A Sensitive and Quantitative Polymerase Chain Reaction-Based Non-Homologous End Joining in Vitro Assay for Hematopoietic Stem Cells

Abstract 4825

Hematopoietic stem cells (HSCs) are longevity cells that are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative in vitro non-homologous end joining (NHEJ) assay using the Bgl II-digested linear pDsRed2ER plasmids and real-time polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 0.25 μg nuclear proteins or nuclear extracts from about 1000 human BM CD34⁺ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34⁺CD38⁻ cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34⁺CD38⁺ cells). In contrast, mouse quiescent HSCs (Pyronin-Y^low LSK⁺ cells) repaired the damage more efficiently than cycling HPCs (LSK⁻ cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as ATM and ligase 4. These findings suggest that the qPCR-based plasmid NHEJ assay can be used to sensitively measure the ability of HSCs to repair DSBs. This assay can also be applied to other rare tissue stem cells as well.