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. 2016 Oct 17;213(11):2259-2267.
doi: 10.1084/jem.20160168. Epub 2016 Oct 10.

Progressive Alterations in Multipotent Hematopoietic Progenitors Underlie Lymphoid Cell Loss in Aging

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Free PMC article

Progressive Alterations in Multipotent Hematopoietic Progenitors Underlie Lymphoid Cell Loss in Aging

Kira Young et al. J Exp Med. .
Free PMC article

Abstract

Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging.

Figures

Figure 1.
Figure 1.
MPP composition is altered with aging. (A) FACS gating showing frequency of HSC and MPP subsets in representative 2-mo, 14-mo, and 28-mo mice. The inset table defines surface markers used for cell isolation. FSC, forward side scatter. (B) Frequency of HSC and MPP subsets in whole BM identified by FACS analysis. Bars denote the mean of 2–4 mo (n = 25), 6 mo (n = 5), 8 mo (n = 7), 12 mo (n = 5), 14 mo (n = 3), and 28 mo (n = 10) assessed in five independent experiments. (C) FACS gating showing frequency of CD150hi, CD150int, and CD150lo HSCs in representative 2-mo and 28-mo mice. (D) Frequency of CD150hi, CD150int, and CD150lo HSCs in whole BM identified by FACS analysis. Error bars denote mean ± SEM of 2–4 mo (n = 25), 6 mo (n = 5), 8 mo (n = 7), 12 mo (n = 5), 14 mo (n = 3), and 28 mo (n = 10) assessed in five independent experiments. (E) Frequency of CLP and CDP subsets in BM identified by FACS analysis. Bars denote the mean of 2–4 mo (n = 25), 6 mo (n = 5), 8 mo (n = 7), 12 mo (n = 5), 14 mo (n = 3), and 28 mo (n = 10) assessed in five independent experiments. (B, D, and E) P-values were generated by one-way ANOVA with Dunnett’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. HSCLT, LT-HSC; HSCST, ST-HSC.
Figure 2.
Figure 2.
Increased cycling and loss of lymphoid priming in aged LMPP/MPP4. (A) Loading plot of principal components (PC) 1–3 from principal component analysis of 4-mo (n = 54) and 14-mo (n = 40) LMPP scRNA-seq data. Percent contribution of each principal component to total variation is shown. (B) Mean expression of G1/S transition genes (x axis) and S + G2/M genes (y axis) in each 4-mo (n = 54) and 14-mo (n = 40) LMPP scRNA-seq library. Boxed groups represent predicted G0, G1, and S + G2/M cell cycle phases. (C) Frequency of MPP4 in G0/G1, S, or G2/M defined by in vivo EdU incorporation and FACS analysis. (D) Frequency of LT-HSCs, MPP2, and MPP3 in G0/G1, S, or G2/M defined by in vivo EdU incorporation and FACS analysis. (C and D) Bars denote the mean of 2 mo (n = 3), 8 mo (n = 3), 14 mo (n = 2), and 28 mo (n = 2) in two independent experiments. (E) Enrichment of lymphoid-priming, myeloid-priming, MkP, CLP, preGM, and preCFU-E gene signatures in 4-mo (n = 54) versus 14-mo (n = 40) LMPP scRNA-seq data. NES, normalized enrichment score. (F) Mean expression of lymphoid-priming driver genes in each scRNA-seq library. Bars denote the mean of 4 mo (n = 54) and 14 mo (n = 40). (C, D, and F) P-values were generated by unpaired, two-tailed Student’s t tests. *, P < 0.05; ***, P < 0.001.
Figure 3.
Figure 3.
Novel in vitro culture conditions supporting concurrent B cell and myeloid cell production. (A) CFUs per 100 de novo isolated HSC and progenitor cell subsets plated into myeloerythroid-promoting M3434 (left) or preB-promoting M3630 (right) methylcellulose media. Results are shown as mean ± SEM of ST-HSCs (n = 3), MPP2 (n = 3), MPP3 (n = 3), MPP4 (n = 3), LMPPs (n = 4), CLPs (n = 4), and CDPs (n = 2) in two independent experiments. (B) Schematic of in vitro assay design. (C) CFUs per 100 LMPPs after 48-h culture with denoted cytokine cocktails. Results are mean ± SEM of n = 3 in three independent experiments. (Right) Representative CFU images. Bars, 200 µm. (D) CFUs per 100 input HSC and progenitor cell subsets after 48-h culture followed by plating in M3434 (left) or M3630 (right). Results are shown as mean ± SEM of ST-HSCs (n = 5), MPP2 (n = 3), MPP3 (n = 3), MPP4 (n = 3), LMPPs (n = 6), CLPs (n = 6), and CDPs (n = 3) in three independent experiments. GEMM, granulocyte, erythroid, macrophage, and megakaryocyte. (E) Representative FACS analysis and Wright-Giemsa staining (inset) of cells isolated from single colonies. Cell surface markers against macrophages (CD11b), granulocytes (Gr-1), and B cells (B220) were used. Bars, 10 µm. L, lymphocyte; Mo, macrophage; PMN, granulocyte.
Figure 4.
Figure 4.
Impaired lymphoid differentiation of aged LMPPs in vitro. (A) Schematic of single-cell assay design. (B) Frequency of wells found to contain one or more cells after 48-h culture of single LMPP cells. Dots represent a single plate of n = 96 single cells. Bars represent the mean of n = 2 independent experiments. (C) Number of cells counted per well after 48-h culture of single LMPP cells. Dots represent individual wells. Bars represent the mean of n = 288 collected in three independent experiments. (D) Frequency of BrdU+ cells after 48-h culture of LMPP cells by FACS analysis. Dots represent independent experiments, and bars represent the mean of n = 3. (E) Frequency of wells found to form CFUs after 48-h culture of single LMPP cells and plating in methylcellulose assays. Dots represent a single plate of n = 96 single cells. Bars represent the mean of n = 2 independent experiments. (B–E) P-values were generated by one-way ANOVA with Holm-Sidak’s multiple comparisons test. (F) Frequency of wells found to form CFU-preB/M, CFU-GM, or CFU-preB/M/G after 48-h culture of single LMPP cells and plating in methylcellulose assays. Results are shown as mean ± SEM of n = 2–4 in two independent experiments. P-values were generated by two-way ANOVA with Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5.
Figure 5.
Cell-autonomous in vivo lymphoid differentiation defect of aged LMPPs. (A) Schematic of experimental design for in vivo analysis of peripheral blood reconstitution by transplanted LMPP cells. (B) Frequency of donor-derived (CD45.2+) cells in peripheral blood of recipient mice at 1–8 wk after transplant by FACS analysis. Results are shown as mean ± SEM of 2-mo donor LMPPs (n = 7), 8-mo donor LMPPs (n = 9), and 28-mo donor LMPPs (n = 7) in three independent experiments. (C–F) Frequency of donor-derived B cells (CD45.2+ B220+), granulocytes (granulo; CD45.2+ CD11b+ Gr1hi), macrophages (macro; CD45.2+ CD11b+ Gr1lo), and T cells (CD45.2+ CD3+) in peripheral blood of recipient mice at 1–4 wk after transplant by FACS analysis. Results are shown as mean ± SEM of 2-mo donor LMPPs (n = 7), 8-mo donor LMPPs (n = 9), and 28-mo donor LMPPs (n = 7) in three independent experiments. P-values were generated by two-way ANOVA with Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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