Angptl4 maintains in vivo repopulation capacity of CD34(+) human cord blood cells.

OBJECTIVES: Methods to expand hematopoietic stem cells (HSCs) ex vivo encompass an attractive approach that would substantially broaden the clinical applicability of HSCs derived from cord blood. Recently, members of the Angiopoietin-like (Angptl) family of growth factors were shown to expand both murine and human HSCs. Specifically, Angptl5 has been implicated in the expansion of human NOD-SCID-repopulating cells (SRCs) ex vivo. Here, we sought to evaluate the potential of additional Angptls to expand human SRCs from cord blood. Additionally, the purpose of this study was to evaluate the reproducibility of Angptl-mediated expansion of SRCs across independent experiments. METHODS: Human CD34(+) cells from cord blood were cultured in vitro for eleven or eight days in the presence or absence of Angptls. The reconstitution capacity of expanded cells was subsequently measured in vivo by transplantation into NOD-SCID or NSG mice, and compared to that of uncultured cells. RESULTS: We report here that Angptl4 functions to maintain SRC-activity of CD34(+) CB-derived cells ex vivo as assayed in NOD-SCID and NSG mice. However, all Angptls tested, including Angptl1, 4, and 5, were associated with variation between experiments. CONCLUSION: Our findings indicate that Angptl4 and Angptl5 can lead to increased engraftment capacity of SRCs, but more frequently these factors are associated with maintenance of SRC-activity during ex vivo culture. Thus, Angptl-mediated expansion of SRCs ex vivo is associated with more inter-experimental variation than previously thought. We conclude that Angptls would be useful in instances where there is a need to maintain HSCs ex vivo, such as during transduction for gene therapy applications

Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-α on Fanconi anemia hematopoietic stem and progenitor cells

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-α (TNF-α) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-α, we studied Fancc−/− HSCs to determine the physiologic effects of HOXB4 on TNF-α sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-α of Fancc−/− HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc−/− but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-α receptors on Fancc−/− HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-α signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution

Cell-intrinsic and Vector-related Properties Cooperate to Determine the Incidence and Consequences of Insertional Mutagenesis

In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient γ-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of ~10−4. Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10−6). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity