IRCCS San Raffaele Scientific Institute Showcase

Hematopoietic stem/progenitor cell (HSPC) aging has long been associated with myeloid skewing, reduced clonal output, and impaired regenerative capacity, but quantitative immunophenotypic and functional analysis across the human lifespan has been lacking. Here, we provide a comprehensive phenotypic, transcriptional, and functional dissection of human hematopoiesis from youth to advanced age. Although primitive hematopoietic stem cell (HSC) numbers were stable during aging, overall cellularity declined, especially for erythroid and lymphoid lineages. HSPC from older individuals exhibited repopulating frequencies comparable to those from younger donors in both primary and secondary xenografts; however, aged HSC displayed impaired differentiation, chromatin and cell-cycle dysregulation, and poor tolerance to activation-induced proliferative stress, resulting in DNA damage and senescence-like features post-xenotransplantation. Importantly, imposing proliferative stress on young human HSPCs in vivo recapitulated key aging-associated phenotypic and functional declines. Together, our findings identify dysregulated activation responses as a defining feature of HSPC aging and establish proliferative stress–based xenotransplantation models as powerful platforms for investigating age-related hematopoietic dysfunctions. This work is published on Journal of Experimental Medicine (doi: 10.1084/jem.20251805) All the data generated in this study have been deposited in the San Raffaele Open Research Data Repository under accession code doi: 10.17632/72ty5v9djn.1 The RNA and ATAC sequencing data generated and discussed in this study have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO SuperSeries GSE311225, which contains the following data: GSE243327 (RNA-seq), GSE311221 (ATAC-seq). The code used to process and to generate the images of RNAseq data in this manuscript is available at: http://www.bioinfotiget.it/gitlab/custom/LetteraScala_Ageing/bulk_RNAseq. WES data generated and discussed in this study are available at the European Nucleotide Archive (ENA) under the following accession code: PRJEB93902

Using a multi-polygenic score approach, we characterized the relationship between genome-wide information and the history of PPD in patients with mood disorders, with the hypothesis that multiple polygenic risk scores (PRSs) could potentially influence the development of PPD. The PLS linear regression in the whole sample defined a model explaining 27.12% of the variance in the presence of PPD history, 56.73% of variance among MDD, and 42.96% of variance in BD. Our findings highlight that multiple genetic factors related to circadian rhythms, inflammation, and psychiatric diagnoses are top contributors to the prediction of PPD. Specifically, in MDD, the top contributing PRS was monocyte count, while in BD, it was chronotype, with PRSs for inflammation and psychiatric diagnoses significantly contributing to both groups.

The neurobiological differences between women who have experienced a peripartum episode and those who have only had episodes outside of this period are not well understood. Our findings demonstrate that women who have experienced a peripartum episode are neurobiologically distinct from women who have no history of PPD in a cluster within the basal ganglia, an area important for motivation, decision making, and emotional processing. Furthermore, we show that the genetic load for estradiol has a differing effect in this area based on PPD status, which supports the claim that PPD is associated with sensitivity to sex steroid hormones.

The introduction of letermovir (LTV) in prophylaxis after allogeneic-hematopoietic stem cell transplantation (allo-HSCT) has reduced the incidence of Cytomegalovirus (CMV) clinically relevant reactivations (CRE), but these events increase after LTV cessation. CMV-specific T cells protect the patients against CRE, but the mechanisms stimulating their emergence during or immediately after LTV treatment need to be further explored, especially in the setting of post-transplant cyclophosphamide (PT-Cy). In this study, we analyzed 42 CMV-seropositive adult patients with hematological malignancies undergoing allo-HSCT with PT-Cy and calcineurin inhibitor (CNI)-free GvHD prophylaxis in a single-center observational study. Fifteen patients received LTV as prophylaxis in the first 100 days after transplant. CMV-specific CD8+ T cells were quantified by flow cytometry using Dextramer® CMV-Kit (IVD, Immudex) in PBMC frozen 90 (D90) and 180 (D180) days after allo-HSCT, and protective anti-viral immunity was defined based on the threshold that we had previously identified of 0.5 CMV-specific cells/ul. Our findings reinforce the protective role of LTV against clinically relevant CMV-CRE and confirm that LTV prophylaxis is associated with a delayed reconstitution of CMV-specific CD8⁺ T cells compared to no-LTV patients. Importantly, our data underscore the pivotal role of antigen exposure—even transient and at low levels, such as during CMV blips—in promoting the expansion of protective levels of CMV-specific T lymphocytes. This suggests that minimal antigenic stimulation is sufficient to boost protective CMV-specific immune responses in the context of ongoing reconstitution.

The dataset includes the row data shown in Tables 1-2 and in graph in Figure 1 of the paper: Lanzani C, Citterio L, Concas MP, Barruscotti A, Zagato L, Simonini M, Brioni E, El Boustani M, Damanti S, Rovere-Querini P, Manfredi AA, Manunta P. The association between CKD and frailty in the FRASNET study: suggestion of a novel eGFR threshold as a key determinant of frailty in the elderly. Journal of Nephrology 2025. doi: 10.1007/s40620-025-02442-y This study deals with frailty, a geriatric syndrome marked by reduced physiological reserves, linked to organ dysfunction. However, the specific contribution of kidney function to frailty remains underexplored. The aim is to assess the impact of kidney function on frailty in a large population of older adults. The FRASNET (Frailty and Sarcopenia Network) cohort included 1183 individuals (59.9% females, age 65–93). Among them, 27.7% of subjects were classified as robust, 37.6% were pre-frail, and 34.7% were frail. The prevalence of frailty increased with age (43% in individuals over 76 years of age) and was associated with obesity (28.3%) and polytherapy (23.2%). Whole estimated glomerular filtration rate (eGFR) was 73.8 (IQR 62.4, 84.7) ml/min/1.73 m2. The prevalence of chronic kidney disease (CKD) increased across frailty classes from 15.2% in robust to 29.0% in frail individuals (P < 0.001). Among young-old subjects (65–75 years old), comorbidity was the main determinant of frailty, whereas in older subjects, when eGFR was below 53.5 ml/min/1.73 m2, it was associated with frailty (P < 0.002). Fractional excretion of sodium progressively increased across frailty classes, from 0.71% in robust individuals (IQR 0.46–1.03) to 0.79% in frail subjects (IQR 0.48–1.17) (P = 0.04). This study revealed a strong relationship between CKD and frailty, identifying a new eGFR threshold associated with frailty in older adults, thus highlighting the potential role of the often-overlooked tubular function in older individuals.

