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Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in "Vav-DTR" mice. Crossing these mice to Flk2-Cre mice results in "HSC-DTR" mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis., (© 2022. The Author(s).)

Hematopoietic stem cells (HSCs) have been studied extensively since their initial functional description in 1961 when Dr. James Till and Dr. Ernest McCulloch developed the first in vivo clonal strategy, termed the spleen colony-forming unit (CFU-S) assay, to assess the functional capacity of bone marrow-derived hematopoietic progenitors at the single-cell level. Through transplantation of bone marrow cells and analysis of the resulting cellular nodules in the spleen, the CFU-S assay revealed both the self-renewal and clonal differentiation capacity of hematopoietic progenitors. Further development and use of this assay have identified highly proliferative, self-renewing, and differentiating HSCs that possess clonal, multilineage differentiation. The CFU-S strategy has also been adapted to interrogating single purified hematopoietic stem and progenitor cell populations, advancing our knowledge of the hematopoietic hierarchy. In this review, we explore the major discoveries made with the CFU-S assay, consider its modern use and recent improvements, and compare it with commonly used long-term transplantation assays to determine the continued value of the CFU-S assay for understanding HSC biology and hematopoiesis.

Hematopoietic stem cells differentiate into all blood cells, including Megakaryocyte progenitors (MkPs) and subsequent platelets. Understanding platelet generation informs cardiovascular and thrombotic disease, a leading global health concern among the aged population. We sought to uncover underappreciated heterogeneity among young, steady-state MkPs, and any changes with age. Recently, our lab uncovered functional differences between MkP populations. We defined two phenotypic subpopulations: canonical (cMkPs), and non-canonical (ncMkPs). We discovered that MkP heterogeneity changes with age, indicated by a dramatic increase in frequency and number of ncMkPs. First, we asked what surface proteins define young MkPs using targeted flow cytometry informed by bulk and single cell RNAseq. We found that specific surface proteins were differentially expressed among steady-state MkPs, indicating greater heterogeneity than previously observed. We combined nine candidate markers that demonstrated variable expression and applied dimensionality reduction analysis (UMAP) to standardized flow cytometry data. We observed heterogeneity among young MkPs and defined clusters of phenotypically-unique subpopulations. Second, we compared old and young MkP heterogeneity in mice. Aged MkPs demonstrated greater heterogeneity at both individual marker and combinatorial (UMAP) levels. Specific subclusters of MkPs were expanded with age. Finally, we assessed human MkPs for features analogous to the mouse. We found that, similar to mice, human MkPs possess phenotypic heterogeneity that changes with age. These new data define phenotypically distinct subpopulations of MkPs in both mice and humans that change with age. Advancing our understanding of platelet generation by specific MkP subpopulations may contribute to understanding the underlying biology of thrombotic and cardiovascular disease. [ABSTRACT FROM AUTHOR]

Hematopoiesis changes upon aging, including expansion of hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs), which undergo changes in phenotype and function. Platelets, the progeny of MkPs, also undergo age-related alterations that drive inflammation and cardiovascular and thrombotic disease; major health concerns. We discovered a non-canonical (ncMkP) population that specifically expands upon aging. Compared to their canonical (cMkP) counterparts, ncMkPs exhibit increased expansion and survival with age. Upon transplantation and serial blood assessment, both young and old cMkPs, and young ncMkPs, contributed to similar levels of platelet formation. In contrast, aged ncMkPs specifically produced greater platelet output. The progressive increase in ncMkPs with age appears to be, in part, originating from HSCs as in vitro generation of ncMkPs was significantly enhanced by aged compared to young HSCs. Further, single-cell functional analysis revealed that this was not driven by individual HSC clones, a finding substantiated by single-cell RNA sequencing of young and old bone marrow (BM). Progressively increasing, chronic inflammation is thought to be a driver of aging. Inducing chronic inflammation in young mice partially phenocopies known features of aged mice, including altered platelet output and expanded HSC numbers, implicating its role in influencing age-related platelet production. Direct assessment of inflammatory modulation of young and old HSCs, cMkPs, and ncMkPs via in vitro culture revealed distinct age- and population-specific responses, uncovering surprisingly distinct and differential roles for direct inflammatory reprogramming. These new data provide mechanistic insight to cell-based causes of aging-related adverse thrombotic events and may offer targets for mitigation and rejuvenation. [ABSTRACT FROM AUTHOR]

Platelets are small and short-lived cell fragments that play a fundamental role in hemostasis; however, age-related platelet dysregulation can contribute to cardiovascular disorders and thrombotic events, which are leading causes of death worldwide. Through use of a dual-fluorescent genetic lineage tracing mouse model, we have uncovered a hematopoietic stem cell differentiation pathway that emerges with age and gives rise to non-canonical, molecularly-distinct megakaryocyte progenitors (MkP) and platelets that are additive to those derived from the canonical differentiation pathway. We have previously shown that while hematopoietic stem cells functionally decline with age, aged MkPs and platelets have enhanced engraftment and thrombotic potential, respectively. Thus, we investigated the phenotypic and functional differences between age-specific and canonically-derived MkPs and platelets. Our findings indicate that age-specific, non-canonically-derived platelets exhibit hyperreactivity and have a distinct molecular profile compared to young and canonically-derived counterparts. These results reveal a unique, differentiation path-specific platelet dysregulation mechanism and suggest that age-specific platelets may be significant drivers of age-associated thrombotic events. [ABSTRACT FROM AUTHOR]