Background: Progesterone (P4) is used as an antiseizure medication (ASM) to treat catamenial epilepsy, refractory to first-line drugs. P4 and other neurosteroids (NSs) are important regulators of multiple nervous system functions, including neuronal excitability and synaptic plasticity. In addition to their antiseizure properties, P4 and other NSs are also anti-inflammatory agents. Neuroinflammation is an important pathophysiological mechanism of epilepsy refractory to ASMs. Accordingly, we evaluated the ability of P4 to modulate neuroinflammation, using human microglia activated by lipopolysaccharide (LPS). Methods: Human microglia (HMC3) were stimulated for 3 h with LPS in the absence or presence of various concentrations of P4. Thereafter, levels of (i) toll-like receptor 4 (TLR4), (ii) the NLRP3 inflammasome, and (iii) pro-inflammatory cytokines were quantitated by real-time PCR and Western blot analyses. Phagocytic activity was also assessed using a phagocytosis assay employing fluorescent beads. Results: P4 treatment significantly reduced the microglial inflammatory state induced by LPS, which was mediated by upregulation of the TLR4- and NLRP3-axes. The protective effects of P4 were mediated by inhibition of Nuclear Factor kappa-light-chain-enhancer of activated B cells (NFκB) phosphorylation and reduced activation of Mitogen-Activated Protein Kinases (MAPK). The effects of P4 included a significant reduction in mRNA levels of the main pro-inflammatory cytokines and a reduction in phagocytic activity of HMC3. Conclusions: P4 is endowed with significant anti-inflammatory properties, which may be involved in the beneficial effects reported for drug-resistant catamenial epilepsy. Further research is required to clarify P4 post-receptor mechanisms of action and to explore the roles of other P4-derived NSs.

The characterization of meniscal extracellular matrix (ECM) is fundamental for tissue engineering to design a functional substitute. However, most of the available data come from animal studies. The meniscus has a complex ECM, characterized by a specific orientation of collagen fibres related to its function. In this work, both pediatric and adult meniscal tissues were examined from morphological, biochemical, and mechanical perspectives to identify the characteristics of physiological tissue and the changes caused by injuries and degeneration. Additionally, a comparison was performed between menisci from patients with normal and valgus knees to observe ECM remodeling associated with altered biomechanics. Obtained results indicatet that, with both aging and pathology, collagen and glycosaminoglycans (GAG) content decreased, while mucins increased. These changes were accompanied by alterations in tissue structure at both the macroscopic and nanoscale levels, including less organized collagen networks and thicker, more fibrotic fibres. Correspondingly, the elastic modulus decreased with age, reflecting a loss of tissue mechanical integrity. The various results from the morphological, biochemical, and mechanical analyses were then correlated in a qualitative and descriptive manner to provide a comprehensive impression of tissue organization and remodelling. Finally, the obtained results were compared with data available in the literature to highlight similarities and differences between humans of various ages and animal models.

BACKGROUND AND PURPOSE:Proteasome inhibitors have been approved for treatment of multiple myeloma but induce significant chemotherapy-related peripheral neurotoxicity in up to one third of patients. Crucial information about the several neurotoxicity mechanisms suggested in the literature and effective triggering events is either missing or controversial, due to heterogeneity of experimental models used to investigate such processes. To fill this knowledge gap, we compared the neurotoxicity of bortezomib (BTZ) and carfilzomib (CFZ), a less neurotoxic drug, by investigating preclinical models and dissecting the underlying molecular mechanisms using a multidimensional approach. EXPERIMENTAL APPROACH:We developed a new mouse model of CFZ-induced neuropathy and compared it with an established BTZ model using behavioural, morphological/morphometric and proteomic analyses of dorsal root ganglia (DRG) tissues. Mitotoxicity and cytoskeleton alterations were compared in terms of onset of altered mitochondrial morphology, functionality and trafficking, alongside cytoskeletal protein expression and axonal degeneration in cultured mouse DRG neurons. KEY RESULTS:BTZ's severe neurotoxicity in vivo correlated with severe loss of nerve fibres and extensive protein expression changes. In vitro, both compounds significantly altered mitochondrial network organization and energy production after 24 h of treatment. However, only BTZ induced accumulation of tubulin post-translational modifications and early axonal degeneration within the first 10 h, severely impacting mitochondrial trafficking after 24 h. CONCLUSIONS AND IMPLICATIONS:These results point to mitochondrial toxicity as a common downstream effect of both treatments, whereas BTZ-specific off-target activity on tubulin hyper-stability may initiate early mitochondrial trafficking alterations. This knowledge may inform future mitigation approaches.

