Single Cell Multiomic Analysis of Bone Marrow Hematopoietic Stem/Progenitor Cells from Individuals with Clonal Hematopoiesis Reveals Potential Mechanisms of Anemia Development
Authors: Buck Michele C , Rivière Jennifer , Navarro Figueredo Anna , Scarcella Daniele , Buser Maximilian , Hecker Judith S. , van der Garde Mark , Marr Carsten , Götze Katharina S. 
Published: December 2024

Clonal hematopoiesis (CH), a precursor to many malignant hematological diseases, is a central research focus for understanding disease development. CH describes a condition where a single hematopoietic stem cell gains a growth advantage through a genetic mutation, outcompeting other stem cells in the bone marrow (BM). While individuals with CH show no overt signs of hematological disease, they may exhibit subtle alterations in blood counts, such as lower hemoglobin levels and shifts in mean corpuscular volume (MCV) values. Changes within the erythroid lineage begin at the hematopoietic stem cell (HSC) level, as demonstrated in our previous study, in which we observed skewing of HSCs towards megakaryocyte-erythroid progenitors (MEP) in CH of various genetic subtypes (Buck and Bast et al., iScience 2023).
In the present study, we aimed to investigate underlying causes of these changes and unravel the potential mechanisms of anemia development, focusing on alterations within the cytoskeleton and chromatin condensation, both processes that precede erythroid enucleation. We conducted single-cell RNA and ATAC sequencing on hematopoietic stem and progenitor cells (HSPCs) from 14 CHIP BM samples harboring either a DNMT3A mutation, a TET2 mutation, or mutations in both genes, and compared these with four age-matched healthy samples, with a particular focus on the erythrocyte progenitor (EryP) compartment. Furthermore, we analyzed in vitro enucleation in CHIP and non-CHIP BM samples. CD34+ cells were purified and cultured in a 2-step erythroid differentiating medium supplemented with erythropoietin, SCF and IL-3 for two weeks. Flow cytometry (FACS) analysis of fixed and permeabilized cells was used to determine differentiation into erythroid progenitors, reticulocytes, and pyrenocytes by staining the nuclei with propidium iodide and highlighting erythroid cells with markers for CD71 and CD235a.
Within our single-cell RNA dataset, we identified 10 cell types corresponding to HSPC subtypes with no difference regarding their distribution in CHIP compared to healthy controls. Differential expression gene (DEG) analysis using a pseudobulk approach revealed an increasing number of deregulated genes with increasing differentiation, particularly highlighting the EryP compartment, with over 1,000 DEGs in CHIP. Many of these genes are involved in the maturation of erythroid cells, which includes enucleation-a multi-step process involving nuclear condensation, cytoskeletal restructuring, and the actual expulsion of the nucleus. Specifically, we found up-regulation of RIOK3, HDAC5, RAC1, RHOA, RHOB, motor proteins, and multiple tubulin- and histone-coding genes. Alterations in the structural components of chromatin could lead to changes in gene accessibility, which will be further analyzed in the ongoing single-cell ATAC sequencing analysis.
The analysis of maturation and enucleation of erythroid progenitors in vitro showed a significantly lower enucleation rate (p=0.0043, calculated by the percentage of reticulocytes in the total erythroid cells except pyrenocytes) at day 11 in CHIP samples (n=6), compared to healthy controls (n=5). This effect was no longer observed at day 14, where both conditions showed a similar enucleation rate, indicating a delayed erythroid maturation in CHIP compared to healthy controls.
In summary, erythroid progenitors from CHIP BM samples show specific transcriptomic changes related to critical characteristics of mature erythroid cells, including cytoskeleton and chromatin structure alterations. These molecular changes can explain lower hemoglobin and higher MCV levels observed in individuals with CH and are likely to contribute to development of anemia in myeloid neoplasia.