Loss of Dnmt3a in HSCs leads to enhanced serial replating capacity. (A) CFU assay of HSCs. A total of 100 control or Dnmt3a-KO HSCs were plated per well 4 weeks after the final pIpC injection. CFUs were scored every 7 days, and 5000 cells were replated each round in triplicate. Mean ± SEM values are shown from 3 independent experiments. (B) FACS analysis of CFU plates. Although control and Dnmt3a-KO colonies showed similar myeloid development after the first plating, by the third plating, most Dnmt3a-KO colonies were negative for mature myeloid markers (Gr-1⁻ Mac-1⁻) and showed an immature c-Kit⁺ CD34⁻ phenotype (red squares). (C) Real-time PCR at the third plating showed Dnmt3a-KO cells were characterized by increased expression of the self-renewal regulators Meis1 and Evi1 and decreased expression of the myeloid differentiation factors CEBPα and Mpo. (D) Serial replating of control or Dnmt3a-KO HSCs from mice previously injected with phosphate-buffered saline (PBS) or 5FU. Mean ± SEM values are shown from 3 independent experiments. *P < .05, **P < .01, ***P < .001.