Title: In Vivo Clonal Tracing of Hematopoietic Stem and Progenitor Cells Reveals Increased Clonal Heterogeneity during Aging, Alongside Critical Changes in Selection Patterns

Aging impacts the bone marrow microenvironment by inducing inflammation. We hypothesize that aging increases heritable epigenetic heterogeneity, leading to transcriptome heterogeneity within hematopoietic stem and progenitor cells (HSPC). This resulting heterogenous state, combined with the aged microenvironment-driven selection, shifts the functional capacity of the hematopoietic system, contributing to the functional decline of the hematopoietic system as well as the evolution of diseases such as myelodysplastic syndrome and leukemias. Our research aims to investigate clonal heterogeneity in both young and old mice and highlight the phenotypic patterns selected in each case.

We employed CellTag indexing and single-cell RNA sequencing to track HSPC, identify the selected clones in young and old mice BM, and characterize their molecular signatures. We generated 10x Genomics single-cell RNA-seq for tagged HSPC after 15-day culturing (in vitro) and two months post-transplantation into mice (in vivo). Tracing the expansion of HSPC clones in young and old BM revealed critical changes in HSPC heterogeneity and selection in older mice.

We observed an increase in transcriptional heterogeneity of old HSPC as compared to young samples (P<0.01, Wilcox test). Genes with higher gene expression variability in older mice coincide with pathways pivotal for HSPC fitness, e.g., ribosome synthesis, inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML, and cancer pathways (FDR<0.1, GSEA). At the clonal composition level, young HSPCs exhibited more oligoclonal reconstitution than old HSPC, indicated by lower Shannon entropy in young HSPCs compared to old (P<0.05 Wilcox test). This result suggests stricter clonal quality control in the young microenvironment, potentially leading to increased purifying selection that removes clones with significant transcriptional profile alterations and reduced fitness. Moreover, comparing the transcriptional profiles of clones showing robust expansion after transplantation (selected or “winner” clones) with negatively selected clones revealed several common pathways shared between young and old selected clones after expansion (FDR<0.1, GSEA), including cell cycle activity, stress response, WNT signaling, and metabolism – identifying candidate mediators of HSPC fitness in vivo. However, major differences between old and young selected clones were evident before expansion. In young, selected clones, signatures related to inflammatory response were observed.Strikingly, we observed a significant positive expression correlation (P<0.0001, Wilcox test) among the selected clones only in young mice, not in old mice. The high transcriptomic similarity among selected clones is consistent with strict clonal quality control in the young BM microenvironment. Notably, we traced the selected clones back among the tagged HSPCs in vitro (scRNA-seq) and observed higher transcriptomic similarity among selected clones from young vs. old donor mice even before transplantation (P<0.0001, Wilcox test). Collectively, these results underscore the selective nature of the young BM, contrasting with the decline of HSPC quality control in old BM, contributing to functional hematopoietic decline and fueling pre-malignant evolution.

These studies reveal striking differences in clonal dynamics and quality control among HSPC in young and old mice. Clonal tracing of hematopoietic clones provides insight into how clonal competition and microenvironmental selection can contribute to hematopoietic decline and malignant evolution. Better understanding of the pathways that contribute to HSPC fitness and functional decline with aging could lead to approaches to track and perhaps even counter these aging-associated changes, which is expected to contribute to improved hematopoietic function and reduced risk of malignancy in the elderly.