Why do stem cells fail? Single-cell and spatial transcriptomics of pediatric myelodysplastic syndromes
Pediatric myelodysplastic syndrome with refractory cytopenia of childhood (pMDS-RCC) is a poorly understood bone marrow (BM) disease, characterized by ineffective hematopoiesis and risk of progression to acute myeloid leukemia. Germline and somatic mutations are present in approximately 40% of pMDS-RCC patients, but the mechanisms leading to BM failure and underlying causes in mutation-free patients remain unclear. In this study, we aimed to comprehensively characterize molecular, cellular and spatial composition of the BM in pMDS-RCC, to gain insight into the disease pathogenesis.
We performed single-cell multimodal sequencing of RNA and surface protein expression on diagnostic BM aspirates from patients with pMDS-RCC without genetic predisposition (n=6; ages 9-17 years) and healthy age-matched controls (n=4; ages 9-17 years). Prior to sequencing, samples were FACS-enriched for hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). Non-enriched samples were also obtained from each individual. We used a customized version of the TotalSeqTM-B panel (Biolegend), targeting 138 cell surface proteins. Combined cDNA and oligo libraries were prepared using the 10X Genomics platform and sequenced. Additionally, we performed spatial transcriptomics on diagnostic trephine biopsies from pMDS-RCC patients (n=4; ages 9-16 years) and non-cancerous controls (n=10; ages 1-17 years), with the 10x Genomics Xenium platform (customized panel of 477 genes). Data preprocessing and analysis were performed in R using the Seurat package.
We successfully sequenced 44747 cells from pMDS-RCC patients and 40400 cells from healthy controls. Cell type composition analysis revealed significantly decreased frequencies of HSPCs and myeloid cells, and significantly increased frequencies of T-cells in pMDS-RCC children. Analysis of genes related to T-cell activation and exhaustion did not show difference between pMDS-RCC and controls, suggesting that the T-cell accumulation is likely due to factors other than increased proliferation. Interestingly, cell cycle analysis revealed a substantial increase in the percentage of HSPCs in the G2M phase in pMDS-RCC compared to controls potentially indicating a differentiation defect.
To explore the molecular pathways underlying this defect, we used differential gene expression analysis. Several genes related to HSPC maintenance (e.g. AREG, SRGN) were significantly downregulated in HSPCs of pMDS-RCC compared to controls. Importantly, cell-cell interaction prediction revealed loss of both known (e.g. KIT-KITL) and novel interactions, indicating a lack of supporting signaling to the HSPCs in the pMDS-RCC BM. To validate these findings, spatial transcriptomics data have been generated for four pMDS-RCC patients and ten healthy controls, for which the analysis is ongoing.
This study provides the first comprehensive single-cell overview of pMDS-RCC BM, revealing distinct cellular features linked to BM failure. Ongoing studies aim to characterize these pathways within the spatial BM architecture and validate the identified targetable factors in vitro.