The overlap between the geographic distribution of COVID-19 outbreaks and pollution levels suggested a strong correlation between exposure to atmospheric particulate matter and an increased risk of developing severe forms of disease. This correlation has been highlighted by several epidemiological studies, indicating the existence of shared molecular mechanisms. Emerging evidence has highlighted the important role of lipid rafts in facilitating viral entry into cells. Specifically, the receptor binding domain of the SARS-CoV-2 spike protein interacts with sialylated glycans of the monosialic ganglioside GM1 and GM2 that are particularly enriched in lipid rafts. This interaction has been proposed to facilitate ACE2 recognition by the spike protein and may contribute to early events involved in viral attachment and entry. Here, we reveal that A549 alveolar lung cells, after DEP exposure, exhibit a significant shift in ACE2 into lipid rafts, accompanied by an increase in the immature form of ADAM17, the sheddase responsible for ACE2 cleavage. Additionally, DEP exposure results in a significant increase in IL-6 release, while no changes were observed in IL-8 and sACE2 release. This treatment does not cause significant alterations in protein levels or membrane redistribution of COX-2 and HO-1, proteins involved in the inflammatory response and oxidative stress following exposure to air pollution, and linked to COVID-19 pathogenesis. Finally, lipidomic analysis by UHPLC-MS revealed that DEP exposure induces a significant increase in GM2 levels, and a concomitant decrease in GM1 and GM3 levels. Together, these results indicate that DEP exposure remodels lipid raft-associated molecular features in A549 cells, including ACE2 membrane redistribution, altered ganglioside composition, and increased IL-6 release. Although these changes may be relevant to cellular mechanisms associated with SARS-CoV-2 susceptibility, the present study does not directly assess viral binding, viral entry, or infection, and further functional studies are required.

The t(4;11) translocation, which drives KMT2A::AFF1 fusion gene expression, is associated with poor prognosis in pediatric and adult B-cell Acute Lymphoblastic Leukemia (B-ALL). KMT2A::AFF1 fusion circular RNAs (f-circRNAs) have been identified in B-ALL, though their contribution to leukemia has not yet been outlined. We identified a novel recurrent KMT2A::AFF1-derived back-splicing junction joining AFF1 exon 7 to KMT2A exon 3 (AK_7_3) in SEM, RS4;11, and ALL-PO B-ALL cell lines, all of which carry the t(4;11)(q24;q23)/KMT2A::AFF1 translocation. This sequence was observed in RT-PCR products encompassing two splicing variants of KMT2A exon 4, which are also present in the linear KMT2A::AFF1 chimera. AK_7_3 was also found in 12/18 pediatric and 17/24 adult t(4;11)-positive B-ALL patients, including three paired diagnosis/relapse samples, whereas it was absent in 23 t(4;11)-negative B-ALL patients. Whole-transcriptome analysis of AK_7_3-knockdown SEM cells identified upregulated genes involved in oxidative stress response and regulation of apoptosis. Cell death analysis confirmed the pro-apoptotic impact of AK_7_3 silencing. Confocal analyses, Seahorse bioenergetic profiling, and electron microscopy revealed an overproduction of reactive oxygen species, increased mitochondrial membrane potential, mitochondrial respiration, and dysmorphic mitochondria after AK_7_3 knockdown in KMT2A::AFF1 cell lines. In summary, we identified AK_7_3 as a novel f-circRNA back-splicing junction recurrent in t(4;11)-positive B-ALL patients at both diagnosis and relapse. Our data provide evidence for a functional role of this molecule as an anti-apoptotic agent that affects mitochondrial function, suggesting its potential involvement in KMT2A::AFF1-driven B-ALL leukemogenesis.

