Clonal Analysis Reveals Functional and Transcriptional Heterogeneity of Self-Renewing Fetal Liver Hematopoietic Stem Cells

The fetal liver (FL) is a key organ supporting the expansion of hematopoietic stem cells (HSCs) during development. The ability to expand transplantable HSCs in vitro is a long-standing challenging; thus, decoding the gene pathways that regulate proliferation and self-renewal during FL-HSC development is highly relevant to engineering HSCs for therapeutic applications. Moreover, while heterogeneity in self-renewal and engraftment properties of HSCs in adult bone marrow has been extensively studied, these properties remain largely uncharacterized in FL-HSCs.

To explore FL-HSC properties at the single cell level, we engineered a serum-free culture platform designed to recapitulate the murine FL endothelial niche (FL-EC) (Figure1), and sorted single FL-HSC (CD45⁺GR1^-F4/80^-SCA1^highEPCR^high) into individual wells containing FL-EC. Following co-culture, we observed emergence of immunophenotypically distinct types of hematopoietic colonies as determined by flow cytometry. Only colonies containing cells predominantly expressing high levels of HSC markers SCA1 and EPCR (Figure1, HSC colonies) provided multilineage engraftment as determined by transplantation assays, whereas colonies containing cells expressing HSC markers and more differentiated progeny (mixed colonies) were unable to provide serial multilineage engraftment. Furthermore, cellular progeny from a single HSC colony transplanted into multiple recipients demonstrated serial engraftment, indicating the FL-EC niche could amplify single FL-HSC with long-term reconstitution capacity. These studies suggest that transplantable FL-HSCs are uniquely distinguished by their propensity for symmetric self-renewal in this assay. However, not all HSC colonies provided engraftment in secondary recipients, suggesting heterogeneity in self-renewal properties of immunophenotypically defined FL-HSCs that required deeper analysis.

Thus, to further assess transcriptional heterogeneity in HSC colonies, we next integrated single-cell RNA sequencing (scRNAseq) into the clonal FL-HSC assay (Figure 2). Residual cells from six HSC colonies were divided for simultaneous scRNAseq and transplantation. Consistent with experiments above, we observed heterogeneity in long-term serial engraftment from individual colonies. Remarkably, cells from the colony capable of serial engraftment were distinctly enriched in genes associated with dormant HSCs in adult marrow (Cabezas-Wallscheid et.al., Cell 2017). Conversely, cells from HSC colonies that lacked serial engraftment capacity exhibited increased expression of Myc target genes by Gene Set Enrichment Analysis (GSEA), and ribosome biogenesis and mitochondrial translation by Gene Ontogeny (GO) analysis, which are hallmarks of biosynthetically activated HSCs in adult marrow that are primed for differentiation to progenitor states.

To further assess the transcriptional heterogeneity of primary FL-HSC, we next performed scRNAseq with freshly isolated FL-HSC. Unsupervised clustering and co-regulated modules of differentially expressed genes identified a rare subset of cells highly enriched for genes associated with HSC dormancy, while the other cells featured higher expression of genes involved in biosynthetic processes via GO analysis (e.g. cytoplasmic translation, rRNA processing). This data further supports transcriptional heterogeneity within the FL-HSC compartment, which includes a unique biosynthetically dormant subset.

Collectively, our studies elucidate previously unrecognized heterogeneity in FL-HSCs, highlighting a state of relative biosynthetic dormancy as a key to symmetric self-renewal of serially engraftable FL-HSCs. These findings complement recent studies demonstrating distinct developmental origins for multipotent progenitors and functional engraftable HSCs in definitive hematopoiesis (Dignum et.al., Cell Reports 2021; Patel et.al., Nature 2022), suggesting functional heterogeneity in immunophenotypically defined FL-HSCs in our study may in part reflect these distinct ontogenies. Building on these studies, we are currently using CRISPR-Cas9 gene knockout to define the roles of candidate genes identified by our scRNAseq analysis that are implicated in regulating dormancy and symmetric self-renewal of FL-HSCs, which would provide essential insight for future strategies for ex vivo amplification of HSCs.

Ishida:Takeda Science Foundation: Other: Grant in aid . Varnum-Finney:Deverra Therapeutics: Patents & Royalties. Bernstein:Lyell Immunopharma: Current equity holder in publicly-traded company, Research Funding; Deverra therapeutics: Current equity holder in private company.