Biology subjects

Laurenti, E.

Publications and source records attributed to Laurenti, E..

17 recordsLinked to original sources

bioRxiv2026

Single-Cell Proteo-Transcriptomic Profiling Uncovers Post-Translational Regulatory Layers in Human Hematopoiesis

Background: Hematopoietic stem cells (HSCs) have the unique ability to produce all mature cell types in the blood and understanding the mechanisms governing their differentiation and renewal is a driving question in stem cell biology. While single-cell RNA sequencing has transformed our view of human hematopoiesis, mRNA abundance represents an incomplete proxy for functional cell states. Here, we apply single-cell multimodal profiling to simultaneously measure 280 surface and intracellular protein targets alongside whole transcriptomes within individual adult human hematopoietic stem and progenitor cells. Results: By directly coupling modalities within single cells, we show that protein-RNA correlations peak dynamically in transitioning intermediate progenitors across lineages, consistent with pervasive post-transcriptional mechanics underlying lineage choice. Furthermore, key transcription factor proteins maintain multilineage co-expression longer into commitment than their transcripts, demarcating a possible window of fate decision reversibility. Moreover, low and flat CTNNB1 mRNA levels mask late-erythroid {beta}-catenin protein enrichment, apparently pairing with E-cadherin expression and suggesting post-translational stabilization at the cell membrane. Conclusions: This dataset enables the observation of translational dynamics and RNA-protein relationships throughout the differentiation landscape at an unprecedented level of granularity. Together, our findings demonstrate that transcriptomics alone misses fundamental regulatory programs, establishing single-cell proteo-transcriptomics as a powerful tool for decoding human stem cell fate.

cell biology↗

bioRxiv2026

Modelling human haematopoietic stem cell commitment ex vivo identifies IL-33 as a regulator of megakaryopoiesis

Commitment events to specific blood lineages arise from single hematopoietic stem cells (HSCs) and are influenced by stress, inflammation and disease. However, the understanding of how such events are regulated in human haematopoiesis is limited by the lack of tractable in vitro models. In this study, we introduce a novel Early Progenitor Differentiation (EPD) assay to study the initial lineage commitment of human 49+ HSCs, in a system faithfully recapitulating cell states observed in vivo. Combining single cell -omics approaches and single cell functional assays, we show that IL-33 acts directly on human 49f+ HSCs activating the MAPK pathway to enhance their commitment towards Megakaryocytic-Erythroid-Mast cell Progenitors and subsequently megakaryopoiesis. This occurs without affecting HSC self-renewal via accelerated establishment of chromatin programmes associated with Erythroid and Megakaryocyte and mast cells lineages. Our findings demonstrate the utility of the EPD model to identify molecular regulators of human HSC differentiation and uncover a new role of IL-33 in haematopoiesis.

cell biology↗

bioRxiv2026

Self-supervised learning for a gene program-centric view of cell states

Single-cell omics has extended the biological interrogation of cell state from examining the expression of individual genes to unbiased profiling of tens of thousands of genes at once. However, extracting biological insights from such high-dimensional data remains challenging. To enable downstream analyses, many computational approaches compress cell state into a single latent representation. This can obscure the structure of underlying gene programs (GP), defined as coordinated sets of biologically related genes, such as signalling pathway response modules or transcription factor targets. Here, we present Tripso, a self-supervised transformer deep learning model which learns multiple GP-specific embeddings from predefined GPs, while also enabling the discovery of novel, data-driven GPs. Tripso facilitates principled comparisons across development, disease, and experimental systems. Firstly, in a dataset of human hematopoietic cells spanning prenatal development through adulthood and aging and including newly generated data, Tripso resolved age-specific GP patterns, including elevated JAK-STAT activity in pediatric hematopoietic cells and postnatal shifts in IKZF1 GP activity during B cell differentiation. Secondly, leveraging Tripso GP embeddings and comparing in vivo to in vitro data, we hypothesized and experimentally validated that inhibition of the SEC61 translocon improved maintenance of hematopoietic stem cells in culture. Finally, Tripsos capacity for data-driven GP discovery revealed a previously uncharacterized tissue-resident memory T cell GP with increased activity in atopic dermatitis. Its spatial co-localization with sebaceous gland-associated immune niches was demonstrated in spatial transcriptomic and proteomic data. Thus, by moving beyond single embeddings of cellular states, Tripso enables interpretable and actionable discoveries, demonstrating how GP-centric modelling can generate hypotheses with substantial biomedical relevance. By anchoring cellular representations in meaningful GPs, Tripso establishes a principled and biologically grounded framework towards the development of interpretable virtual cell models.