Triple Negative Breast Cancers (TNBCs) are heterogeneous and aggressive tumors with a median overall survival of less than two years. Despite the availability of new drugs, the prognosis remains poor, implicating a more aggressive clinical course in the metastatic setting. This study investigated the effects of metronomic treatment (mCHT) with 5-fluorouracil (5-FU) plus vinorelbine (VNR) on spheroids derived from two different TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186). mCHT significantly reduced spheroid growth and altered spheroid architecture, with a pronounced effect in second-generation spheroids, enriched in self-renewing cancer stem cells (CSCs). Expression of CSC-related markers (CD44, CD133, NOTCH-1, and MYC) was more significantly altered-both at the mRNA and protein levels-by mCHT than by standard treatment (STD). In MS-186-derived spheroids, mCHT downregulated EZH2 and STAT3, key regulators of CSC maintenance, and reduced H3K27ac, suggesting a global epigenetic reprogramming. Unlike STD, which partially and transiently reduced stemness markers, mCHT achieved sustained suppression, indicating preferential targeting of therapy-resistant CSCs. These results indicate mCHT as a promising strategy for specifically aiming at the CSC-like compartment in TNBC, underscoring a therapeutic approach that reprograms key epigenetic networks and overcomes resistance to treatment.

Purpose PI4KA-related disorder is a highly clinically variable condition characterized by neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus) and gastrointestinal (inflammatory bowel disease and multiple intestinal atresia) manifestations. Although features consistent with immunodeficiency (autoimmunity/autoinflammation and recurrent infections) have been reported in a subset of patients, the burden of B-cell deficiency and hypogammaglobulinemia has not been extensively investigated. We sought to describe the clinical presentation and manifestations of patients with PI4KA-related disorder and to investigate the metabolic consequences of biallelic PI4KA variants in B cells. Methods Clinical data from patients with PI4KA variants were obtained. Multi-omics analyses combining transcriptome, proteome, lipidome and metabolome analyses in conjunction with functional assays were performed in EBV-transformed B cells. Results Clinical and laboratory data of 13 patients were collected. Recurrent infections (7/13), autoimmune/autoinflammatory manifestations (5/13), B-cell deficiency (8/13) and hypogammaglobulinemia (8/13) were frequently observed. Patients' B cells frequently showed increased transitional and decreased switched memory B-cell subsets. Pathway analyses based on differentially expressed transcripts and proteins confirmed the central role of PI4KA in B cell differentiation with altered B-cell receptor (BCR) complex and signalling. By altering lipids production and tricarboxylic acid cycle regulation, and causing increased endoplasmic reticulum stress, biallelic PI4KA mutations disrupt B cell metabolism inducing mitochondrial dysfunction. As a result, B cells show hyperactive PI3K/mTOR pathway, increased autophagy and deranged cytoskeleton organization. Conclusion By altering lipid metabolism and TCA cycle, impairing mitochondrial activity, hyperactivating mTOR pathway and increasing autophagy, PI4KA-related disorder causes a syndromic inborn error of immunity presenting with B-cell deficiency and hypogammaglobulinemia.

BackgroundAnaplastic Large Cell Lymphoma (ALCL) is a rare and aggressive T-cell lymphoma, classified into ALK-positive and ALK-negative subtypes, based on the presence of chromosomal translocations involving the ALK gene. The current standard of treatment for ALCL is polychemotherapy, with a high overall survival rate. However, a subset of patients does not respond to or develops resistance to these therapies, posing a serious challenge for clinicians. Recent targeted treatments such as ALK kinase inhibitors and anti-CD30 antibody-drug conjugates have shown promise but, for a fraction of patients, the prognosis is still unsatisfactory.MethodsWe investigated the genetic landscape of ALK + ALCL by whole-exome sequencing; recurring mutations were characterized in vitro and in vivo using transduced ALCL cellular models.ResultsRecurrent mutations in FAT family genes and the transcription factor RUNX1T1 were found. These mutations induced changes in ALCL cells morphology, growth, and migration, shedding light on potential factors contributing to treatment resistance. In particular, FAT4 silencing in ALCL cells activated the beta-catenin and YAP1 pathways, which play crucial roles in tumor growth, and conferred resistance to chemotherapy. Furthermore, STAT1 and STAT3 were hyper-activated in these cells. Gene expression profiling showed global changes in pathways related to cell adhesion, cytoskeletal organization, and oncogenic signaling. Notably, FAT mutations associated with poor outcome in patients.ConclusionsThese findings provide novel insights into the molecular portrait of ALCL, that could help improve treatment strategies and the prognosis for ALCL patients.

