Functional heterogeneity of mouse fetal liver hematopoietic stem cells revealed by clonal lineage tracing and single-cell transcriptomics

The relationship between lineage output and transcriptional features of hematopoietic stem cells (HSCs) has been reported in adult bone marrow, yet it remains unclear in fetal liver, given their distinct developmental stage and microenvironment. Here, we systematically characterized the functions of E14.5 mouse fetal liver HSCs by lentiviral barcode labeling followed by transplantation, with subsequent single‑cell RNA sequencing and clonal analysis performed on HSCs derived from recipient bone marrow. We identified three HSC subtypes based on the lineage bias, with myeloid-biased and balanced subtypes predominating. Importantly, within the same subtype, HSCs and their progeny share several transcriptional programs, and these programs show minimal overlap between subtypes. We term the retention of subtype-specific transcriptional features across lineages as ‘transcriptional persistence’. Notably, this phenomenon was not observed in adult bone marrow. Alternatively, classification by progeny output activity revealed the presence of low-output and high-output subtypes within fetal liver HSCs. Although these two subtypes showed no significant difference in stemness features, they still exhibited distinct transcriptional features and signaling activation states, which also differed from their counterparts in bone marrow, indicating that the biological characteristics of HSCs with different output activities vary by developmental stage. Of note, in both fetal liver and adult bone marrow, transcriptional persistence showed no obvious correlation with output activity, suggesting a specific coupling relationship between transcriptional persistence and lineage bias. Collectively, our study provides a clonal-resolution view of fetal liver HSC heterogeneity, enhancing our understanding of HSC diversity across different developmental stages.