bioinformatics↗

bioRxiv2026

Predicting how perturbations reshape cellular trajectories with PerturbGen

A major challenge in biology is predicting how cells transition between states over time and how perturbations disrupt these transitions. Understanding such dynamics is critical for identifying interventions that reverse pathological programs or reprogram cells toward desired states. Although recent computational approaches can predict single-cell perturbation responses in silico, they cannot predict responses across dynamic cell trajectories, for example how early perturbations reconfigure later cell states. To address this gap, we introduce PerturbGen, a generative foundation model trained on over 100 million single-cell transcriptomes that predicts perturbation responses along cellular trajectories. PerturbGen predicts how genetic perturbation at source state shapes downstream states, alters gene programs and trajectories across time, for example in differentiation or disease progression. We apply PerturbGen to three newly generated multi-condition human single-cell datasets spanning immune responses, hematopoiesis and skin development. In an in vivo immune challenge, PerturbGen predicts that knocking out an IL1B signal in myeloid cells attenuates later cytokine-interferon programs, with downstream changes consistent with a reversal of IL-1{beta} stimulation signature. In hematopoiesis, anchoring perturbation-induced programs to human genetics enables simulation of monogenic blood disorders, recapitulates established disease-associated biology whilst systematically revealing lineage-specific programs, including in lineages where this was not previously possible. In skin organoids, PerturbGen predicted that Wnt activation enhances stromal differentiation recapitulating the trajectory observed in human prenatal skin, findings that were functionally validated by experimentally activating Wnt signaling. Together, PerturbGen extends modeling of gene perturbations from static to dynamic cellular systems. We envision PerturbGen enabling the creation of in silico, trajectory-aware perturbation atlases and virtual cells across diverse biological scenarios, supporting optimization of disease models and prioritization of candidate molecular interventions for therapeutic discovery.

bioinformatics↗

bioRxiv2025

DNMT3A-R882 mutations intrinsically drive dysfunctional neutropoiesis from human haematopoietic stem cells

Clonal haematopoiesis (CH) arises from the expansion of hematopoietic stem cells (HSCs) carrying leukaemia-associated somatic mutations. CH is linked to pathological immune dysregulation and a greater risk of age-related inflammatory diseases. Yet, how CH mutations impact HSC differentiation into immune effector cells remains understudied. Here, we report a single-cell resolution functional and multi-omic investigation of HSC clonal and differentiation dynamics in individuals with DNMT3A-R882 CH. DNMT3A-R882 reshapes the clonal architecture of haematopoiesis towards an aged phylogenetic structure. Functionally, DNMT3A-R882 HSCs produce decreased monocytic output but more abundant and mature neutrophil progeny compared to WT HSCs in the same individual. Whereas DNMT3A-R882 myeloid progenitors display attenuated inflammatory transcriptional programmes, DNMT3A- R882 mature neutrophils acquire proinflammatory and immunomodulatory features typical of maladaptive immunity and CH co-morbidities. Our findings, validated in humanised mice, identify aberrant DNMT3A-R882 HSC-driven neutropoiesis as a key link between CH, immune dysregulation and risk of inflammatory disease.

cell biology↗

bioRxiv2025

Delayed Trp53 activation protects Dnmt3a-mutant hematopoietic stem cells from inflammatory attrition.