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis , suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2 . Only 2 patients presented high mutation burden somatic variants, involving DNMT3A , TET2 , ASXL1 and WT1 . We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT , Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE. Graphical abstract

Background FNIP1 (Folliculin Interacting Protein 1) deficiency is a rare inborn error of immunity characterized by heart defects and B-cell deficiency. Neutropenia is frequently reported in FNIP1-deficient patients, but its underlying mechanism has not been elucidated, with no evidence of neutropenia in the Fnip1 mouse model. Aims To test the hypothesis that FNIP1 deficiency could be directly associated with neutropenia we collected updated clinical and laboratory data of FNIP1-deficient patients. To unveil the role of FNIP1 in neutrophil development and function, we employed CRISPR/Cas9 genome editing to create a FNIP1-deficient model in HL-60 cells. Methods Ten (8 published and 2 unpublished) patients were enrolled. CRISPR/Cas9 was used to knock out (KO) FNIP1 in HL-60 cells. Whole RNA-sequencing, expression of pAKT, pS6, p4EBP1, mitochondrial abundance and membrane potential (MMP), oxygen consumption rate (OCR), ATP production and extracellular acidification rate (ECAR) of FNIP1-deficient and wild-type HL-60 cells were performed to assess effects of FNIP1 deficiency. Results All the patients (male:female = 5:5, age range <1-40 years, median 5.5 years) showed hypogammaglobulinemia/agammaglobulinemia with B-cell deficiency, heart defects, severe/recurrent infections and were receiving immunoglobulin replacement treatment. Crohn disease and seronegative arthritis were diagnosed in 1 individual each. Eight out of 10 patients showed decreased absolute neutrophil count (ANC; four chronic and four intermittent neutropenia). All the patients with chronic neutropenia had ANC <500/µl (severe neutropenia). Neutrophil oxidative burst was normal in 2 patients while chemotaxis resulted defective in 1 case. Bone marrow (BM) analysis showed left-shifted granulopoiesis with no promyelocyte/myelocyte arrest in 3/3 cases. Monocytosis and monocytopenia were present in 4 and 1 out of 10 patients, respectively. G-CSF was started in 3 patients, who responded to <5 mcg/kg/day. To date, no individual has developed a clonal hematopoietic disorder. Six patients were alive at the last follow-up. Whole RNA-seq in FNIP1 KO HL-60 cells showed aberrant transcriptional control of differentiation from HSC to myeloid cells (increased C/EBPα and decreased MEIS1 and HOXA9). Enrichment in gene sets related to cell differentiation (Notch Transcriptional and post-Translation regulation), motility/cytoskeletal remodeling (Rap1, Rac1, Rho, RhoA and Cdc42 pathways), mitochondrial (oxidative stress induced senescence) and neutrophil (degranulation, chemotaxis and extracellular trap formation) function was observed in FNIP1 KO HL-60 cells. FNIP1 KO HL-60 cells did not show differences in total mitochondria but MMP was significantly reduced (p<0.0001). Although transcripts involved in mitochondrial electron transport chain (COX16, COX19, COX6C, COX7A2 and COX7B) and OXPHOS (ATP5F1) were dysregulated, FNIP1 KO HL-60 cells showed no differences in ATP production and OCR, a measure of OXPHOS. Decrease of PPARγ and increase of key glycolytic enzymes (HK2 and LDHA) suggested altered glucose metabolism. ECAR, a measure of glycolysis, was increased in FNIP1 KO HL-60 cells (p<0.0001). We examined the PI3K/AKT pathway and did not observe differences in pAKT473, p4EBP1 and pS6 between WT and FNIP1 KO HL-60 cells. Conclusions Neutropenia is a frequent finding and part of the phenotypic spectrum of FNIP1 deficiency. FNIP1-deficient patients do not lack mature neutrophils in the BM and respond to G-CSF treatment. FNIP1 deficiency profoundly impacts the mechanisms underlying critical neutrophil activities, ultimately impairing neutrophil differentiation and function. Similarly to FNIP1-deficienct B cells, FNIP1 acts in neutrophil's progenitors regulating glycolysis. Our work shows that FNIP1 deficiency alters previously unrecognized pathways (cytoskeletal dynamics/cell migration). We have previously described increased MMP and phosphorylation of pAKT473 and p4EBP in FNIP1-deficient B cells. As neutropenia is not associated with promyelocyte/myelocyte arrest, FNIP1 may have a prominent role in later stages of granulocyte's differentiation, thus explaining the difference between B cells and HL-60 observed in MMP and PI3K pathway. Future work will focus on targeted interventions to restore neutrophil function in FNIP1 deficiency.

