Characterization of multipotent hematopoietic progenitors isolated from spleens of miR-17-92–overexpressing mice. (A) Quantitative RT-PCR analysis of precursor miR-17-92 expression in CSC1 and CSC2 cell lines. Results were normalized relative to β-actin expression in all experimental samples. Each experiment was performed in quadruplicate, where error bars represent SD; ***P < .0005. (B) Frequency of hematopoietic cell surface marker expression, as a percentage of total CSC1 and CSC2 cell lines (n = 4), empty vector control (n = 4), and miR-17-92 mice (n = 4). Values represent the mean % ± SD. (C) Numbers of CSC1 colonies (CFU-GEMM, BFU-E, and CFU-GM) observed in an in vitro colony formation assay on methylcellulose. Colony counts were performed in triplicate after 5 days of culture. Values represent the mean ± SD detected in 3 separate experiments. (D) Average spleen weights of PBS control (n = 6) and CSC1 transplanted NOD/SCID mice (n = 8), where error bars represent SD and **P < .005. (E) Flow cytometric analysis using the indicated hematopoietic markers revealed reconstitution of T (CD4⁺/CD8⁺) and B (B220⁺/CD19⁺) lymphocytes in NOD/SCID mice injected with 5 × 10⁶ CSC1 cells (n = 4), 4 months after injection. Uninfected (n = 3), and PBS-injected mice (n = 4) were used as controls. Values represent the mean % ± SD; *P < .05. (F) Quantitative RT-PCR analysis of CSC1 donor DNA in 3 female NOD/SCID mice transplanted with CSC1 cells. Serial dilutions of CSC1 DNA were added to normal spleen DNA and used to quantify the percentage of donor DNA, by examination of the Y chromosome specific SRY gene, where error bars represent SD.