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homooligomerization and ALK2–ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12–ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression. Complete data files relative to the manuscript "Pettinato M. et al., FKBP12 inhibits hepcidin expression by modulating BMP receptors interaction and ligand responsiveness in hepatocytes. Am J Hematol. 2023 Aug;98(8):1223-1235. doi: 10.1002/ajh.26961. Epub 2023 May 18. PMID: 37199280".

TEM images of substantia nigra pars compacta of WT and PrknR275W mice at 6 months of age

A dynamic protein network at the leading edge of motile cells is needed to coordinate events required for efficient cell motility. Previous work has shown that the Ser/Thr kinase DYRK3 affects the assembly of this network, and phosphorylates its component Liprin-α1, a scaffold protein regulating adhesion turnover and cell motility. We have looked for phospho-sites of Liprin-α1 relevant for the regulation of cell motility, by examining the role played by serine/threonine residues phosphorylated within the intrinsically disordered regions of Liprin-α1. Phospho-null mutations within either the amino-terminal or the carboxy-terminal disordered regions affect Liprin-α1 phosphorylation induced by DYRK3. Functional analysis shows that mutations within the amino-terminal region do not affect cell motility, while a set of carboxy-terminal mutations reduces the positive effects of Liprin-α1 on cell spreading on the extracellular matrix. Among several candidate phospho-sites in this protein region, we identify Thr701 as one of the potential main targets of DYRK3 activity in Liprin-1. The phospho-null mutation of Thr701 specifically inhibits Liprin-1–induced potentiation of cell spreading on fibronectin. Our findings contribute to highlight the complexity of the regulation of Liprin-αprotein functions by phosphorylation/dephosphorylation events. Given the involvement of Liprin-α proteins in tumor cell motility and invasion, in-depth understanding of this regulatory complexity may highlight new possibilities for therapeutic intervention.

"Chimeric enzymes enhance treatment potential for globoid cell leukodystrophy through hematopoietic stem cell gene therapy" - PMID: 40988335 - DOI: 10.1016/j.ymthe.2025.09.030 Globoid cell leukodystrophy (GLD) is a fatal lysosomal storage disorder caused by a deficiency in the β-galactosylceramidase (GALC) enzyme, leading to severe demyelination and neurodegeneration, and often death before the age of 2 years. Hematopoietic stem/progenitor cell transplantation (HSPC-T) has limited efficacy due to inadequate GALC delivery to the central (CNS) and peripheral nervous systems (PNS) and associated risks. In vivo gene therapy (GT) using adeno-associated viral vectors shows promise, but safety concerns persist. This research presents a strategy using lentiviral (LV) vector-mediated ex vivo HSPC-GT with a chimeric GALC enzyme that incorporates peptides from α-L-iduronidase (IDUA) and apolipoprotein E II (APO) to enhance expression and blood-brain barrier penetration. The chimeric IDUAsp.GALC.APO enzyme exhibited superior production and secretion compared to native GALC and previous chimeric variants in LV-transduced HSPCs, resulting in improved cross-correction and normalization of GALC activity in GLD neural cells. Proof-of-concept studies demonstrated effective enzyme production, secretion, and cross-correction capability of macrophages from GLD patients. In vivo results showed stable gene marking, sustained enzyme production, and efficient delivery of the chimeric GALC in affected organs, including the CNS and PNS. These findings highlight the potential of HSPC-GT using chimeric GALC enzymes as an innovative therapeutic approach for treating GLD. Data related to the paper are available in the folder with the same name. "Enhancement of expression, bioavailability and cross-correction of chimeric GALC enzyme to refine gene therapy approaches for globoid cell leukodystrophy" - GR 2019-12369357 - Final Report In this project, we propose a lentiviral vector-mediated gene therapy with a chimeric GALC enzyme engineered for improved expression and blood-brain barrier penetration as therapeutic approach for GLD. Our findings demonstrate that the chimeric enzyme, when expressed in LV-transduced human HSPCs and GLD iPSC derived NPC progeny, exhibits enhanced production and secretion compared to native GALC and prior chimeric variants. This leads to significant cross-correction of GALC activity in GLD neural cells. In vivo studies in immunodeficient mice and a severe GLD mouse model confirmed stable gene marking and sustained enzyme production, emphasizing our strategy's potential for effective therapeutic delivery and cross-correction of affected tissues. The data related to the final report of GR 2019-12369357 are available in the folder with the same name.

Data generated under the umbrella of the research grant “RF-2019-12368847” have been included in the pubblication "Specific types of male infertility are correlated with T cell exhaustion or senescence signatures" (https://doi.org/10.1038/s41467-025-56193-2). Below are the links to the open-access data repository where the data have been published. The first link to GEO Repository contains all the data generated by single cell RNA sequencing The second link to the San Raffaele Open Research Data Repository contains all the other data generated and used to build manuscript figures.