Key Points: After induced HSPC depletion, HSC numbers remain at low levels whereas progenitors show robust recovery. Despite low HSC numbers, hematopoiesis proceeds normally without increased proliferation of the few residual HSCs.

Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48^hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48^−/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.

Blood cell generation depends on continuous cellular output by the sequential hierarchy of hematopoietic stem cell (HSC) and progenitor populations that all contain quiescent and actively cycling cells. Hematopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem cell antigen 1 (SCA-1/LY6A). Using histone 2B-red fluorescent fusion protein label retention and cell-cycle reporter mice, we demonstrate that high SCA-1 expression (SCA-1hi) identifies not only quiescent HSCs but quiescent cells on all hierarchical levels within the lineage-SCA-1+KIT+ (LSK) population. Each transplanted SCA-1hi HSPC population also displayed self-renewal potential superior to that of the respective SCA-1lo population. SCA-1 expression is inducible by type I interferon (IFN). We show, however, that quiescence and high self-renewal capacity of cells with brighter SCA-1 expression at steady state were independent of type I IFN signaling. We conclude that SCA-1 expression levels can be used to prospectively isolate functionally heterogeneous HSPC subpopulations.

The proliferative activity of aging hematopoietic stem cells (HSCs) is controversially discussed. Inducible fluorescent histone 2B fusion protein (H2B-FP) transgenic mice are important tools for tracking the mitotic history of murine HSCs in label dilution experiments. A recent study proposed that primitive HSCs symmetrically divide only four times to then enter permanent quiescence. We observed that background fluorescence due to leaky H2B-FP expression, occurring in all H2B-FP transgenes independent of label induction, accumulated with age in HSCs with high repopulation potential. We argue that this background had been misinterpreted as stable retention of induced label. We found cell division–independent half-lives of H2B-FPs to be short, which had led to overestimation of HSC divisional activity. Our data do not support abrupt entry of HSCs into permanent quiescence or sudden loss of regeneration potential after four divisions, but show that primitive HSCs of adult mice continue to cycle rarely.

A novel series of dimeric melatonin analogues obtained by connecting two melatonin molecules through N1 with spacers of 15–24 atoms was synthesized and characterized in 2-[¹²⁵-I]-iodomelatonin binding and bioluminescence resonance energy transfer (BRET) experiments at MT₁ and MT₂ receptors. Compounds 4 (16 atoms spacer) and 13 (24 atoms spacer) are among the ligands inducing the maximal BRET at MT₂-homodimers as well as at both types of MT₁/MT₂ heterodimers. Notably, ligand-induced BRET changes observed for compounds linked through spacers of 22–24 atoms could be attributed to ligand-induced conformational changes between the two protomers of MT₁ and MT₂ homo- and heterodimers providing evidence for the binding of both pharmacophores of dimeric melatonin analogues to the two protomers of receptor dimers.

PAX5 acts as a master regulator of B-cell proliferation and differentiation. Its germline and somatic deregulation have both been implicated in the development of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the process how reduced PAX5 transcriptional activity mediates progression to BCP-ALL, is still poorly understood. Here, we characterized the longitudinal effects of PAX5 reduction on healthy, pre-leukemic and BCP-ALL cells at the single-cell level. Cell-surface marker analysis revealed a genotype-driven enrichment of the pre-BII population in healthy Pax5 ^± mice. This population showed downregulated B-cell receptor signaling, while DNA replication/repair and cell-cycle signaling pathways were upregulated. Moreover, we observed a shift in the kappa/lambda light chain ratio toward lambda rearranged B-cells. Transplantation experiments further validated a delay of Pax5 ^± pre-BII cells in maturation and transition to IgM-positivity. Additionally, single-cell RNA-Sequencing and bulk ATAC-Sequencing of different stages of BCP-ALL evolution showed that Pax5 ^± pre-leukemic cells lose their B-cell identity and display Myc activation. Subsequently, BCP-ALLs acquired additional RAG-mediated aberrations and driver mutations in JAK-STAT and RAS-signaling pathways. Together, this study elucidates molecular and functional checkpoints in PAX5-mediated pre-leukemic cell progression exploitable for therapeutic intervention and demonstrates that PAX5 reduction is sufficient to initiate clonal evolution to BCP-ALL through activation of MYC.