The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.

Mobilization of mesenchymal stem cells (MSCs) is a promising strategy for tissue repair and regenerative medicine. The establishment of an appropriate animal model and clarification of the underlying mechanisms are beneficial to develop the mobilization regimens for therapeutic use. In this study, we therefore established a rat MSC mobilization model and investigated the related mechanisms, using continuous hypoxia as the mobilizing stimulus. We found that MSCs could be mobilized into peripheral blood of rats exposed to short-term hypoxia (2 days) and the mobilization efficiency increased in a time-dependent manner (2-14 days). Hypoxia-inducible factor-1α (HIF-1α) was upregulated during hypoxic exposure and was expressed continuously in bone marrow. Inhibition of HIF-1α expression by YC-1 remarkably reduced the number of mobilized MSCs, suggesting that HIF-1α is essential for hypoxia-induced MSC mobilization. Further, we investigated the potential role of HIF-1α target genes, vascular endothelial growth factor (VEGF), and stromal cell-derived factor-1α (SDF-1α). VEGF expression was elevated from day 2 to day 7 of hypoxia, stimulating an increase in bone marrow sinusoidal vessels and possibly facilitating the egress of MSCs. SDF-1α protein levels were increased in the peripheral blood of rats during MSC mobilization and promoted the migration of MSCs under hypoxic conditions in vitro. These results suggest that HIF-1α plays a pivotal role in hypoxia-induced MSC mobilization, possibly acting via its downstream genes VEGF and SDF-1α. These data provide a novel insight into the mechanisms responsible for MSC mobilization and may help in the development of clinically useful therapeutic agents.

Donor cell leukemia after allogeneic hematopoietic stem cell transplantation might provide a unique human model for our understanding of leukemogenesis in vivo. We hypothesized that the "2-genetic-hits model" may contribute to the "leukemization" of donor cells and first evaluated these genetic mutations that are implicated in the development of acute myeloid leukemia in a donor cell leukemia patient and donor. The patient and his donor-sister both harbored a germline mutation in CEBPA (584_589dup). Susceptible donor hematopoietic cells evolved to overt acute myeloid leukemia by developing 2 somatic CEBPA mutations (247dupC and 914_916dup) in the patient's microenvironment. These were identical to the acquired mutations identified in leukemic cells that originated from the patient during de novo acute myeloid leukemia. Our results provide the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype and provide clues to support the multiplegenetic-hits mechanism of donor cell leukemia. (Blood. 2011;117(19):5257-5260) IntroductionLeukemia relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) that arises in cells of donor origin in the transplant recipient, so-called donor cell leukemia (DCL), is a rare disease entity, and only 51 cases were reported since 1971. [1][2][3][4] The precise etiologic mechanisms of DCL remain unknown, and no common mechanism can be identified in most of the cases reported in the literature. Careful analysis of the mechanisms with respect to the oncogenic transformation of donor-derived cells might provide a unique human model for our understanding of leukemogenesis in vivo.According to the "2-genetic-hits model," cooperation between 2 classes of genetic mutations contributes to leukemogenesis. 5 One group (class 1) comprises mutations in the fms-related tyrosine kinase 3 gene (FLT3) or the neuroblastoma RAS viral oncogene homolog gene (NRAS), which increase the proliferation and/or survival of hematopoietic stem/progenitor cells. 6,7 The other complementation group (class 2) comprises mutations in CEBPA, the gene that encodes the CCAAT enhancer-binding protein␣ (C/EBP␣); the myeloid-lymphoid or mixed-lineage leukemia gene (MLL); or the nucleophosmin gene (NPM1), which cause impaired differentiation. [8][9][10] The most common mutations, which include internal tandem duplications restricted to exons 14 and 15 and point mutation of Asp 835 within the TK domain of FLT3, codon 12/13 in exon 1 and codon 61 in exon 2 of NRAS, the entire coding region of CEBPA, partial tandem duplications that span exons 2-6 or exons 2-8 of MLL, and mutations in exon 12 of NPM1, have been described extensively in acute myeloid leukemia (AML). [11][12][13][14] We hypothesized that the 2-genetic-hits model may contribute to the "leukemization" of donor cells in DCL. We screened these genetic mutations implicated in the development of common forms of AML in a DCL patient and donor. MethodsThe study was approved by the Zhejiang U...