Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells

Abstract

Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening ( approximately 7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.

Figure 1

FIGE analysis of TRF length for hematopoietic cells from different mus musculus strains. DNA was isolated from bone marrow cells (BM) and splenocytes (SP) from young adult mice, as well as murine embryonic fibroblasts (MEF) established from the BA strain, and digested with the restriction enzymes as described (see Materials and Methods). 1 μg of each DNA sample was resolved in a 0.75% agarose gel by FIGE (pulse conditions, 0.8 V forward and 0.4 V reverse for 12 h). The gel was dried and the DNA hybridized to a ³²P-end labeled telomeric oligomer as described (see Materials and Methods). Sizes of molecular weight standards (in kb) are shown on the left.

Figure 2

Analysis of cell cycle status of HSCs after transplantation. (A) Detection of HSCs in the S/G2/M phase of the cell cycle by FACS^® analysis. Bone marrow was isolated from adult BA mice and primary recipients (1 mo after transplant) and immunostained for HSC detection with Hoechst dye (see Materials and Methods). Shown are sample side scatter (y-axis) and Hoechst fluorescence (x-axis) density plots of HSCs (Sca1⁺c-kit⁺Lin⁻Thy1.1^lo cells) from a typical adult BA mouse (top) and a primary recipient reconstituted with 3 × 10⁵ bone marrow cells (∼30 HSCs) (bottom). Boxes indicate the subpopulation of HSCs in the S/G2/M phase of the cell cycle, showing the percentage of S/G2/M phase HSCs. (B) Analysis of the frequency of cycling HSCs. The average relative frequency of HSCs in S/G2/M for adult BA mice and primary recipients reconstituted with ∼3 × 10⁵ or ∼3 × 10⁷ bone marrow cells (30 and 3,000 HSCs, respectively) is shown at 4 wk after transplant (top) and 4 mo after transplant (bottom). Error bars (standard deviation) and P value (Student's t test) are shown.

Figure 2

Analysis of cell cycle status of HSCs after transplantation. (A) Detection of HSCs in the S/G2/M phase of the cell cycle by FACS^® analysis. Bone marrow was isolated from adult BA mice and primary recipients (1 mo after transplant) and immunostained for HSC detection with Hoechst dye (see Materials and Methods). Shown are sample side scatter (y-axis) and Hoechst fluorescence (x-axis) density plots of HSCs (Sca1⁺c-kit⁺Lin⁻Thy1.1^lo cells) from a typical adult BA mouse (top) and a primary recipient reconstituted with 3 × 10⁵ bone marrow cells (∼30 HSCs) (bottom). Boxes indicate the subpopulation of HSCs in the S/G2/M phase of the cell cycle, showing the percentage of S/G2/M phase HSCs. (B) Analysis of the frequency of cycling HSCs. The average relative frequency of HSCs in S/G2/M for adult BA mice and primary recipients reconstituted with ∼3 × 10⁵ or ∼3 × 10⁷ bone marrow cells (30 and 3,000 HSCs, respectively) is shown at 4 wk after transplant (top) and 4 mo after transplant (bottom). Error bars (standard deviation) and P value (Student's t test) are shown.

Figure 3

FIGE analysis of TRF length of bone marrow cells during serial HSC transplantation. (A) Southern blot analysis of TRF length by FIGE. In this sample analysis, all primary recipients and two secondary recipients, represented by the rightmost two lanes, were reconstituted with 100 pure HSCs. The other two secondary recipients were reconstituted with 2 × 10⁶ bone marrow cells. Whole bone marrow cells were isolated from adult BA mice and recipient mice and digested with restriction enzymes as described (see Materials and Methods). 0.5 μg of each digested DNA sample was resolved in a 0.75% agarose gel by FIGE (pulse conditions, 0.8 V forward and 0.4 V reverse for 12 h). The bone marrow sample of the donor mouse in each round of transplantation is indicated by an arrow (↓) at the top. The gel was dried and the DNA hybridized to a ³²P-end labeled telomeric oligomer as described (see Materials and Methods, and reference 23). Sizes of molecular weight standards (in kb) are shown on the left. (B) Measurement of mean TRF length during serial transplantation. The mean TRF length was calculated as described previously (references 23, 29) for a total of 9 sibling adult BA mice, 11 primary recipients, and 10 secondary recipients and averaged for all experiments. Error bars (standard deviation) and P values (Student's t test) are shown.