Extracellular vesicles (EVs) are a new mechanism of cellular communication, by delivering their cargo into target cells to modulate molecular pathways. EV-mediated crosstalk contributes to tumor survival and resistance to cellular stress. However, the role of EVs in B-cell Acute Lymphoblastic Leukaemia (B-ALL) awaits to be thoroughly investigated. We recently published that ActivinA increases intracellular calcium levels and promotes actin polymerization in B-ALL cells. These biological processes guide cytoskeleton reorganization, which is a crucial event for EV secretion and internalization. Hence, we investigated the role of EVs in the context of B-ALL and the impact of ActivinA on this phenomenon. We demonstrated that leukemic cells release a higher number of EVs in response to ActivinA treatment, and they can actively uptake EVs released by other B-ALL cells. Under culture-induced stress conditions, EVs coculture promoted cell survival in B-ALL cells in a dose-dependent manner. Direct stimulation of B-ALL cells with ActivinA or with EVs isolated from ActivinA-stimulated cells was even more effective in preventing cell death. This effect can be possibly ascribed to the increase of vesiculation and modifications of EV-associated microRNAs induced by ActivinA. These data demonstrate that ActivinA boosts EV-mediated B-ALL crosstalk, improving leukemia survival in stress conditions.

Inflammatory bowel disease (IBD) incidence has increased in the last decades due to changes in dietary habits. IBDs are characterized by intestinal epithelial barrier disruption, increased inflammatory mediator production and excessive tissue injury. Since the current treatments are not sufficient to achieve and maintain remission, complementary and alternative medicine (CAM) becomes a primary practice as a co-adjuvant for the therapy. Thus, the intake of functional food enriched in vegetal extracts represents a promising nutritional strategy. This study evaluates the anti-inflammatory effects of artichoke, caihua and fenugreek vegetal extract original blend (ACFB) in an in vitro model of gut barrier mimicking the early acute phases of the disease. Caco2 cells cultured on transwell supports were treated with digested ACFB before exposure to pro-inflammatory cytokines. The pre-treatment counteracts the increase in barrier permeability induced by the inflammatory stimulus, as demonstrated by the evaluation of TEER and CLDN-2 parameters. In parallel, ACFB reduces p65NF-κB pro-inflammatory pathway activation that results in the decrement of COX-2 expression as PGE2 and IL-8 secretion. ACFB properties might be due to the synergistic effects of different flavonoids, indicating it as a valid candidate for new formulation in the prevention/mitigation of non-communicable diseases.

Background: Anaplastic lymphoma kinase (ALK) plays a role in the development of lymphoma, lung cancer and neuroblastoma. While tyrosine kinase inhibitors (TKIs) have improved treatment outcomes, relapse remains a challenge due to on-target mutations and off-target resistance mechanisms. ALK-positive (ALK+) tumors can evade the immune system, partly through tumor-associated macrophages (TAMs) that facilitate immune escape. Cancer cells use “don’t eat me” signals (DEMs), such as CD47, to resist TAMs-mediated phagocytosis. TKIs may upregulate pro-phagocytic stimuli (i.e., calreticulin, CALR), suggesting a potential therapeutic benefit in combining TKIs with an anti-CD47 monoclonal antibody (mAb). However, the impact of this combination on both TKIs-sensitive and resistant ALK+ tumors requires further investigation. Methods: A panel of TKIs-sensitive and resistant ALK+ cancer subtypes was assessed for CALR and CD47 expression over time using flow cytometry. Flow cytometry co-culture and fluorescent microscopy assays were employed to evaluate phagocytosis under various treatment conditions. Results: ALK inhibitors increased CALR expression in both TKIs-sensitive and off-target resistant ALK+ cancer cells. Prolonged TKIs exposure also led to CD47 upregulation. The combination of ALK inhibitors and anti-CD47 mAb significantly enhanced phagocytosis compared to anti-CD47 alone, as confirmed by flow cytometry and fluorescent microscopy. Conclusions: Anti-CD47 mAb can quench DEMs while exposing pro-phagocytic signals, promoting tumor cell phagocytosis. ALK inhibitors induced immunogenic cell damage by upregulating CALR in both sensitive and off-target resistant tumors. Continuous TKIs exposure in off-target resistant settings also resulted in the upregulation of CD47 over time. Combining TKIs with a CD47 blockade may offer therapeutic benefits in ALK+ cancers, especially in overcoming off-target resistance where TKIs alone are less effective.

