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Securin controls hematopoietic progenitor function
the frequency of cells in the S-phase of the cell division cycle. It has been thus concluded that the underlying mechanism of the distinct susceptibility of HSPC from the inbred strains is due to distinct S-phase frequencies. BM cells with the Lin-cKit+ surface marker combination (hematopoietic progenitor cells, LK cells) are highly enriched for CAFC day 7 cells (Online Supplementary Figure S2C). However, analysis of the frequency of LK cells from the inbred and the congenic strains in different stages of the cell division cycle by in vivo BrdU incorporation and flow cytometry, as well as that of hematopoietic stem cells (HSC) and less primitive progenitors (LSK), revealed almost identical patterns and especially almost identical frequencies of cells in S-phase among all the strains tested (Figure 2B). HU susceptibility in HSPC does therefore not correlate with the frequency of HSPC in S-phase, which
excludes differences in cycling frequencies as the underly- ing mechanism for the phenotype observed, as well as in general HU susceptibility as surrogate for the frequency of cells in S-phase. Consistent with that finding was the fact that HSPC from all groups had similar telomere lengths. Short telomeres can be seen as a surrogate marker for high levels of proliferation (Online Supplementary Figure S2D). In addition, the frequency of LK and LSK was very similar in all strains, while D2-derived mice displayed a general higher HSC frequency, as already reported,1 which is, however, not mirrored in B6/line A mice and thus locus- independent. That finding excludes a difference in the number of these cells as a factor contributing to the phe- notype (Figure 2C). Furthermore, the frequencies of HSPC undergoing apoptosis upon ex vivo HU treatment and under steady state conditions in vivo were at a low level
AB
CD
Figure 1. Quantitative trait locus (QTL) analysis of hydroxyurea (HU) responses and mean life spans of BXD mice and generation of mice congenic for the corre- sponding chromosome 11 locus. (A) WebQTL analysis of HU sensitivity rates and mean life spans of hematopoietic stem and progenitor cells (HSPC) isolated from various BXD and parental strains, identifying a proximal part of chromosome 11, among others, involved in this phenotype. Values are in Likelihood Ratio Statistics (LRS). (B) QTL analysis of mean life spans and HU responses of the various BXD strains for chromosome 11. (C) Schematic illustration showing the generation of the congenic mouse strains line A and K. Briefly, after crossing B6 with D2 mice, F1 littermates were backcrossed with the corresponding parental strains (B6/D2). Offspring were backcrossed in four rounds with parental strains reciprocal for the corresponding chromosome 11 specific SNP D11Mit20 to finally obtain B6 or D2 mice congenic for the proximal locus on chromosome 11 of D2 or B6, respectively. (D) SNP analysis of chromosome 11 from strains B6, D2, A and K.
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