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.
Hematopoietic stem cells (HSCs) produce a highly diverse array of cell lineages. To assay hematopoietic differentiation with minimal experimental perturbation, non-invasive methods for heritable labeling (1-3) or barcoding (4-7) of HSCs in vivo have recently been developed and used to study lineage fate of HSCs in physiological conditions. However, the differentiation pathways leading from HSCs to mature cells remain controversial (8), with suggested models ranging from gradual lineage restriction in a branching cascade of progenitors to HSCs already making ultimate lineage decisions. Here we show, by iterating HSC fate-mapping, mitotic history tracking, single-cell RNA-sequencing and computational inference, that the major differentiation routes to megakaryocytes, erythro-myeloid cells and lymphocytes split within HSCs. We identify the hitherto elusive self-renewing source of physiological hematopoiesis as an HSC subpopulation co-expressing high levels of Sca-1 and CD201. Downstream, HSCs reduce Sca-1 expression and enter into either thrombopoiesis or erythro-myelopoiesis, or retain high Sca-1 levels and the ability to generate lymphocytes. Moreover, we show that a distinct population of CD48-/lo megakaryocyte progenitors links HSCs to megakaryocytes. This direct thrombopoiesis pathway is independent of the classical pathway of megakaryocyte differentiation via multipotent progenitors and becomes the dominant platelet production line upon enhanced thrombopoietin signaling. Our results define a hierarchy of self-renewal and lineage decisions within HSCs in native hematopoiesis. Methodologically, we provide a blueprint for mapping physiological differentiation pathways of stem cells and probing their regulation.
Hematopoietic stem cells (HSCs) are the ultimate source of blood and immune cells. Under homeostatic conditions, these cells are considered a quiescent reserve population. However, it is not clear to what extent HSCs participate in emergency responses. Herein, we use fate mapping and proliferation tracking mouse models, which cumulatively record HSC activity in situ. We observed no direct contribution of HSCs to mature blood cell regeneration in response to common hematopoietic emergencies, including inflammation or blood loss. Innate immune training, in which HSCs were proposed to store and integrate information on previous infections, did not alter HSC activity upon secondary exposure. Only severe myeloablation resulted in a robust increase of HSC contribution. Our data demonstrates that HSCs do not directly participate in the regeneration of mature blood cells and therefore do not represent a reserve population to compensate for physiological hematopoietic perturbations.
Genome damage is a main driver of malignant transformation, but it also induces aberrant inflammation via the cGAS/STING DNA sensing pathway. Activation of cGAS/STING can trigger cell death and senescence, thereby potentially eliminating genome-damaged cells and preventing against malignant transformation. Here, we report that defective ribonucleotide excision repair (RER) in the hematopoietic system caused genome instability with concomitant activation of the cGAS/STING axis and compromised hematopoietic stem cell function, ultimately resulting in leukemogenesis. Additional inactivation of cGAS, STING, or type I IFN signaling, however, had no detectable effect on blood cell generation and leukemia development in RER-deficient hematopoietic cells. In wild-type mice, hematopoiesis under steady-state conditions and in response to genome damage was not affected by loss of cGAS. Together, this data challenges a role of the cGAS/STING pathway in protecting the hematopoietic system against DNA damage and leukemic transformation.
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