Hematopoietic stem cells (HSCs) accumulate somatic mutations over time, some conferring a fitness advantage that can lead to clonal hematopoiesis (CH). Mutations in DNMT3A, particularly at hotspot R882, are the most prevalent in CH and carry an increased risk of acute myeloid leukemia (AML). Although DNMT3A R882 mutations are linked to global DNA hypomethylation, the mechanisms underlying their selective advantage remain unclear. Here, we show that Dnmt3a-R882H mutant HSCs exhibit resilience under inflammatory and genotoxic stress. During IL-1{beta}-induced emergency granulopoiesis, Dnmt3a R882H/+ HSCs uncouple increased proliferation from stem cell exhaustion. In contrast, wild-type HSCs rapidly progress to terminal differentiation. We link this phenotype to a delayed activation of the p53-p21-DREAM axis, that allows mutant HSCs to avoid attrition, despite increased replication stress. Similarly, mutant HSCs exhibit delayed Trp53 activation following irradiation, but eventually recover a physiological Trp53 response. Analysis of patient data reveals shared phenotypic features between DNMT3A and monoallelic TP53 mutations in CH and myeloid neoplasms, highlighting potential functional similarities. Collectively, these findings suggest that the expansion of DNMT3A-mutant clones is affected by impaired TP53 signaling, which confers resilience against stressors. Therapeutic strategies targeting inflammatory pathways or the p53-p21-DREAM axis may reduce DNMT3A-CH expansion and/or progression and its associated risks.

molecular biology↗

bioRxiv2024

Unveiling Clonal Cell Fate and Differentiation Dynamics: A Hybrid NeuralODE-Gillespie Approach

Recent lineage tracing single-cell techniques (LT-scSeq), e.g., the Lineage And RNA RecoverY (LARRY) barcoding system, have enabled clonally resolved interpretation of differentiation trajectories. However, the heterogeneity of clone-specific kinetics remains understudied, both quantitatively and in terms of interpretability, thus limiting the power of bar-coding systems to unravel how heterogeneous stem cell clones drive overall cell population dynamics. Here, we present CLADES, a NeuralODE-based framework to faithfully estimate clone-specific kinetics of cell states from newly generated and publicly available human cord blood LARRY LT-scSeq data. By incorporating a stochastic simulation algorithm (SSA) and differential expression gene (DEGs) analysis, CLADES yields cell division dynamics across differentiation timecourses and fate bias predictions for the early progenitor cells. Moreover, clone-level quantitative behaviours can be grouped into characteristic types by pooling individual clones into meta-clones. By benchmarking with CoSpar, we found that CLADES improves fate bias prediction accuracy at the meta-clone level. In conclusion, we report a broadly applicable approach to robustly quantify differentiation kinetics using meta-clones while providing valuable insights into the fate bias of cellular populations for any organ system maintained by a pool of heterogeneous stem and progenitor cells.

bioinformatics↗

bioRxiv2024

Maintenance of haematopoietic stem cells by JAK inhibition and increased tyrosine-unphosphorylated STAT5

Normal and malignant hematopoietic stem cells (HSCs) are controlled by extracellular cues including cytokine signalling through the JAK/STAT pathway. Here, we show that STAT5-deficient HSCs exhibit an unusual phenotype: while reduced multi-lineage repopulation and reduced self-renewal are commonly associated with overproliferation and exhaustion, they are instead associated with reduced cell-cycle progression and increased differentiation in STAT5-deficient HSCs. Mechanistic studies show that unphosphorylated-STAT5 (uSTAT5) contributes to this phenotype by constraining HSC differentiation, promoting HSC maintenance and upregulating transcriptional programs associated with stemness. The JAK1/2 inhibitor ruxolitinib increases levels of uSTAT5, constrains differentiation and proliferation of murine HSCs, promotes their maintenance and upregulates transcriptional programs associated with stemness. Ruxolitinib also enhances clonogenicity of normal human HSPCs, CALR-mutant murine HSCs and HSPCs from patients with myelofibrosis. Our results therefore reveal a previously unrecognized role for uSTAT5 in controlling HSC function, highlight JAK inhibition as a strategy for enhancing HSC function and provide insights into the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.

