Rossi et al. 10.1073/pnas.0503280102. |
Fig. 4.
Peripheral blood analysis to determine contribution of donor derived to B, T, and myeloid cells from recipients of young and old LT-HSCs. Representative flow cytometric profiles showing the staining and gating strategy used to determine donor contribution to peripheral B, T, and myeloid cells after transplantation. Cells were stained with fluorescence-conjugated antibodies as described in Materials and Methods and gated through SSC vs. Ter119 and PI to exclude erythroid cells and dead cells, then gating on donor cells (CD45.2 positive), followed by gating on B220+ B-cells, Mac1+ myeloid cells and TCRb + T-cells.
Supporting Figure 5
Fig. 5.
Phenotypic and functional analysis of young and old side population (SP) and KLSflk2-CD34-SP tip cells. (A) Representative flow cytometric plots of young and old BM stained for SP and KLS, flk2 and CD34 cell surface phenotype. SP cells were gated as shown (Left) and analyzed for cell surface phenotype (Right). Murine BM cells from young or old mice (n = 5 in each group) were stained with Hoechst 33342 as described (1) followed by cell surface staining with fluorescence-conjugated antibodies against c-kit, ScaI, flk2 and CD34, and lineage (CD3, CD4, CD8, Mac-1, Gr-1, B220, and Ter119). (B) Frequencies of KLS, flk2, and CD34 subsets exhibiting a SP phenotype in the BM of young and old mice. (C) Relative distributions of KLS, flk2, and CD34 subsets exhibiting a SP phenotype in the BM of young and old mice illustrating the changes in the cellular composition of the SP with age. (D) Representative flow cytometric plots of young (Left) and old (Right) BM showing gating strategy for KLSflk2-CD34-SP"tip" cells. Murine BM cells from young or old mice were stained with Hoechst 33342 as described (1) followed by cell surface staining with fluorescence-conjugated antibodies against c-kit, ScaI, flk2, CD34, and lineage (CD3, CD4, CD8, Mac-1, Gr-1, B220, and Ter119). Cells were gated through ForSc vs. lineage negative, followed by c-kit high vs. ScaI high, then flk2 negative vs. CD34 negative (as shown in Fig. 1A) and finally Hoechst as shown (gated in red). (E) Multilineage reconstitution (Left) and lineage potential (Right) from competitive transplant experiments of KLSflk2-CD34-SP tip cells from young or old C57BL/6 mice. Twenty KLSflk2-CD34-SP tip cells purified (double FACS sorted) from young or old CD45.2 donors were transplanted into congenic (CD45.1/CD45.2) lethally irradiated young recipients against 2 × 105 congenic (CD45.1) competitor BM cells. Peripheral blood analysis was done at the indicated time points and donor-derived repopulating ability is presented as the percent of total white blood cells (Left). Donor-derived contribution to lymphoid and myeloid lineages is presented as the percent of donor-derived B cell (B220+), T cell (TCRb+), and myeloid cells (Mac1+) (Right). In these experiments five of five recipients transplanted with young LT-HSCs, and five of five transplanted with old LT-HSCs, were multilineage reconstituted. Significantly higher levels of reconstitution between young and old donors (P < 0.05 by Student’s t test) are indicated by *. BM cells with the capacity to efflux the dye Hoechst 33342 (SP) are enriched for HSC (2). To assay the impact of aging on SP cells, we determined the frequency of these cells in young and old animals and found that cells with SP activity increased dramatically with age (4.6-fold, P = 0.001, A and B). Moreover, by combining cell surface staining with SP analysis we found that it was predominantly KLSflk2-CD34- cells within the SP that accounted for the expansion of this population with advanced age (A). Thus, similar to KLS cells (Fig. 1B), the cellular composition of the SP changes significantly with age (Fig. 5C). Taken together, these data underscore the importance of using additional markers to purify LT-HSCs from the KLS or SP subpopulations, as a comparison of LT-HSC phenotype/function based solely on cells with KLS or SP phenotype would, to a large extent, be confounded by the differential cellular composition of these compartment as a consequence of age. A recent study suggested that a highly purified population of long-term repopulating cells could be obtained by combining KLSCD34- surface marking together with the isolation of cells with the highest capacity to efflux Hoechst 33342 (so-called SP tip cells) (1). This finding prompted us to investigate whether stem cells isolated by this technique would also exhibit age-dependent functional decline. To this end, we isolated SP tip KLSCD34- cells from young and old donors (D), and competitively transplanted them into young hosts (E). Compared with their young counterparts, old SP tip KLSCD34- cells showed significantly reduced total reconstituting ability and a proportional decrease in their contribution to the B cell lineage at all time points measured (E). These experiments established that the competitive repopulating ability of SP tip KLSCD34- cells was not significantly different to cells isolated as KLSCD34-, and hence that the two populations in our hands were functionally equivalent.1. Matsuzaki, Y., Kinjo, K., Mulligan, R. C. & Okano, H. (2004) Immunity 20, 87-93.
2. Goodell, M. A., Brose, K., Paradis, G., Conner, A. S. & Mulligan, R. C. (1996) J. Exp. Med. 183, 1797-1806.
Supporting Figure 6
Fig. 6.
Aging is associated with a stem cell intrinsic decrease in CLPs and an increase in GMPs. (A) Representative flow cytometric profiles of committed myeloid progenitor cells from the BM of young and old mice showing CMPs, GMPs, and MEPs. (B) Representative flow cytometric profiles of CLP cells from the BM of young and old mice.
Supporting Figure 7
Fig. 7.
Expression profile of characterized and uncharacterized genes in aging LT-HSCs. Graphical representation of the 907 genes found to be age-regulated in old compared with young LT-HSCs showing the contribution of annotated and characterized genes compared with the contribution of uncharacterized genes in the total age-regulated set, the age-down-regulated set, and the age-up-regulated set.