Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Stem Cell Compartment

Abstract

Aging is linked to functional deterioration and hematological diseases. The hematopoietic system is maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC compartment is thought to be a key mechanism underlying age-related hematopoietic perturbations. Using single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, we determined the age-related functional changes to the HSC compartment using over 400 single-cell transplantation assays. Notably, MyRP frequency increased dramatically with age, while multipotent HSCs expanded modestly within the bone marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in secondary recipients. We have termed this cell type latent-HSCs, which appear exclusive to the aged HSC compartment. These results question the traditional dogma of HSC aging and our current approaches to assay and define HSCs.

Keywords: HSC; aging; clonal analysis; hematopoiesis; hematopoietic stem cell; multipotency; self-renewal; single cell; stem cell aging.

Graphical abstract

Figure 1

The Phenotypic HSC Compartment Changes with Age (A) Representative flow cytometric data of young and aged bone marrow (BM): MPP, multipotent progenitor; LMPP, lymphoid-primed multipotent progenitor; Fr 1, fraction 1; Fr 2, fraction 2; Fr 3, fraction 3. (B) Frequency of the HSC/MPP population (left) and HSC subpopulations (right) in young and aged BM (as detailed in A). Dots represent individual mice, and horizontal lines indicate median ± SD. (C) Summary of primary and secondary transplantation experiments to test potential of young and aged single phenotypic HSCs. Single CD34⁻KSL, fraction 1, fraction 2, or fraction 3 cells were sorted from BM cells of Kusabira Orange (KuO) mice and were individually transplanted with 2 × 10⁵ BM competitor cells from Ly5.1/Ly5.2-F1 mice into lethally irradiated Ly5.2 mice. Chimerism of KuO⁺ neutrophils/monocytes, erythrocytes, platelets, B cells, and T cells in peripheral blood (PB) was analyzed at 2, 3, 4, 8, (12), 16, (20), and 24 weeks after primary transplantation. Secondary transplantation assays were performed by transferring 1 × 10⁷ whole BM cells from primary recipient mice. PB chimerism was analyzed 4, 12, 16, 20, (and 21–22) weeks in secondary recipients. (D) PB chimerism of individual single young and aged HSCs in a total of 421 primary recipients (as described in C), separated based on lineage output. (E) Estimated frequency of functional HSCs, CMRPs, MERPs, MkRPs, and “other” within the young and aged pHSC compartment, derived from single-cell transplantation assays (Table 1). CMRPs, MERPs, and MkRPs are subsets of MyRPs. “Non-reconstituting” denotes no PB reconstitution of KuO⁺ cells in primary recipients.

Figure 2

Functional Comparison of Young and Aged HSCs by Single-Cell Transplantation (A) Average chimerism of young and aged HSCs and MyRP/MySC subsets within primary recipients over 24 weeks. Functional cell types subdivided based on reconstitution duration into long-term (LT) or intermediate-term (IT) and short-term (ST) RCs. Data points indicate mean ± SD. (B) Estimated frequency of each functional cell type (HSCs, CMRPs/CMSCs, MERPs/MESCs, MkRPs/MkSCs, and “other”) divided into LT/IT- and ST-repopulating subsets within the young and aged pHSC compartment, derived from single-cell transplantation assays (Table 1). (C) PB chimerism of individual single young and aged LT- and IT-HSCs in primary and secondary recipients. Blood lineage chimerism in primary recipients at 24 weeks was compared using an unpaired t test (^∗p < 0.05).

Figure 3

A Subset of Aged MyRPs Display a Latent-HSC Phenotype (A) Average chimerism kinetics of young and aged LT-HSCs (n = 24 and 6, respectively), IT-HSCs (n = 24 and 10, respectively), LT-CMSCs (n = 1 and 0, respectively), IT-CMSCs (n = 7 and 3, respectively), and latent-HSCs (n = 0 and 7, respectively) using all single-cell transplantation datasets (including our data published in Yamamoto et al., 2013). Each line represents the frequency of donor-derived cells in the blood of a single recipient after primary transplantation and secondary transplantation. (B) Representative PB and BM chimerism in primary and secondary recipients for latent-HSCs (1–3), young LT-HSCs (4), and aged LT-HSCs (5). The frequency of KuO⁺ phenotypic stem and progenitor cells within the BM was determined at 24 weeks after primary transplantation and 20 weeks after secondary transplantation. Chimerism of KuO⁺ phenotypic HSCs, fraction 1, fraction 2, fraction 3 (highlighted in purple), MPPs, LMPPs (highlighted in blue), CMPs, GMPs, MEPs, MkPs (highlighted in gray), or CLPs (highlighted in green) is show in the bar graph. n.d. denotes no data. BM cells from mouse 1-1 and 1-2 and mouse 5-1 and 5-2 were pooled and analyzed. (C) Schematic of age-related changes to the mouse HSC compartment. Circle size/number represents the frequency of each cell type per 10⁶ BM cells. With age, the pHSC compartment expands ∼10-fold, largely due to a large increase in MyRPs/MySCs and non-reconstituting cells. Absolute numbers of functional HSCs (fHSCs) only increases modestly and therefore become less frequent within the pHSC compartment. Cumulatively, this leads to the reduced function of the HSC compartment including loss of lymphoid (B and T cell) lineage output. However, the aged pHSC compartment also contains latent HSCs, which display myeloid-restricted output in primary recipients but multipotent (five blood-lineage) output in secondary recipients. We have not detected latent-HSCs in the young pHSC compartment.