cell biology↗

bioRxiv2024

The long-term effects of chemotherapy on normal blood cells

In developed countries, [~]10% of individuals are exposed to systemic chemotherapy for cancer and other diseases. Many chemotherapeutic agents act by increasing DNA damage in cancer cells, triggering cell death. However, there is limited understanding of the extent and long-term consequences of collateral DNA damage to normal tissues. To investigate the impact of chemotherapy on mutation burdens and cell population structure of a normal tissue we sequenced blood cell genomes from 23 individuals, aged 3-80 years, treated with a range of chemotherapy regimens. Substantial additional mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but there were differences in burden between different classes of agent, different agents of the same class and different blood cell types. Chemotherapy also induced premature changes in the cell population structure of normal blood, similar to those of normal ageing. The results constitute an initial survey of the long-term biological consequences of cytotoxic agents to which a substantial fraction of the population is exposed during the course of their disease management, raising mechanistic questions and highlighting opportunities for mitigation of adverse effects.

cell biology↗

bioRxiv2023

Preleukemic clonal evolution revealed using single-cell DNA sequencing and computational modelling

The representation of driver mutations in preleukemic haematopoietic stem cells (pHSCs) provides a window into the somatic evolution that precedes Acute Myeloid Leukemia (AML). Here, we isolate pHSCs from the bone marrow of 16 patients diagnosed with AML and perform single-cell DNA sequencing on thousands of cells to reconstruct phylogenetic trees of the major driver clones in each patient. We develop a computational framework that can infer levels of positive selection operating during preleukemic evolution from the statistical properties of these phylogenetic trees. Combining these data with 67 previously published phylogenetic trees, we find that the highly variable structures of preleukemic trees emerge naturally from a simple model of somatic evolution in which there is pervasive positive selection acting throughout the disease trajectory. We infer that selective advantages of preleukemic clones are typically in the range of 9%-24% per year, but vary considerably between individuals. At these level of positive selection, we show that the identification of early multiple-mutant clones identifies individuals at risk of future AML.

cancer biology↗

bioRxiv2022

Adaptation to ex vivo culture drives human haematopoietic stem cell loss of repopulation capacity in a cell cycle independent manner

Loss of long-term haematopoietic stem cell function (LT-HSC) hampers the success of ex vivo HSC gene therapy and expansion procedures, but the kinetics and the mechanisms by which this occurs remain incompletely characterized. Here through time-resolved scRNA-Seq, matched in vivo functional analysis and the use of a reversible in vitro system of early G1 arrest, we define the sequence of transcriptional and functional events occurring during the first ex vivo division of human LT-HSCs. We demonstrate that contrary to current assumptions, loss of long-term repopulation capacity during culture is independent of cell cycle progression. Instead it is a rapid event that follows an early period of adaptation to culture, characterised by transient gene expression dynamics and constrained global variability in gene expression. Cell cycle progression however contributes to the establishment of differentiation programmes in culture. Our data have important implications for improving HSC gene therapy and expansion protocols.

cell biology↗

bioRxiv2022

Human HLFneg placental erythro-myeloid progenitors give rise to HLA Class IIneg Hofbauer cells

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. This process is still poorly understood in humans but is generally thought to be spatially restricted to the yolk sac. Human fetal placental macrophages, Hofbauer cells (HBC), arise during the primitive haematopoietic wave, yet are unlikely to be yolk sac derived as they appear prior to placental vascularisation. Here we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that give rise to HBC. PEMP are fetal CD34+CD43+ progenitors found exclusively at early gestational timepoints. Transcriptomic analyses reveal that PEMP have a unique transcriptome with some conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro single-cell culture experiments we show that PEMP generate HBC-like cells which lack HLA-DR expression, a conserved feature of all fetal primitive macrophages in humans. These findings indicate that HBC are derived locally from PEMP and demonstrate that human primitive haematopoiesis is not restricted to the yolk sac, occurring also in the placenta.

