IRCCS San Raffaele Scientific Institute Showcase

Images and dataset of densitometric analysis used for the generation of the figure 1 of the paper by Ravasi G, Pelucchi S, Canonico F, Mariani R, Piperno A. Atypical phenotype in a patient with ceruloplasmin mutations in the compound heterozygous state. Meta Gene. 2021, 29: 100905. doi.org/10.1016/j.mgene.2021.100905. This paper is associated to the project "Blood-cerebrospinal fluid-barrie defect as gate for targeting by liver-directed ceruloplasmin gene replacement therapy in the rare disease aceruloplasminemia" supported by Italian Ministry of Health, with the grant RF-2018-1236671 to Massimo ALESSIO as principal investigator at the IRCCS-San Raffaele Hospital, Milano, Italy.

Raw images and dataset of densitometric analysis used for the generation of the figure 1 of the paper by Pelucchi S, Ravasi G, Piperno A. Ceruloplasmin variants might have different effects in different iron overload disorders. J Hepatol. 2021, 75:1003-1004. doi: 10.1016/j.jhep.2021.05.005. This paper is associated to the project "Blood-cerebrospinal fluid-barrie defect as gate for targeting by liver-directed ceruloplasmin gene replacement therapy in the rare disease aceruloplasminemia" supported by Italian Ministry of Health, with the grant RF-2018-1236671 to Massimo ALESSIO as principal investigator at the IRCCS-San Raffaele Hospital, Milano, Italy.

The dataset includes 172 subjects for a total of 519 fetal rs-fMRI scans with respective brain segmentations. Brain segmentations were obtained with the RS-FetMRI package (https://github.com/NicoloPecco/RS-FetMRI). For each subject the gestational week at scan is reported as the last two digits of the file name. The RF-2016-02364081 DL dataset has been used to train and test deep learning architectures (i.e. Swin-UNETR, UNTER, CNN, GAN) on a fetal brain extraction task for the study titled ‘Optimizing performance of transformer-based models for fetal brain MR image segmentation’. Pretrain weights of Swin-UNETR best model can be found on the ‘weight’ folder (Fetal_pretrain.pth).

Preliminary data for the selection of housekeeping genes suitable for our cells to be used as normaliser for the full experiment that will allow us to identify genes potentially modified by the infection with ZIKV and counteracted with the heparin treatment. Instead of performing RNAseq analyses, we decided to opt for QuantiGene Plex (Thermofisher). This test incorporate branched DNA technology for accurate gene expression profiling. Branched DNA assays allow direct measurement of RNA transcripts using signal amplification (Luminex 200 technology) rather than target amplification. We decided to include 4 conditions: uninfected, uninfected + heparin, ZIKV and ZIKV + heparin, and three time points: 4, 24, 48 h post infection, for a total of 12 samples. We performed three 1:4 serial dilutions of all samples. Then, we load the plate with all undiluted samples + all dilutions made. We performed the experiment according to the manufacturer’s instructions (Thermofisher). Thanks to the Thermofisher free online software, we performed the analyses of the data we obtained from the Luminex reading. From the analyses file, we highlighted the data that were saturated (over 20000 reads), then we remove data from "uninfected + heparin 4h" and "ZIKV 4h" since the preparation was not perfect and the data were not reliable (incorrect serial dilution). We selected the samples in which genes are not saturated (dilution 1:64), and we performed the geometric mean (GEOMEAN) of all genes expressed by the same sample. We divided each values by its GEOMEAN and we calculated the standard deviation (SD). We selected the genes with the lower SD. This experiment allowed us to identify the best 4 housekeeping genes: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), ubiquitin C (UBC) and the ATPase H+ transporting the A subunit V1 (ATP6V1A) .

Preliminary data of ZIKV infection in brain organoids. We perform immunostaining to characterise organoids grown in culture for 5 weeks. We identified neurons (MAP2) and astrocytes (GFAP). We tested the cytopathic effect of ZIKV in this 3D-system after infection with 1.000.000 infectious particles. We took pictures every 3 days post-infection by optic microscope and collected the supernatant for the retrotitration through plaque forming assay to check viral replication.

Raw data of the figures reported in the published related article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10609960/

Raw data of the figures reported in the published related article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9555206/

This dataset represents findings obtained within a project supported by the Italian Ministry of Health (project code: RF-2019-12369841) and focused on investigating the mechanism of myeloid differentiation blockade by the transcription factor HIF-2alpha in acute myeloid leukemia (AML) and the consequences of its inhibition with novel targeted therapies. We found that in AML HIF-2alpha regulates a specific set of target genes involved in transcriptional repression, leading to inhibition of entire gene clusters linked to myeloid difefrentiation. As a consequence, HIF-2alpha blockade via genetic or pharmacologic approaches leads to myeloid differentiation and leukemia debulking. Also, we positioned HIF-2alpha under direct transcriptional control by the prodifferentiation agent all-trans retinoic acid (ATRA) and demonstrated that HIF-2alpha blockade cooperates with ATRA to trigger AML cell differentiation. Links related to this dataset contain: 1. A publication describing part of the results funded by this grant; 2. Source data linked to this publication and deposited in the BioStudies repository (accession number: S-SCDT-10_15252-EMMM_202317810); 3. Sequencing data deposited in the GEO repository.

During embryonic development, blood cells emerge from specialized endothelial cells, named hemogenic endothelial cells (HECs). As HECs are rare and transiently found during very early development, it remains difficult to distinguish them from endothelial cells. Here, we performed transcriptomic analysis of 28-32 day human embryos, and observed that the expression of Fc receptor CD32 (FCGR2B), is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and hPSC-derived endothelial cells revealed that robust multilineage hematopoietic potential is harbored within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a hematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and hPSC cultures, thus allowing the efficient generation of hematopoietic cells in vitro.

In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPC) are present in the peripheral blood but their contribution to hematopoietic homeostasis in humans remain unsolved. By integrating advanced immunophenotyping, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), functional single-cell assays and integration site (IS) clonal tracking, we unveiled the phenotypic composition, the transcriptional features and the biological role of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPC progressively reduced in cell count over aging and are enriched for primitive, lymphoid and erythroid subpopulations, showing pre-activated transcriptional and functional state. Moreover, cHSPC have low expression of multiple BM-retention molecules, but maintain their homing potential after xenotransplantation. By generating a comprehensive Human Organ-Resident HSPC (HuOR) dataset based on scRNAseq data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Of note, circulating multi-lymphoid progenitors (MLP) are primed for seeding the thymus and actively contribute to T-cell production at steady state in patients treated with HSPC-gene therapy (GT). Human clonal tracking data from GT patients also showed that cHSPC connect distant BM niches and participate to steady-state hematopoietic production, with primitive cHSPC having the highest re-circulation capability to travel in and out the BM. Finally, in case of hematopoietic impairment, cHSPC composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis.