics analysis of dorsoventral polarized signaling in the aorta with gene expression profiling of sorted cell populations and single cells. Our analysis revealed a subset of aortic endothelial cells with a downregulated arterial signature and a predicted lineage relationship with the emerging HSC/progenitor population. Analysis of the ventrally polarized molecular landscape identified endothelin 1 as an important secreted regulator of human HSC development. The obtained gene expression datasets will inform future studies on mechanisms of HSC development in vivo and on generation of clinically relevant HSCs in vitro

Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs

Haematopoietic stem and progenitor cells (HSPCs) develop through distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) is able to recapitulate some of these processes, however, it has proven difficult to generate functional haematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we exploited single cell RNA sequencing (scRNAseq) in combination with single cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we identified CD44, CD326, ICAM2/CD9 and CD18 as markers of these progenitors, respectively. Using an artificial neural network (ANN), that we trained on a scRNAseq derived from human fetal liver, we were able to identify a wide range of hPSCs-derived HPSC phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded and was completely missing in the dataset that had been generated using cells selected on the basis of CD43expression. By comparing the single cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver we identified transcription factors and molecular pathways that can be exploited in the future to improve the in vitro production of HSCs

Slégami Open Access - Manuale d'uso per ricercatori

Il seguente documento nasce nell’ambito delle attività svolte dal Gruppo di Lavoro (GdL) APRE dedicato al tema dell’Open Science e si sviluppa come un manuale d’uso per i ricercatori, con specifico riguardo all’Open Access e all’Open Data. La sua redazione ha coinvolto attivamente tutti i membri del GdL, i cui membri sono rappresentanti delle biblioteche e degli uffici di supporto alla ricerca di diverse università e centri di ricerca italiani (è possibile consultare la lista dei partecipanti nell’ultima pagina di questo documento). Il lavoro è un aggiornamento del manuale originariamente pubblicato nel 2019 e la cui prima edizione era il risultato di un lavoro svolto in 3 fasi: 1) un’iniziale raccolta delle domande più comuni poste dai ricercatori presso le strutture di supporto (siano esse biblioteche o uffici di supporto alla ricerca) degli enti partecipanti in materia di Open Access e Open Data; 2) una fase di consolidamento e classificazione delle domande raccolte in 6 categorie; 3) un’ultima fase di redazione, da parte di alcuni membri del GdL, delle risposte alle domande poste e successivamente emendate a più riprese dall’intero gruppo. Nel 2021 il GdL si è riunito nuovamente per lavorare ad un aggiornamento del manuale in ottica Horizon Europe. Seguendo lo stesso schema di lavoro in 3 fasi (raccolta, classificazione ed elaborazione), il gruppo ha identificato 76 domande aggiuntive rispetto al documento originale, le quali a loro volta sono state successivamente raggruppate e classificate in 10 categorie

A molecular roadmap of the AGM region reveals BMP ER as a novel regulator of HSC maturation

In the developing embryo, hematopoietic stem cells (HSCs) emerge from the aorta-gonad-mesonephros (AGM) region, but the molecular regulation of this process is poorly understood. Recently, the progression from E9.5 to E10.5 and polarity along the dorso-ventral axis have been identified as clear demarcations of the supportive HSC niche. To identify novel secreted regulators of HSC maturation, we performed RNA sequencing over these spatiotemporal transitions in the AGM region and supportive OP9 cell line. Screening several proteins through an ex vivo reaggregate culture system, we identify BMP ER as a novel positive regulator of HSC development. We demonstrate that BMP ER is associated with BMP signaling inhibition, but is transcriptionally induced by BMP4, suggesting that BMP ER contributes to the precise control of BMP activity within the AGM region, enabling the maturation of HSCs within a BMP-negative environment. These findings and the availability of our transcriptional data through an accessible interface should provide insight into the maintenance and potential derivation of HSCs in culture.Peer reviewe

Competence Centre ICDI per Open Science, FAIR, ed EOSC - Mission, Strategia e piano d'azione

This document presents the mission and strategy of the Italian Competence Centre on Open Science, FAIR, and EOSC. The Competence Centre is an initiative born within the Italian Computing and Data Infrastructure (ICDI), a forum created by representatives of major Italian Research Infrastructures and e-Infrastructures, with the aim of promoting sinergies at the national level, and optimising the Italian participation to European and global challenges in this field, including the European Open Science Cloud (EOSC), the European Data Infrastructure (EDI) and HPC. This working paper depicts the mission and objectives of the ICDI Competence Centre, a network of experts with various skills and competences that are supporting the national stakeholders on topics related to Open Science, FAIR principles application and participation to the EOSC. The different actors and roles are described in the document as well as the activities and services offered, and the added value each stakeholder can find the in Competence Centre. The tools and services provided, in particular the concept for the portal, though which the Centre will connect to the national landscape and users, are also presented

Characterization of the role of angiopoietin-tie signalling in haematopoietic stem cell development in the murine embryo

Haematopoietic stem cells (HSCs) are capable of self-renewing and multi-lineage reconstitution of the haematopoietic system of irradiated recipient mice. In the mouse embryo, HSCs originate in a step-wise manner from the haematogenic endothelium. The first HSC precursor has been detected at E9.5 in the dorsal aorta, while HSCs emerge in the aorta-gonad-mesonephros (AGM) region around E11. To date, the molecular mechanisms regulating these events are poorly characterized. Through the activating role of Angiopoietin1 (Ang1) on Tie2 receptor, the Ang-Tie signalling pathway plays a critical role in HSC maintenance in the adult bone marrow niche. Tie2 ligand Angiopoietin2 (Ang2) is described as being a Tie2 inhibitor, however its role is unknown. The aim of this thesis was to characterise the role of Ang-Tie signalling pathway in HSC formation in the mouse embryo. First, I used an ex vivo aggregate system to culture with angiopoietins cells derived from the AGM region at stages of development preceding HSC formation (E9.5-E11). Ang2- treated cells were able to reconstitute the peripheral blood of recipient mice to a higher extent compared to control, indicating a role for Ang2 in promoting HSC maturation. Then, I characterized the expression pattern of Ang-Tie molecules in the AGM region. Ang2-expressing cells were identified as perivascular and sub-aortic mesenchymal cells located in the ventral side of the aorta and in proximity of intra-aortic haematopoietic clusters. Finally, I performed an RNA-seq analysis with the aim of unravelling the molecular mechanisms involved in Ang2-mediated HSC maturation. Pre-HSC-I were cultured in presence or absence of Ang2 and their transcriptional profiles were compared, revealing a number of genes and pathways up-regulated or down-regulated in presence of Ang2, which might indicate a role for Ang2 in increasing cell proliferation, favouring cell migration, and regulation of other signalling pathways involved in HSC development. All together, these data support Ang2 as a novel regulator for HSC formation

Stroke and Amyloid-\u3b2 Downregulate TREM-2 and Uch-L1 Expression that Synergistically Promote the Inflammatory Response

 Neuroinflammation is involved in the pathogenesis of Alzheimer's disease, and the transcription factor NF-\u3baB is a player in this event. We found here that the ischemic damage alone or in association with A\u3b21-42 activates the NF-\u3baB pathway, induces an increase of BACE1 and a parallel inhibition of Uch-L1 and TREM2, both in vitro and in vivo, in Tg 5XFAD and in human brains of sporadic AD. This mechanism creates a synergistic loop that fosters inflammation. We also demonstrated a significant protection exerted by the restoration of Uch-L1 activity. The rescue of the enzyme is able to abolish the decrease of TREM2 and the parameters of neuroinflammation