Hematopoietic stem cells: concepts, definitions, and the new reality

Abstract

Hematopoietic stem cell (HSC) research took hold in the 1950s with the demonstration that intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production. Attempts to quantify the cells responsible led to the discovery of serially transplantable, donor-derived, macroscopic, multilineage colonies detectable on the spleen surface 1 to 2 weeks posttransplant. The concept of self-renewing multipotent HSCs was born, but accompanied by perplexing evidence of great variability in the outcomes of HSC self-renewal divisions. The next 60 years saw an explosion in the development and use of more refined tools for assessing the behavior of prospectively purified subsets of hematopoietic cells with blood cell-producing capacity. These developments have led to the formulation of increasingly complex hierarchical models of hematopoiesis and a growing list of intrinsic and extrinsic elements that regulate HSC cycling status, viability, self-renewal, and lineage outputs. More recent examination of these properties in individual, highly purified HSCs and analyses of their perpetuation in clonally generated progeny HSCs have now provided definitive evidence of linearly transmitted heterogeneity in HSC states. These results anticipate the need and use of emerging new technologies to establish models that will accommodate such pluralistic features of HSCs and their control mechanisms.

Figure 1

Historical sequence of methods used to detect and quantify mouse HSCs in vivo. (A) Development of LDA approaches to identify the transplantable cells that can rescue mice permanently from radiation-induced lethality by regenerating the inactivated blood-forming system of the host. (B) Genetic approaches to track HSCs by detection of their long-lived clonal outputs in transplanted recipients. Random sites of vector integration into the DNA of the regenerated progeny of transduced transplanted cells were the first unique DNA identifiers used.^, More recently, uptake of a single vector encoding a short unique “barcode” sequence from a diverse vector library has been used as a clonal tracking strategy.^- (C) Advances in LTRC purification enabling single-cell transplants to reveal the diversity of long-term clonal white blood cell outputs of individual HSCs as previously suggested by limiting dilution transplants and vector-marking experiments. Data shown are for purified Rho⁻SP⁺ LTRCs (HSCs) adapted from Uchida et al (with permission from Experimental Hematology.) BM, bone marrow; L+M, lymphoid + myeloid; mo, months; LAM-PCR, linear amplification mediated polymerase chain reaction; SP, side population; UV, ultraviolet; WBCs, white blood cells.

Figure 2

Hierarchical models of HSC self-renewal and differentiation. Increasingly complex models of HSC differentiation hierarchies reflecting historically changing information about the numbers and types of lineages obtained from single-mouse “HSCs” in various in vitro and in vivo systems. (A) Initial view showing all lymphoid and all myeloid potentialities as the first lineage groupings to be segregated. (B) A more detailed view of the compartmentalization of intermediate, lineage-restricted progenitor subsets based on their behavior in short-term in vitro colony assays and properties allowing their separate isolation. (C) Map of early lineage restriction events based on the predominant functional activities of particular cell phenotypes (redrawn from Figure 1 of Seita and Weissman. (D) Concept of multiple origins of GM cells derived from examination of the lymphoid and myeloid activities elicited from different subsets of mouse hematopoietic cells in vitro. The hierarchy shown in this case has been adapted from Figure 1E of Kawamoto et al. (E) Differences in lineage potentialities exhibited by LTRCs with durable (serially transplantable) self-renewal activity (HSCs) as reported by Dykstra et al and Benz et al. BFU-E, burst-forming unit-erythroid; E, erythroid; G, granulocyte; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; Mk, megakaryocyte; NK, natural killer; DC, dendritic cell; T, T-lymphoid; B, B-lymphoid; STRC, short-term repopulating cell.

Figure 3

HSC viability, mitogenesis and self-maintenance can be separately regulated by different external cues. (A) Schema showing the multiple responses that can affect HSC numbers. (B) Demonstration that different extrinsic conditions can separately regulate the survival, proliferation, and self-renewal responses of highly purified, durable LTRCs assessed in single-cell cultures. Shown are examples of different in vitro conditions in which full LTRC activity of the surviving input cells was maintained for 7 days either in the absence of cell death or division, or under different conditions that variably kept the input cells alive but maximally stimulated mitogenesis of the survivors. Also shown are conditions that supported full survival and mitogenesis of the same input cells, but with substantial loss of their original LTRC activity (redrawn from data published in Wohrer et al with permission). ESLAM, EPCR⁺CD48⁻CD150⁺; FBS, fetal bovine serum; EP, erythropoietin; SF, Steel factor; CM, UG26 stromal cell conditioned medium; LDA, limiting dilution assay.