Embryonic hematopoiesis consists of distinct waves originating in rapid succession from different anatomical locations. Hematopoietic progenitors appearing earlier than definitive hematopoietic stem cells (HSCs) play key roles in fetal and postnatal life. However, their precise origin, identity and the extent of their contribution need further clarification. To this aim, we took advantage of a genetic fate-mapping strategy in mice that allows labeling and tracking of distinct subsets of hemogenic endothelium (HE). Time-course labeling of hematopoietic progenitors emerging from HE between E8.5 and E9.5, before intra-embryonic definitive HSC generation, revealed a major fetal lympho-myeloid contribution which declined in the adult. Lineage tracing coupled with whole-mount imaging and single-cell RNA sequencing located its source within hematopoietic clusters of vitelline and umbilical arteries. Functional assays confirmed the transient nature of these progenitors. We therefore unveiled a hitherto unidentified early wave of fetal-restricted hematopoietic stem/progenitor cells poised for differentiation that provide a major contribution to pre-natal hematopoiesis. ### Competing Interest Statement The authors have declared no competing interest.

Primers used and mutations identified by WES

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: In mammals, embryonic hematopoiesis takes place in discrete but overlapping waves. Hematopoietic Stem Cells (HSC) generation is preceded by the appearance of HSC-independent progenitors, both emerging from a specialized transient population of endothelial cells termed hemogenic endothelium (HE). Recently, several reports showed that HSC contribution to fetal hematopoiesis is limited and that, in contrast, HSC-independent progenitor play key roles in fetal and postnatal life. Understanding the dynamics of fetal hematopoiesis has a number of important implications. Indeed, gaining a better knowledge of the cellular and molecular processes underlying the production of hematopoietic cells in the embryo would aid establishing new methodologies for the in vitro generation of different hematopoietic cells from pluripotent stem cells. Moreover, given the prenatal origin of many pediatric blood cancers, a better understanding of fetal hematopoiesis could lead to the identification of potential cell(s) of origin and relevant therapeutic vulnerabilities. Aims: To precisely define the origin, identity and extent of contribution of the distinct waves of embryonic hematopoietic stem and progenitor cells. Methods: To this aim, we took advantage of a genetic fate-mapping strategy in mouse that allows in vivo labeling and tracking of distinct subsets of HE. We combined this strategy with whole-mount embryo imaging, single-cell RNA sequencing and a range of functional assays. Results: Time-course labeling of HE revealed that the major lympho-myeloid contribution towards the end of gestation was derived from progenitors appearing between E8.5 and E9.5, a time window in which dorsal aorta definitive-type HSCs have not yet emerged. This contribution was transient and fetal-restricted, as it exhibited a sharp decline during postnatal life. Remarkably, we were able to localize the emergence of fetal-restricted hematopoietic stem/progenitor cells (HSPCs) to Kit+ hematopoietic clusters emerging from HE in the vitelline and umbilical (extraembryonic) arteries. Lineage tracing using a different, myeloid-specific, transgenic mouse line established that these clusters contained cells other than erythro-myeloid progenitors (EMPs). Moreover, single-cell RNA sequencing showed that fetal-restricted HSPCs express a transcriptional signature characteristic of HSCs. Accordingly, ex vivo co-cultures showed that B- and T-lymphoid potential were enriched in these progenitors. Finally, transplantation assays demonstrated that fetal-restricted HSPCs are endowed with in vivo multi-lineage repopulation potential. Summary/Conclusion: In summary, here we identify a wave of fetal-restricted HSPCs that physiologically contribute the majority of lymphoid and myeloid cells other than macrophages during fetal development, and we demonstrate that its emergence is segregated in space and time from that of EMPs and adult definitive-type HSCs. Keywords: Hematopoietic stem and progenitor cells, Hematopoiesis, Development, Mouse model