Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion

Abstract

Aging of the hematopoietic stem cell compartment is believed to contribute to the onset of a variety of age-dependent blood cell pathophysiologies. Mechanistic drivers of hematopoietic stem cell (HSC) aging include DNA damage accumulation and induction of tumor suppressor pathways that combine to reduce the regenerative capacity of aged HSCs. Such mechanisms do not however account for the change in lymphoid and myeloid lineage potential characteristic of HSC aging, which is believed to be central to the decline of immune competence and predisposition to myelogenous diseases in the elderly. Here we have prospectively isolated functionally distinct HSC clonal subtypes, based on cell surface phenotype, bearing intrinsically different capacities to differentiate toward lymphoid and myeloid effector cells mediated by quantitative differences in lineage priming. Finally, we present data supporting a model in which clonal expansion of a class of intrinsically myeloid-biased HSCs with robust self-renewal potential is a central component of hematopoietic aging.

Fig. 1.

Prospective identification of lineage-biased hematopoietic stem cells. (A) Representative FACS plot of lineage⁻Sca1⁺c-kit⁺ cells separated based on CD34 and flt3 expression and subsequently investigated for cell surface expression of Slamf1. (B) FACS purification of Slamf1^high and Slamf1^low HSCs. (C) Slamf1^high and Slamf1^low HSCs were isolated and competitively transplanted at 10 cells or 180 cells per mouse. Bars (Upper) represent the overall donor chimerism. Bars (Lower) show lineage chimerism ratios of individual recipients of Slamf1^high (Upper) and Slamf1^low (Lower) HSCs 20 or 17 weeks, respectively, posttransplantation. (D) Quantitative real-time PCR data of a panel of myeloid and lymphoid lineage specification genes on FACS-sorted Slamf1^high and Slamf1^low HSCs. Expression is shown relative to the expression of beta actin. Results represent mean (SD) values from three independent experiments, with qRT-PCR analysis in each experiment performed in duplicate.

Fig. 2.

Slamf1^high and Slamf1^low HSCs differentially regenerate primitive hematopoietic subsets. (A) Representative histograms and (B) average donor contribution to the primitive BM stem and multipotent progenitor cell compartment of mice competitively transplanted with either 180 Slamf1^high (Left) or Slamf1^low HSCs (Right) analyzed 20 weeks posttransplantation. In all panels, n = 4 recipients for each of Slamf1^high and Slamf1^low HSC donors. Error bars denote SEM.

Fig. 3.

Slamf1^high myeloid-biased HSCs predominate the stem cell pool with age. (A) Lineage potential of aged HSCs reveal myeloid bias in vivo. Either 10 or 50 LSKCD34⁻flt3⁻ HSCs from 21- to 24-month-old donors, and 50 LSKCD34⁻flt3⁻ HSCs from 3-month-old donors were competitively transplanted and peripheral blood chimeric ratios of B, T, and myeloid cells was determined 16–20 weeks posttransplantation. (B) Test-cell lineage chimerism ratios in serially transplanted mice in primary (1°), secondary (2°), and tertiary (3°) recipients. Columns depict individual recipients. (C) Representative FACS plots showing the primitive HSC compartment stained for Slamf1 expression in young adult (4 months), midaged (12 months), and old (24 months) mice. Cells were pregated on live (PI-negative), lineage-negative cells that were discriminated for doublets. (D) Pie charts showing average contribution to each of the Slamf1 populations within the LSKCD34⁻flt3⁻ compartment (n = 3 for each age). (E) Representative FACS plots of cell cycle analysis on purified Slamf1^high HSCs, Slamf1^low HSCs, and Lin⁻Sca1⁻ckit⁺ myeloid progenitors with the average frequency of cells in S/G₂/M shown with SD (n = 5 mice for each population).

Fig. 4.

Slamf1^high and Slamf1^low HSCs maintain lineage potential during aging. (A) A total of 125 Slamf1^high or Slamf1^low HSCs were isolated from either young adult (4 months) or old (23 months) mice and competitively transplanted against 2 × 10⁵ unfractionated BM cells. Peripheral blood analysis was performed 17 weeks posttransplantation. Black bars (Upper) represent total donor chimerism; colored bars (Lower) show lineage chimerism ratios in individual recipients. (B) Average total peripheral blood donor chimerism for the experiments presented in A is shown, with error bars denoting SEM.