Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells

Abstract

To test the hypothesis that aging has negative effects on stem-cell homing and engraftment, young or old C57BL/6 bone marrow (BM) cells were injected, using a limiting-dilution, competitive transplantation method, into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age, the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly, the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However, the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage, indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes, erythrocytes, and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively, these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients.

Figure 1.

Reduced homing capacity of HPCs with donor and recipient age. Lethally irradiated young or old Ly-5.1 mice were injected with 2 × 10⁶ old or young Ly-5.2 donor cells. Three hours later, HPCs that had homed to the BM (□) or spleen (▪) were measured by hematopoietic colony formation in vitro. Shown are the mean (± SEM) percentages of donor-derived HPCs recovered per organ, relative to numbers injected. Data are pooled from 3 independent experiments with 3 to 5 mice per group. Differences between young cells injected into young recipients and either young cells transplanted into old recipients or old cells transplanted into young recipients are significant (P < .05).

Figure 2.

Experimental design for competitive repopulation studies. Lethally irradiated young or old Ly-5.1 mice were intravenously injected with 3 × 10⁶ old or young Ly-5.2 BM cells. Twenty-four hours later, marrow from the primary recipients was harvested to assay the number of Ly-5.2⁺ stem cells that had homed there after transplantation. The frequencies of CRUs in the initial BM suspensions and in the BM of primary mice after homing were determined by competitive repopulation of 2 sets of Ly-5.1 mice that underwent transplantation with graded numbers of fresh or homed BM cells admixed with 2 × 10⁵ Ly-5.1 competitor BM cells. Donor (Ly-5.2)-derived lymphoid and myeloid cells in PB were then measured at 5, 10, 17, and 26 weeks after transplantation. CRU frequencies were calculated from the proportions of negative mice in each cell-dose group, as described in “Materials and methods.”

Figure 3.

Age-related decline in the homing efficiency of murine HSCs. Lethally irradiated young or old Ly-5.1 mice were intravenously injected with 2-3 × 10⁶ old or young Ly-5.2 donor cells, as depicted in Figure 2. The number of CRUs recovered from the BM 24 hours later was determined by limiting-dilution competitive repopulation assays conducted in young secondary recipients assessed for donor engraftment at 5, 10, 17, and 26 weeks after transplantation (Tables 1, 2). The homing efficiency of HSCs was calculated by dividing the number of CRUs recovered in the primary BM 24 hours after transplantation by the number of CRUs initially injected and then multiplying by 100%. Three different transplantation groups—young cells into young recipients (□), young cells into old recipients (), and old cells into young recipients (▪)—are depicted. Data for young-to-old and old-to-young groups are derived from the experiments described in Tables 1 and 2. Data for the young-to-young group, though collected contemporaneously with those for the other transplantation groups, were reported previously by Szilvassy et al and are shown for comparison. All values are mean ± SEM, and statistically significant differences are designated with an asterisk (P < .05).

Figure 4.

Effects of aging and previous transplantation on the proliferation potential of CRUs. After determination of the CRU frequency in fresh and homed BM (Tables 1, 2), it was possible to retrospectively identify mice that had been injected with less than 0.3 CRU and in which the lymphoid and the myeloid compartments were subsequently repopulated with donor stem cells. On the basis of Poisson statistics, it is 95% probable that such mice were engrafted with a single HSC. Clone sizes generated by single, fresh (□) or homed () CRUs as a function of donor and recipient age are represented as the mean ± SEM percentage of donor (Ly-5.2+)-derived PB leukocytes assessed 5 to 26 weeks after transplantation (4-8 mice per group from 2 pooled experiments). Note that data for the young-to-young group were collected contemporaneously with those for the other groups but were, in part, reported in Szilvassy et al.

Figure 5.

Effect of aging and transplantation on the differentiation potential of CRUs. Mice that had been repopulated with a single, fresh (□) or homed () CRU were identified as described in the Figure 4 legend and in “Materials and methods.” The lineage composition of clones generated by single, young, or old HSCs in old or young hosts was defined as the proportion of donor (Ly-5.2⁺)-derived leukocytes expressing markers for B (CD45R/B220⁺) or T (CD90/Thy-1.2⁺) lymphocytes or for myeloid (Gr-1/Ly6G⁺ and Mac-1/CD11b⁺) cells. Values shown for the 3 combinations of variably aged donors and recipients represent the mean ± SEM of 2 pooled experiments with 3 to 6 mice per group. Note that data for the young-to-young group were collected contemporaneously with those for other groups but were, in part, reported in Szilvassy et al.

Figure 6.

Differential contribution of single BM CRU to hematopoietic engraftment. Recipient mice that were repopulated with a single, fresh (□) or homed () CRU were identified as described in the Figure 4 legend and in “Materials and methods.” The differential contribution of individual CRUs to engraftment was defined as the proportion of all circulating B (CD45R/B220⁺) or T (CD90/Thy-1.2⁺) lymphocytes or myeloid (Gr-1/Ly6G⁺ and Mac-1/CD11b⁺) cells that were Ly-5.2⁺. Values shown for the 3 combinations of variably aged donors and recipients represent the mean ± SEM of 2 pooled experiments with 3 to 6 mice per group. Note that data for the young-to-young group were collected contemporaneously with those for other groups but were, in part, reported in Szilvassy et al.

Figure 7.

Differential engraftment kinetics of young or old BM cells in old or young recipients. 2 × 10⁶ young (⋄) or old (▪) Ly-5.2 BM cells were injected into lethally irradiated young mice, or 3 × 10⁶ young Ly-5.2 BM cells were injected into lethally irradiated old Ly-5.1 mice (○). Shown are the mean ± SEM number of PB leukocytes (A), erythrocytes (B), and platelets (C) counted on the indicated days after transplantation (pooled data from 2 experiments with 10 mice per group). The absence of error bars for specific data points indicates that fewer than 3 animals were available for analysis. Note that the time after transplantation is not depicted on a linear scale. Shaded areas indicate normal ranges of blood cell counts in age-matched B6.SJL control mice. Data for the young-to-young group were collected contemporaneously with those of other groups but were, in part, reported in Szilvassy et al.