The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)–derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34+cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34+ cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34+population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400 000 cord blood CD34+ cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP+ lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells.

Retinoic acid (RA) stimulates the clonal proliferation of mature bone marrow progenitor cells and inhibits the growth of leukemic progenitors, whereas its effects on normal primitive hematopoietic progenitors have not yet been investigated. This study investigated the ability of all-trans- and 9-cis-RA to modulate the proliferation and differentiation of murine Lin-Sca-1+ bone marrow progenitor cells. Both RA isoforms inhibited in a reversible and dose-dependent fashion, the proliferation of multi- but not single-factor responsive Lin-Sca-1+ progenitor cells. The 50% effective dose was 10 nM for both all-trans- and 9-cis-RA. Maximum inhibition was observed at 100-1,000 nM RA, resulting in a 50-75% reduction in the number of proliferative clones. Lin-Sca-1+ cells with high proliferative potential were preferentially inhibited by RA, resulting in a 80-100% inhibition depending on the hematopoietic growth factors stimulating their growth. The inhibitory effects of RA were directly mediated on the target cell, since the effects were observed at the single cell level. Furthermore, autocrine transforming growth factor beta (TGF-beta) production can probably not account for the observed inhibitory effects of RA, since a TGF-beta neutralizing antibody did not block RA-induced inhibition. Whereas RA, in general, is a differentiation-inducing agent, treatment of Lin-Sca-1+ progenitors resulted in the accumulation of an increased fraction of blasts and immature myeloid cells. Thus, RA inhibits the proliferation as well as differentiation of normal primitive hematopoietic progenitor cells.

Tumor necrosis factor-alpha (TNF-alpha) is a bifunctional regulator of hematopoiesis, and its cellular responses are mediated by two distinct cell surface receptors. TNF-alpha generally inhibits the growth of primitive murine hematopoietic progenitor cells (Lin-Scal+) in response to multiple cytokine combinations, and the p75 TNF receptor is essential in signaling such inhibition. In the present study we show the reverse phenomenon in that TNF-alpha on the same progenitor cell population in combination with stem cell factor (SCF) and interleukin-7 (IL-7) through the p55 TNF receptor can recruit additional progenitors to proliferate. In contrast, TGF-beta 1, another bifunctional regulator of hematopoietic progenitor cell growth, completely blocked SCF plus IL- 7-induced proliferation. TNF-alpha increased the number of responding progenitors, as well as the size of the colonies formed. The synergistic effects of TNF-alpha were seen at the single cell level, suggesting that its effects are directly mediated. Finally, whereas SCF plus IL-7 promoted primarily granulopoiesis, the addition of TNF-alpha switched the differentiation toward the production of almost exclusively macrophages.