26 research outputs found
Identification of human and mouse hematopoietic stem cell populations expressing high levels of mRNA encoding retrovirus receptors
One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in Lin- CD34+ CD38- cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin- CD34+ CD38- cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM
AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates
AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors
Amphotropic or Gibbon Ape Leukemia Virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines
The low level of amphotropic retrovirus mediated gene transfer into human hematopoietic stem cells (HSC) has been an impediment to gene therapy for hematopoietic diseases (1). We have previously shown that mouse and human HSC have low levels of the mRNA encoding PiT-2, the amphotropic retrovirus receptor. We hypothesized that the low level of PiT-2 mRNA was responsible for the low frequency of transduction of HSC by amphotropic retroviral vectors (2). In this study we compared the level of PiT-2 and PiT-1, the Gibbon Ape Leukemia Virus receptor (GaLV), in 5 human tissue culture cell lines. PiT-2 and PiT-1 mRNA levels were highest in K562 cells and lowest in HL60 cells. In hematopoietic cell lines, the level of PiT-2 or PiT-1 mRNA correlated directly with retrovirus binding and transduction with the appropriate (amphotropic or GaLV) retrovirus vector. The level of expression of PiT-2 and PiT-1 mRNA could be increased by treatment of HL60 cells with either PMA or Interleukin-1α. The increase in the level of PiT-2 and PiT-1 mRNA correlated with increased transduction with both amphotropic and GaLV retroviral vectors. We conclude hat the improved transduction was a direct effect of the increased levels of receptor mRNA and unrelated to changes in the cell cycle status