Loss of Ku70 Results in a HSC Repopulating Defect.

Genomic integrity is essential for organism development and longevity and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is a specific pathway essential for DNA double-strand break repair in all cells and VDJ processing in B and T cells; and it is crucial for maintenance of mammalian genome. We hypothesize that NHEJ is also critical for hematopoietic stem cells (HSC) maintenance and function. Ku70 is a key component of the NHEJ pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Ku70-deficient mice were used to assess the importance of NHEJ pathway in HSC function. Ku70-deficient mice have decreased bone marrow cells (BMC), and significant reductions in the frequency of colony-forming units in culture (CFU-C) in the presence of cytokines including IL-3, IL-6, SCF and Epo. SKL (Sca1⁺, c-Kit⁺, Lin⁻) cells from Ku70-deficeint mice showed a similar proliferation rate compared with SKL cells from WT mice when cultured in vitro in the presence of cytokines; single cell culture experiment showed that SKL cells from Ku70-deficient mice and WT mice have similar clonogenic ability. Thus, Ku70-deficient SKL cells retain proliferation capacity although there is a deficiency in overall hematopoietic homeostasis. To address Ku70-deficient HSC function in vivo, Ku70-deficient BMCs were serially transplanted into lethally irradiated congenic wild-type mice. These Ku70-deficient marrow cells were able to reconstitute the myeloid lineages in both primary and secondary recipients, while over 95% of the T cells in the recipients were derived from the host. However, the tertiary whole marrow recipients died within 2 weeks of transplantation, while the WT BMCs recipients survived tertiary transplantation. Competitive repopulation assays were also performed between WT and Ku70-deficient BMCs. The results showed that when Ku70-deficient BMCs were mixed with WT competitor BMCs at a 1:1 ratio, Ku70-deficient BMCs were completely outcompeted by the WT BMCs. These data demonstrate that Ku70, a key component of NHEJ, is required for HSC function. Ku70 deficiency in the hematopoietic compartment results in a long-term repopulating defect and loss of competitive repopulation ability.