Deciphering the intrinsic and extrinsic mechanisms shaping aging and leukemic evolution of hematopoietic stem cells at the single cell level

Información del proyecto

PRELEUKMEC

Identificador del acuerdo de subvención: 101106929

DOI

10.3030/101106929

Proyecto cerrado

Fecha de la firma de la CE 20 Marzo 2023

Fecha de inicio 1 Abril 2023

Fecha de finalización 31 Marzo 2025

Financiado con arreglo a

• Marie Skłodowska-Curie Actions (MSCA)

Coste total

Sin datos

Aportación de la UE

€ 200 538,24

Coordinado por

WEIZMANN INSTITUTE OF SCIENCE
Israel

Periodic Reporting for period 1 - PRELEUKMEC (Deciphering the intrinsic and extrinsic mechanisms shaping aging and leukemic evolution of hematopoietic stem cells at the single cell level)

Período documentado: 2023-04-01 hasta 2025-03-31

Resumen del contexto y de los objetivos generales del proyecto

Hematopoiesis is the production of all blood cellular components, which are derived from hematopoietic stem and progenitor cells (HSPCs) in a stepwise process. Proper regulation of HSPCs behavior is essential for lifelong blood production, immune function and tissue oxygenation. As individuals age, somatic mutations accumulate in HSPCs. Some mutations will confer a proliferative advantage to certain clones in the hematopoietic hierarchy, allowing them to expand disproportionately. This phenomena known as, Clonal Hematopoiesis (CH) is defined as the expansion of HSPCs, harboring specific, disruptive, and recurrent genetic variants, among individuals without clear diagnosis of hematological malignancies. CH is a significant risk factor for hematological malignancies such as Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS), but also for cardiovascular diseases and all-causes mortality.
The objective of this project was to investigate the impact of aging and of disease-associated mutations on human HSPCs, focusing on how they affect lineage commitment and transcriptional programs under varying microenvironmental conditions. As, the project faced challenges related to geopolitical instability, we mainly focused on developing a robust experimental and computational system to deeply characterize normal human HSPC differentiation at single-cell and clonal resolution.Using an innovative lineage-tracing assay combined with single-cell RNA sequencing, we generated the most comprehensive reference map of healthy HSPCs differentiation to date. Establishing this robust baseline is a necessary prerequisite for confidently detecting and interpreting how disease-associated mutations alter hematopoiesis in patient-derived samples.
This map captures dynamic clonal trajectories, reveals novel progenitor populations, and uncovers key features of HSPCs biology. Specifically, we showed that HSPCs clones are stochastically driven as they frequently bifurcate into multiple myeloid lineages amid homogeneous signals. At the molecular level, we uncovered evidence of clonal memory extending beyond the differentiation state, including a striking inter-clonal variability in the absolute rate of differentiation. Altogether, we provide an essential baseline for better understanding the dynamics of human HSPCs, with broad application to both healthy individual and patients with hematological disorders.
This foundational work not only advances scientific understanding of human blood development but also establishes a critical resource for future studies of hematological diseases. By equipping researchers with high-resolution tools to dissect HSPC fate decisions, the project paves the way for improved modeling of clonal hematopoiesis and blood cancers. The results contribute to European strategies aiming to promote health research innovation and strengthen personalized medicine approaches. Ultimately, this work supports long-term goals of enhancing diagnosis, treatment, and patient outcomes for blood disorders, addressing a significant societal and healthcare challenge.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

To complete our initial objective, we successfully developed a single-cell clonal tracing assay using peripheral blood-derived HSPCs to quantitatively track the differentiation dynamics of individual clones in vitro under various cytokine combinations. By integrating longitudinal sampling, barcode-based lineag tracing and transcriptional profiling, our assay enabled high-resolution analysis of lineage output and gene expression states within each clone.
On the computational side, Dror Brook from Dr Amos Tanay’s lab developed all the advanced analytical pipelines to cluster clones based on barcode sequences, link clone states to microenvironmental conditions and perform detailed lineage tracing and transcriptomic analysis. In total, we profiled approximately 80,000 cells derived from 17 healthy donors across nine differentiation time points, and grown under different culture conditions. We investigated the clonal dynamics of HSPCs across the manifold, focusing on how they choose and diversify their differentiation trajectories, providing unprecedented resolution into human HSPCs clonal dynamics.

We successfully optimized in vitro differentiation of single CD34⁺ HSPCs into all major myeloid lineages. Longitudinal sampling captured cells at key developmental stages across each lineage. This enabled reconstruction of full clonal trajectories at temporal and transcriptional resolution. Using amplicon-based barcode sequencing, we reliably linked each single-cell transcriptome to its original cytokine condition. The result is a scalable, high-throughput framework to systematically interrogate HSPC fate decisions under defined microenvironmental inputs. Importantly, comparison to published bone marrow scRNA-seq datasets showed strong transcriptional trajectoris concordance, validating the physiological relevance of our in vitro model.
By combining clonal lineage tracing with dense temporal sampling, we identified a previously uncharacterized progenitor population with dual potential for basophil and eosinophil fates, which we termed Baso-Eosinophil Progenitor (BEP).
As expected, HSPCs were highly responsive to microenvironmental signals, showing biased differentiation in response to different cytokines. Strikingly, we observed that clones followed stochastic differentiation paths, frequently bifurcating into multiple myeloid lineages even under homogeneous culture conditions. At the molecular level, we uncovered evidence of clonal memory extending beyond differentiation state, including notable inter-clonal variability in the absolute rate of differentiation. Together, these findings provides both a robust dataset and robust methodology for dissecting the dynamics of human HSPCs, with broad application to both physiological haematopoiesis and haematological disorders.

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Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

The project delivered several advances beyond the current state of the art in human hematopoiesis research. First, by integrating clonal barcoding with single-cell transcriptomics during in vitro differentiation, the project provided a unique view into how individual human HSPCs commit to specific lineages over time, information that cannot be inferred from snapshot dataset.
Secondly, the reference dataset generated as part of the project represents one of the most detailed time-resolved single-cell atlases of human hematopoietic differentiation to date. This dataset will be valuable for identifying molecular events that precede lineage commitment and for for comparing normal and diseased states
These results address critical gaps in the field, including the lack of standardized reference data for human HSPCs dynamic and the need for scalable systems to study cell-intrinsic fate decisions alongside the impact of cell extrinsic factors. The insights gained will directly support future translational work, particularly studies aiming to:

-Projection of patient samples against the healthy atlas for disease research
-Preclinical studies of therapies that alter clonal expansion or lineage bias
-Develop new diagnostic criteria based on deviations from reference clonal behavior

To maximize the impact and broader applicability of this work, further research is needed to extend the system to patient samples and disease models. However, the methodological tools, datasets, and conceptual frameworks developed during the project are already enabling follow-up studies.