American Society of Hematology

Figure 2.

$Figure 2. Analysis of telomere length and replicative capacity during serial transplantation of HSCs from telomerase-deficient mice. (A) At each stage of transplantation, approximately 2000 HSCs were purified via FACS from a total of 1 to 2 mice and cytospun onto glass slides. All HSC samples were collected at 15 months (mTR+/+ donor) or 7 months (mTR–/– donor) after the initial transplantation. Detection of telomeres by FISH was performed using an FITC-tagged peptide nucleic acid telomeric probe as previously described.14 Individual interphase nuclei are indicated by arrowheads and the size scale (5 μm; bottom right) is shown in the top left panel. Original magnification, × 400. (B) The fluorescent signal intensity was measured for 25 nuclei at each stage of transplantation using a Zeiss confocal microscope. During serial transplantation of HSCs from both mTR–/– and mTR+/+ mice, the change in fluorescent signal intensity was significant (P < .05) for all stages except 3° → 4° for mTR+/+ HSCs (P = .21; Student t test). (C) The average frequency of donor-derived HSCs from long-term (> 4 months) reconstituted mice (n = 5) was measured at each stage of serial transplantation. The HSC frequency is shown relative to the fraction of donor-derived cells. Error bars (standard deviation) are shown.All mTR+/+ and mTR–/– mice used in this analysis and FISH analysis of telomere length were also used in the analysis of frequency of donor-derived BM cells (Figure 1). (D-E) FISH analysis of telomere length and analysis of donor-derived HSC frequency were performed during serial transplantation of HSCs from mTERT+/+ mice and mTERT–/– mice as described above.At least 4 mice were used in the assessment of donor HSCs at each stage of transplantation. Signal intensity was significantly diminished in HSCs from 1° recipients and 2° recipients that were reconstituted with mTERT–/– HSCs compared with 1° (P = .006) and 2° (P = .009) recipient mice reconstituted with mTERT+/+ HSCs. The analysis of telomere signal intensity and HSC frequency for the 2° mTERT–/– HSC donor and successive 3° and 4° recipients in which telomere length and replicative life span were observed to increase are indicated by the open circle and dashed line. Error bars show SD. (F) HSCs from 2° recipients of either mTR+/+ or mTR–/– HSCs were FACS sorted into media containing 10% fetal bovine serum, interleukin-3 (IL-3), IL-6, IL-11, and Steel factor (all 10 ng/mL) and cultured for 5 days. Metaphase spreads were then prepared using standard procedures, and telomeres were detected using FISH. End-to-end-chromosome fusions are indicated by the arrowheads. Original magnification, × 1009,$

Analysis of telomere length and replicative capacity during serial transplantation of HSCs from telomerase-deficient mice. (A) At each stage of transplantation, approximately 2000 HSCs were purified via FACS from a total of 1 to 2 mice and cytospun onto glass slides. All HSC samples were collected at 15 months (mTR^+/+ donor) or 7 months (mTR^–/– donor) after the initial transplantation. Detection of telomeres by FISH was performed using an FITC-tagged peptide nucleic acid telomeric probe as previously described.¹⁴ Individual interphase nuclei are indicated by arrowheads and the size scale (5 μm; bottom right) is shown in the top left panel. Original magnification, × 400. (B) The fluorescent signal intensity was measured for 25 nuclei at each stage of transplantation using a Zeiss confocal microscope. During serial transplantation of HSCs from both mTR^–/– and mTR^+/+ mice, the change in fluorescent signal intensity was significant (P < .05) for all stages except 3° → 4° for mTR^+/+ HSCs (P = .21; Student t test). (C) The average frequency of donor-derived HSCs from long-term (> 4 months) reconstituted mice (n = 5) was measured at each stage of serial transplantation. The HSC frequency is shown relative to the fraction of donor-derived cells. Error bars (standard deviation) are shown.All mTR^+/+ and mTR^–/– mice used in this analysis and FISH analysis of telomere length were also used in the analysis of frequency of donor-derived BM cells (Figure 1). (D-E) FISH analysis of telomere length and analysis of donor-derived HSC frequency were performed during serial transplantation of HSCs from mTERT^+/+ mice and mTERT^–/– mice as described above.At least 4 mice were used in the assessment of donor HSCs at each stage of transplantation. Signal intensity was significantly diminished in HSCs from 1° recipients and 2° recipients that were reconstituted with mTERT^–/– HSCs compared with 1° (P = .006) and 2° (P = .009) recipient mice reconstituted with mTERT^+/+ HSCs. The analysis of telomere signal intensity and HSC frequency for the 2° mTERT^–/– HSC donor and successive 3° and 4° recipients in which telomere length and replicative life span were observed to increase are indicated by the open circle and dashed line. Error bars show SD. (F) HSCs from 2° recipients of either mTR^+/+ or mTR^–/– HSCs were FACS sorted into media containing 10% fetal bovine serum, interleukin-3 (IL-3), IL-6, IL-11, and Steel factor (all 10 ng/mL) and cultured for 5 days. Metaphase spreads were then prepared using standard procedures, and telomeres were detected using FISH. End-to-end-chromosome fusions are indicated by the arrowheads. Original magnification, × 1009,

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