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. 2011 Aug 18;118(7):1766-73.
doi: 10.1182/blood-2010-11-319632. Epub 2011 Jul 5.

Prolonged Self-Renewal Activity Unmasks Telomerase Control of Telomere Homeostasis and Function of Mouse Hematopoietic Stem Cells

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Prolonged Self-Renewal Activity Unmasks Telomerase Control of Telomere Homeostasis and Function of Mouse Hematopoietic Stem Cells

Sanja Sekulovic et al. Blood. .
Free PMC article

Abstract

Strategies for expanding hematopoietic stem cells (HSCs) could have significant utility for transplantation-based therapies. However, deleterious consequences of such manipulations remain unknown. Here we examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using a NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. We observed ~ 10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert(-/-) HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type (WT) or Tert(-/-) HSCs, suggesting that HSC replication posttransplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity on prolonged self-renewal stimulation.

Figures

Figure 1
Figure 1
Mouse HSCs have a reservoir of telomeres sufficient to sustain their self-renewal during several cycles of serial transplantation. (A) Experimental protocol in which whole bone marrow (WBM) cells from either WT or Tert−/− mice were transplanted into lethally irradiated primary recipients and their telomere length monitored at various time points post transplantation (PT). (B) The average telomere length of donor-derived WT (blue bars) or Tert−/− (red bars) BM cells 6 weeks, 3 and 9 months PT within primary recipients. Dotted lines indicate the telomere length of donor WT (blue) or Tert−/− (red) BM cells on the day of the primary transplantation. Error bars denote SD. Nwt = 5 and Ntert−/− = 5 at 6-week, 3- and 9-month time point analysis. (C) Serial transplantation protocol of either WT or Tert−/− WBM cells. (D) The average telomere length of donor-derived WT (blue bars) or Tert−/− (red bars) BM cells measured at the time of each transplantation. Dotted lines indicate the telomere length of donor WT (blue) or Tert−/− (red) BM cells on the day of the primary transplantation. Error bars denote SD. Nwt = 6; 6; 4 and Ntert−/− = 6; 4; 2 at 3-month PT time point analysis of 1°, 2°, and 3° recipients, respectively. (E) Percent donor reconstitution in PB of primary, secondary and tertiary recipients (generated as described in panel C) transplanted with unmanipulated WT (blue triangles) and Tert−/− (red triangles) BM cells. Each triangle represents an individual recipient. Each horizontal line represents the mean percent donor reconstitution of at least 3 recipients.
Figure 2
Figure 2
NA10hd effect on telomere maintenance and reconstitution activity of WT and Tert−/− HSCs. (A) Experimental protocol in which wbm cells from 5-FU pre-treated WT or Tert−/− mice were transduced with NA10hd and transplanted into lethally irradiated primary recipients. Six months later, donor-derived WBM cells from the primary recipients were transplanted into secondary recipients. (B) Average telomere lengths of donor-derived WT (blue bars) or Tert−/− (red bars) BM cells obtained 3 and 6 months PT from the primary recipients and 3 months PT from the secondary recipients. Dotted lines indicate the telomere length of donor WT (blue) or Tert−/− (red) BM cells before infection and primary transplantation. Error bars denote SD. Nwt = 2; 2; 2 and Ntert−/− = 2; 2; 3 at 3- and 6-month PT time point analysis of 1° and 3-month PT time point analysis of 2° recipients, respectively. (C) Percent donor reconstitution in the PB of primary and secondary recipients (generated as described in panel A) transplanted with NA10hd-transduced WT (blue triangles) and Tert−/− (red triangles) BM cells. Statistical significance was determined by application of the paired Student t test and is shown as *P < .05 or **P < .01. Each triangle represents an individual recipient. Each horizontal line represents the mean percent donor reconstitution of at least 3 recipients. (D) Experimental protocol in which WBM cells from 5-FU pre-treated WT or Tert−/− mice were transduced with NA10hd, expanded for 6 days in vitro and transplanted into lethally irradiated primary recipients. Six months later, donor-derived WBM cells from the primary recipients were transplanted into secondary recipients. (E) Average telomere lengths of donor-derived WT (blue bars) or Tert−/− (red bars) BM cells were obtained at the end of expansion in vitro, 3 and 6 months PT from the primary recipients and 3 months PT from the secondary recipients. Dotted lines indicate telomere lengths of donor WT (blue) or Tert−/− (red) BM cells before infection, in vitro expansion and primary transplantation. Error bars denote SD. Nwt = 2; 4; 4; 4 and Ntert−/− = 2; 4; 4; 5 at 10-day time point analysis of cultured cells, 3- and 6-month PT time point analysis of 1° and 3-month PT time point analysis of 2° recipients, respectively. Statistical significance was determined by application of the paired Student t test and is shown as **P < .01 or ***P < .005. (F) Percent donor reconstitution in the PB of primary and secondary recipients (generated as described in panel D) transplanted with NA10hd-transduced and in vitro expanded WT (blue triangles) and Tert−/− (red triangles) BM cells. Statistical significance was determined by the application of the paired Student t test and is shown as *P < .05 or ***P < .005. Each triangle represents an individual recipient. Each horizontal line represents the mean percent donor reconstitution of at least 3 recipients.
Figure 3
Figure 3
Absence of telomerase activity blunts NA10hd-induced self-renewal of myeloid progenitors and HSCs in vitro. Cultures were initiated with WT or Tert−/− BM cells from 5-FU pre-treated mice. Cells were transduced with NA10hd, expanded for 6 days in vitro and either plated in methylcellulose medium (A) or transplanted into lethally irradiated recipients (B). (A) Each 7 days of methylcellulose culture, generated colonies were counted, cells harvested, pooled and equal cell numbers replated for a total of 3 times to calculate the yield of granulocyte-macrophage colonies formed. Statistical significance was determined by application of the paired Student t test and is shown as *P < .05. (B) Mice were transplanted with limiting dilutions of cells used to initiate the cultures and with cells harvested at the end of expansion in vitro. Proportions of circulating B, T, and myeloid donor-derived (CD45.2+ or GFP+) WBCs were determined 4-6 months later. Fold in vitro expansions of WT and Tert−/− HSCs stimulated by NA10hd was estimated by determining the frequency, and hence HSC content in suspensions used to initiate and harvested from in vitro cultures. Results are expressed as the mean ± SEM of 2 independent experiments.
Figure 4
Figure 4
Primitive Tert−/− hematopoietic cells express elevated levels of γ-H2AX. Expression of γ-H2AX and DNA content were analyzed by flow-cytometry, within LSK subset of (A) Nonmanipulated WT and Tert−/− BM. (B) Recipient- (CD45.2) and donor-derived (CD45.2+) BM from recipients reconstituted with nonmanipulated WT BM or Tert−/− BM; (C) Recipient- (GFP) and donor-derived (GFP+) BM from recipients reconstituted with NA10hd-transduced and in vitro expanded either WT or Tert−/− BM cells.

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