How is blood (re-)made? Regeneration of human hematopoietic stem cells after transplantation

Project Information

RESTART

Grant agreement ID: 101114895

DOI

10.3030/101114895

EC signature date 12 October 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

PRINSES MAXIMA CENTRUM VOOR KINDERONCOLOGIE BV
Netherlands

Periodic Reporting for period 1 - RESTART (How is blood (re-)made? Regeneration of human hematopoietic stem cells after transplantation)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

Understanding how human hematopoietic stem and progenitor cells (HSPCs) regenerate the blood system remains a major unresolved challenge, limiting their effective therapeutic use. Each year, more than 40,000 patients undergo HSPC transplantation (HCT) as a last-resort treatment for life-threatening conditions such as leukemia. However, approximately 40% of recipients die due to poor donor cell engraftment, inflammatory complications, or disease relapse. Strategies to predict and prevent these adverse outcomes are urgently needed.

In mouse models, single-cell technologies have transformed our understanding of hematopoietic organization, allowing us to control the outcome of murine HCT in detail. In contrast, understanding of human hematopoietic regeneration, and our capacity to control it, is lagging behind. Bridging this gap is essential for improving HCT success.

RESTART aims to comprehensively characterize the cellular and molecular mechanisms guiding hematopoietic regeneration in humans. Building on pioneering work using single-cell methods to study human HSPC biology, this project will employ state-of-the-art multi-omics to dissect the identities and functional states of thousands of HSPCs and their surrounding niche cells in human bone marrow. Embedded in Europe's largest pediatric cancer center, we will apply these methods to a unique, longitudinal collection of bone marrow samples of pediatric HCT recipients and their donors, collected from pre-transplantation to one year post-transplantation.

Our objectives are:
1. Define the cellular and molecular composition of HSPC populations during successful hematopoietic regeneration in HCT recipients;
2. Determine how transplantation-induced changes in the bone marrow niche influence HSPC fate;
3. Identify and validate single-cell states or trajectories that predict adverse clinical outcomes, including graft failure and relapse.

By integrating fundamental biology with clinical data, RESTART will advance understanding of human tissue regeneration and support the development of predictive strategies to improve HCT outcomes and patient survival.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the first two years, three major achievements were realized:

1. Generation of a single-cell and spatial reference atlas of healthy pediatric donor bone marrow.
Using single-cell multiomics on residual bone marrow cells of pediatric HCT donos, we generated a high-resolution atlas of healthy pediatric bone marrow. This atlas encompasses single-cell transcriptomics, surface proteomics, and spatial transcriptomics across developmental stages from early childhood to young adulthood.We demonstrate that young pediatric BM is distinct from bone marrow of adolescents and young adults, in terms of cellular composition, molecular cell states and lineage fate decisions, indicating that age-matched references are crucial to distinguish age- from disease associated differences. In ongoing research, this atlas forms an essential baseline for interpreting post-transplant regeneration data, and supporting Objectives 1 and 3. In addition (beyond this ERC-work), the dataset provides a useful reference for studies on (pediatric) hematologic, oncologic and immunologic research.

2. Optimization of technologies to characterize bone marrow mesenchymal stromal cells and the niche.
Given the technical challenges of recovering mesenchymal stromal cells (MSCs) from cryopreserved aspirates, enrichment strategies were systematically evaluated and optimized, revealing pre-analytical losses as a major limiting factor. To enable robust niche characterization, we optimized and implemented spatial transcriptomics of bone marrow biopsies, yielding several-fold more niche cells (including populations not captured in aspirates), while preserving tissue architecture. High-quality transcriptional profiles of MSC subsets were generated, and analyses of post-transplant biopsies are ongoing. Integration of single-cell multiomic and spatial data will allow systematic assessment of transplantation-induced niche alterations and their effects on HSPC regeneration (Objective 2).

3. Establishment of a longitudinal single-cell profiling pipeline for post-transplant regeneration.
A standardized and reproducible workflow was developed for longitudinal single-cell analysis of post-HCT bone marrow samples, including sample processing, library preparation, and computational analysis. Initial datasets from early post-transplant timepoints reveal dynamic changes in HSPC states and immune reconstitution, with persistent alterations in bone marrow composition months after transplantation. These data provide the first patient-derived single-cell view of human hematopoietic regeneration after allo-HCT and establish the foundation for identifying predictive cellular states associated with graft failure or relapse (Objective 3).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project delivers beyond-state-of-the-art results by generating the first comprehensive single-cell reference atlas of healthy pediatric bone marrow, filling a longstanding gap in hematology, immunology, and regenerative medicine. Until now, studies of pediatric hematologic disease have relied on adult reference datasets, assuming comparable biology despite limited evidence.

Using single-cell multiomics across postnatal development, we mapped cellular composition, stem cell states, and niche interactions in healthy pediatric bone marrow, with specific enrichment of rare but critical hematopoietic stem and progenitor cells and mesenchymal stromal cells. The data demonstrate that pediatric bone marrow differs fundamentally from adolescent and young adult marrow, with age-dependent stem cell programs and remodeling of the microenvironment that shape hematopoietic function.

These findings establish the necessity of age-matched reference maps to distinguish disease from normal development, provide a widely accessible community resource, and enable new research into how stem cells adapt to their niche after transplantation.