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.

Summary The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo . Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.

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 CD48hi 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.

Hematopoietic stem cells (HSCs) are the ultimate source of blood and immune cells and transplantation reveals their unique potential to regenerate all blood lineages lifelong. HSCs are considered a quiescent reserve population under homeostatic conditions, which can be rapidly activated by perturbations to fuel blood regeneration. In accordance with this concept, inflammation and loss of blood cells were reported to stimulate proliferation of HSCs, which is associated with a decline in their transplantation potential. To investigate the contribution of primitive HSC to the hematopoietic stress response in the native environment, we use Fgd5-driven fate mapping and H2B-GFP proliferation tracking mouse models. While primitive HSCs were robustly activated by severe myeloablation, they did not contribute to the regeneration of mature blood cells in response to prototypic hematopoietic emergencies such as acute inflammation or blood loss. Even chronic inflammatory stimulation, which triggered vigorous HSC proliferation, only resulted in weak contribution of HSCs to mature blood cell production. Thus, our data demonstrates that primitive HSCs do not participate in the hematopoietic recovery from common perturbations and calls for the re-evaluation of the concept of HSC-driven stress responses.