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Single-cell suspensions of granulocyte colony-stimulating factor (G- CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) cultured in alpha minimal essential medium (alphaMEM) containing 10% fetal bovine serum formed multicellular aggregates within 24 hours. In six separate experiments, formation of aggregates appeared to be dependent on cell density per surface area, so that 5.8 +/- 1.3 aggregates formed per 1 x 10(5) cells when G-PBMC were cultured at densities greater than or equal to 1 x 10(5) cells/cm2. The frequency of aggregate formation was less than 1 per 10(5) cells when G-PBMC were cultured at densities less than 1 x 10(5) cells/cm2. Once formed, aggregates became adherent within 72 hours, and then, over the course of 21 days, released CD3/CD4/CD25-positive cells into the supernatant. This T-cell production peaked between days 7 and 14, reaching a total of 1,269 +/- 125.9 cells released per aggregate by day 21. Between days 14 and 21, the aggregates also generated macroscopic clusters of adherent mononuclear and giant multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). At 4 weeks, the macroscopic foci coalesced into monolayers. Multinucleated TRAP-positive cells were distinguished from macrophage polykaryons by the absence of CD14 expression and the presence of osteoclast-specific membrane receptors for calcitonin and alphavbeta3-vitronectin. The osteoclast nature of these cells was further demonstrated by their ability to form resorption lacunae on dentine slices. Comparable osteoclast formation was not detected in cultures of normal marrow or normal nonmobilized peripheral blood.

The retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. We previously reported that pharmacologic levels (1 micromol) of ATRA enhanced the generation of colony-forming cell (CFC) and colony-forming unit-spleen (CFU-S) in liquid suspension cultures of lin- c-kit+ Sca-1+ murine hematopoietic precursors. In this study, we further investigated the effects of ATRA as well as an RAR antagonist, AGN 193109, on the generation of transplantable cells, including pre-CFU-S, short-term repopulating stem cells (STRCs), and long-term repopulating stem cells (LTRCs). ATRA enhanced the ex vivo maintenance and production of competitive repopulating STRCs and LTRCs from lin- c-kit+ Sca-1+ cells cultured in liquid suspension for 14 days. In addition, ATRA prevented the differentiation of these primitive stem cells into more mature pre-CFU-S during the 14 days of culture. In marked contrast, lin- c-kit+ Sca-1+ cells cultured with AGN 193109 for 7 days had virtually no short- or long-term repopulating ability, but displayed an approximately 6-fold increase in the pre-CFU-S population. The data suggest that the RAR agonist ATRA enhances the maintenance and self-renewal of short- and long-term repopulating stem cells. In contrast, the RAR antagonist AGN 193109 abrogates reconstituting ability, most likely by promoting the differentiation of the primitive stem cells. These results imply an important and unexpected role of retinoids in regulating hematopoietic stem cell differentiation. (Blood. 2000;95:470-477)

All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin-c-kit+Sca-1(+)) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin- cells: 31.5% at day 7 and 4% at day 14; Sca-1(+): 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin-c-kit+Sca-1(+) hematopoietic precursors, ATRA did not exert the same effects on the more committed lin-c-kit+Sca-1(-) progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.