Figure 3

FIGE analysis of TRF length of bone marrow cells during serial HSC transplantation. (A) Southern blot analysis of TRF length by FIGE. In this sample analysis, all primary recipients and two secondary recipients, represented by the rightmost two lanes, were reconstituted with 100 pure HSCs. The other two secondary recipients were reconstituted with 2 × 10⁶ bone marrow cells. Whole bone marrow cells were isolated from adult BA mice and recipient mice and digested with restriction enzymes as described (see Materials and Methods). 0.5 μg of each digested DNA sample was resolved in a 0.75% agarose gel by FIGE (pulse conditions, 0.8 V forward and 0.4 V reverse for 12 h). The bone marrow sample of the donor mouse in each round of transplantation is indicated by an arrow (↓) at the top. The gel was dried and the DNA hybridized to a ³²P-end labeled telomeric oligomer as described (see Materials and Methods, and reference 23). Sizes of molecular weight standards (in kb) are shown on the left. (B) Measurement of mean TRF length during serial transplantation. The mean TRF length was calculated as described previously (references 23, 29) for a total of 9 sibling adult BA mice, 11 primary recipients, and 10 secondary recipients and averaged for all experiments. Error bars (standard deviation) and P values (Student's t test) are shown.

Figure 4

FISH analysis of telomere length of HSCs from adult BA mice and secondary recipients. (A) Confocal microscope image of individual HSC nuclei after hybridization to a fluorescent telomeric probe. 1,000 HSCs were isolated from the pooled bone marrow from two BA mice and two secondary recipients by FACS^®, cytospun onto glass slides, and fixed as described (see Materials and Methods). The telomeres were detected by FISH using an FITC-conjugated PNA telomeric oligomer as described by Lansdorp et al. (reference 33). Individual interphase nuclei are indicated by arrowheads. Image was collected using a 60× objective. The size scale (in μm) is indicated in the lower left. (B) Fluorescent signal intensity measurements of HSC nuclei. After FISH, the fluorescent telomeric signal intensity was calculated and corrected for background for 30 well-isolated individual HSC nuclei from both adult BA mice and secondary recipients. The mean fluorescent signal intensity and corresponding standard deviation are shown. P value was calculated by Student's t test.

Figure 4

FISH analysis of telomere length of HSCs from adult BA mice and secondary recipients. (A) Confocal microscope image of individual HSC nuclei after hybridization to a fluorescent telomeric probe. 1,000 HSCs were isolated from the pooled bone marrow from two BA mice and two secondary recipients by FACS^®, cytospun onto glass slides, and fixed as described (see Materials and Methods). The telomeres were detected by FISH using an FITC-conjugated PNA telomeric oligomer as described by Lansdorp et al. (reference 33). Individual interphase nuclei are indicated by arrowheads. Image was collected using a 60× objective. The size scale (in μm) is indicated in the lower left. (B) Fluorescent signal intensity measurements of HSC nuclei. After FISH, the fluorescent telomeric signal intensity was calculated and corrected for background for 30 well-isolated individual HSC nuclei from both adult BA mice and secondary recipients. The mean fluorescent signal intensity and corresponding standard deviation are shown. P value was calculated by Student's t test.

Figure 5

Hypothetical model to explain the reduction in telomere length during serial transplantation of HSCs. In adult mice, HSCs are predominantly in a nonstressed, resting state (indicated by the small arrows) where they function mainly to sustain homeostatic levels of hematopoietic cells. Since telomere shortening is primarily dependent on cell division (reference 41), there is no discernible telomere shortening over a time period of a few months in adult BA mice (data not shown). After transplantation into primary recipients, the cycling activity of the donor HSCs increases (thicker arrows), primarily to allow reconstitution of all hematopoietic lineages including the HSC pool. Consequently, a modest amount of telomere shortening occurs. The cycling activity of the donor HSCs is still elevated when they are once again isolated for a second round of transplantation. This results in a considerable drop in reconstitutive capacity because of poor engraftment in the secondary recipients and a further increase in the rate of HSC turnover (thickest arrows) so that regeneration of the HSC pool and hematopoietic reconstitution can be completed once again. It is also possible that, after transplantation, HSCs from primary recipients may be more prone to cell death or replicative exhaustion than HSCs taken directly from adult mice, which could also influence the rate of HSC division in secondary recipients. Thus, during hematopoietic reconstitution in the secondary recipients, telomeres in the HSCs undergo an even greater degree of shortening.