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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.

The generation of distinct hematopoietic cell types, including tissue-resident immune cells, distinguishes fetal from adult hematopoiesis. However, the mechanisms underlying differential cell production to generate a layered immune system during hematopoietic development are unclear. Using an irreversible lineage-tracing model, we identify a definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients but does not persist into adulthood in situ. These HSCs are fully multipotent, yet they display both higher lymphoid cell production and greater capacity to generate innate-like B and T lymphocytes as compared to coexisting fetal HSCs and adult HSCs. Thus, these developmentally restricted HSCs (drHSCs) define the origin and generation of early lymphoid cells that play essential roles in establishing self-recognition and tolerance, with important implications for understanding autoimmune disease, allergy, and rejection of transplanted organs.

During development, transient fetal hematopoietic stem cells (HSCs) are responsible for the production of "unconventional" innate-like immune cells that contribute to adult immunity. Whereas dysregulation of fetal-derived immune cells contributes to pathogenesis in a variety of immune tolerance disorders, the cellular and molecular drivers of pathogenesis are unknown. We have previously identified a transient developmentally-restricted HSC (drHSC) that specifically gives rise to innate-like lymphocytes during the perinatal period1. Under homeostatic conditions, the drHSC disappears postnatally, but its innate-like lymphocyte progeny persist into adulthood. Our discovery of a transient cell-of-origin for a specialized component of adult immunity underscores a “critical window” of immune development, during which phenotype of the adult immune system can be shaped by extrinsic inputs in early life. Here, we tested how developmental perturbation induced in utero by a single low-dose injection of poly(I:C) at mid-gestation drives both immediate and lasting changes to hematopoiesis and immunity in offspring. In utero perturbation disrupted the entrance of fetal HSCs into quiescence, causing disproportionate expansion of drHSCs, and a resultant shift in multipotent progenitor output. Postnatally, we observed sustained expansion and inappropriate persistence of the drHSC population into adulthood. These fundamental changes to the postnatal HSC compartment resulted in parallel expansion and hyperactivation of innate-like lymphocytes, thereby altering immune landscape and function in offspring. Our work validates the existence of a critical window of immune development by revealing how early perturbation of distinct fetal HSCs can drive long-term changes to immunity and disease susceptibility in offspring.

Our goal is to define the cellular and molecular mechanisms that control aging of the hematopoietic system. Given the increasing global life expectancy of people and the prevalence of age-related hematological diseases, understanding alterations to stem and progenitor cell function during aging is critical for developing preventative and restorative therapies. Previous reports have suggested that age-related morbidity is determined by functional decline of hematopoietic stem cells (HSCs). While HSCs themselves have been associated with hematopoietic aging, the role of progenitor cells during aging remains a mystery. Progenitor cells carry varied capacity to proliferate and differentiate in order to meet the demands of the blood system. Therefore, we hypothesize that progenitor cells play a significant role in the homeostatic decline associated with aging. Indeed, Megakaryocyte Progenitors (MkPs) have been shown to be consistently enriched in the aging bone marrow. Therefore, we specifically tackled the question – how might aging alter the functional and molecular regulation of MkPs? Surprisingly, and in stark contrast to the functional decline of aging HSCs, we found that old MkPs displayed greater capacity to generate platelets compared to young MkPs. These functional differences were apparent by both in vivo reconstitution and in vitro proliferation assays. To gain further insight into the changes in molecular regulation of MkP populations, we performed RNA sequencing (RNA-seq) analysis of young and old MkPs. Interestingly, their functional differences are paralleled by unique molecular regulators of old MkPs. These data demonstrate that megakaryopoiesis and MkP maintenance is fundamentally different between young and old mice, and that old MkPs gain expansion capacity whereas old HSCs functionally decline. Understanding these cellular and molecular events provides a promise for development of effective therapeutic strategies aimed at guiding differentiation and maturation toward a more youthful megakaryopoiesis.

SummaryAlthough classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

Tissue-resident macrophages play critical roles in tissue homeostasis and disease. Many populations of tissue-resident macrophages derive from fetal progenitors and self-maintain across the lifespan through in situ proliferation, independent of bone marrow hematopoiesis. However, the developmental mechanisms that specify fetal-derived tissue-resident macrophages are poorly understood. Here, we have identified a novel cytokine regulating tissue-resident macrophage development using an IL7Ra-cre lineage tracing model. Adult tissue resident macrophages in the brain, skin, liver, and lung were extensively labeled by IL7ra-cre, in the absence of IL7ra message or protein expression. To gain insight into developmental expression of IL7ra, we profiled IL7Ra surface expression, mRNA expression, and IL7ra-cre-driven labeling across fetal myeloid development. We observed rapid upregulation of IL7ra surface expression during a limited window of tissue macrophage establishment, and dynamic regulation of IL7ra message and protein levels in monocyte precursors, suggesting that IL7ra regulated the transition from fetal liver monocytes into tissue macrophages. Blockade of the IL7R using a monoclonal antibody decreased cellularity of liver, lung and skin tissue resident macrophages at birth, and increased cellularity of fetal liver monocytes. These data suggest that late in gestation, IL7Ra is upregulated as fetal monocytes exit the fetal liver and differentiate into macrophages, and provides evidence that IL-7R signaling regulates fetal tissue-resident macrophage development. Ongoing work addresses the function of IL-7 signaling in myeloid development, and the function of IL-7R marked macrophages in adult immunity.

SummaryA model of hematopoietic development wherein multipotentiality is conserved until segregation of myeloid and lymphoid potential has recently been challenged, proposing that megakaryocyte/erythrocyte (MegE) potential is lost in Flk2/Flt3-expressing early progenitors. Here, we used sensitive in vivo approaches to quantitatively and kinetically assess the MegE potential of hematopoietic stem cells and various Flk2+ early progenitors and compared it with the MegE potential of downstream committed myeloid and lymphoid progenitors and with their ability to give rise to mature myelomonocytic and lymphoid cells. We demonstrate that Flk2+ early progenitors retain MegE potential in vivo both at the population and clonal levels. These results indicate that Flk2 expression by early progenitors is not at the expense of full multipotency and support the current model of hematopoietic development with segregation of myeloid and lymphoid lineages from multipotent progenitors.