immunology↗

bioRxiv2021

Clonal dynamics of haematopoiesis across the human lifespan

Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer. The cellular alterations that underpin the abruptness of this functional decline after the age of 70 years remain elusive. We sequenced 3579 genomes from single-cell-derived colonies of haematopoietic stem cell/multipotent progenitors (HSC/MPPs) across 10 haematologically normal subjects aged 0-81 years. HSC/MPPs accumulated 17 mutations/year after birth and lost 30bp/year of telomere length. Haematopoiesis in adults aged <65 was massively polyclonal, with high indices of clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. In contrast, haematopoiesis in individuals aged >75 showed profoundly decreased clonal diversity. In each elderly subject, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before age 40, but only 22% had known driver mutations. Genome-wide selection analysis estimated that 1/34 to 1/12 non-synonymous mutations were drivers, occurring at a constant rate throughout life, affecting a wider pool of genes than identified in blood cancers. Loss of Y chromosome conferred selective benefits on HSC/MPPs in males. Simulations from a simple model of haematopoiesis, with constant HSC population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.

genomics↗

bioRxiv2021

Genome-wide mutational signatures of immunological diversification in normal lymphocytes

A lymphocyte suffers many threats to its genome, including programmed mutation during differentiation, antigen-driven proliferation and residency in diverse microenvironments. After developing protocols for single-cell lymphocyte expansions, we sequenced whole genomes from 717 normal naive and memory B and T lymphocytes and hematopoietic stem cells. Lymphocytes carried more point mutations and structural variation than stem cells, accruing at higher rates in T than B cells, attributable to both exogenous and endogenous mutational processes. Ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every one on-target IGV mutation during the germinal center reaction. Structural variation was 16-fold higher in lymphocytes than stem cells, with ~15% of deletions being attributable to off-target RAG activity. One Sentence SummaryThe mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

genomics↗

bioRxiv2020

Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

In the current understanding of adult bone marrow hematopoiesis, megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. Here, we use single cell RNA sequencing to characterize HSCs and MKs from human bone marrow, to investigate MK lineage commitment and maturation. We identify two MK primed HSC clusters exhibiting unique differentiation kinetics, at least one of which is used in steady state and stress thrombopoiesis. By analyzing transcriptional signatures we show that human bone marrow MKs originate from MK primed HSC subpopulations, supporting the notion that these display exclusive priming for MK differentiation. We show that transcriptional programs change with increasing MK ploidy, where genes upregulated in high ploidy states may have functional relevance in platelet production. Finally, we highlight the presence of a specific transcriptional signature in MKs from individuals with myocardial infarction, supporting the aberration of MK differentiation in this thrombotic state.

cell biology↗

bioRxiv2020

Quantitative and molecular differences distinguish adult human medullary and extramedullary haematopoietic stem and progenitor cell landscapes

In adults, the bone marrow (BM) is the main site of haematopoietic stem and progenitor cells (HSPCs) maintenance and differentiation. It is known that other anatomical sites can contribute significantly to blood production under stress conditions. However limited tissue availability restricts our knowledge on the cellular, molecular and functional composition of extramedullary HSPC pools in humans at steady state or under stress. Here we describe the landscape of human HSPC differentiation across the three major haematopoietic anatomical sites: BM, spleen and peripheral blood (PB), using matched tissues isolated from the same individuals. Single cell RNA-seq of 30,000 HSPCs and 700 phenotypic haematopoietic stem cells and multipotent progenitors (HSC/MPP) demonstrates significantly different dynamics of haematopoiesis between BM and extramedullary tissues. Lineage-committed progenitors of spleen and PB do not actively divide, whereas BM is the primary site of progenitor proliferation. The balance of differentiation in spleen and PB is skewed towards the lymphoid and erythroid lineages, whereas in BM it is tilted towards megakaryocytic and myeloid progenitors. Extramedullary tissues also harbour a molecularly defined subset of HSC/MPP not found in the BM, which is marked by a specific acto-myosin cytoskeletal signature and transcriptional priming for division and lineage differentiation. Collectively, our findings define a unique cellular and molecular structure of the haematopoietic landscape in extramedullary organs, positioned for rapid lineage-primed demand-adapted haematopoiesis. These data also provide a framework for better understanding of human extramedullary haematopoiesis in health and disease.

cell biology↗

bioRxiv2019

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis

Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a-CD7- subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options.

cancer biology↗