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Myelopoiesis following phorbol ester exposure in human long-term bone marrow cell culture
Insights
Phorbol esters irreversibly reduce bone marrow colony-forming cells (CFC-GM) but enhance accessory cell support for hematopoietic stem cell recovery in vitro.
Area of Science:
- Hematology
- Cell Biology
- Stem Cell Research
Background:
- Phorbol esters exhibit dual effects on bone marrow granulocyte/macrophage colony-forming cells (CFC-GM), inhibiting their response to colony-stimulating activity (CSA) while also stimulating CSA release from accessory cells.
- The reversibility of phorbol ester's direct inhibitory effect on CFC-GM and the potential for phorbol ester-treated accessory cells to enhance in vitro recovery remain key questions.
Purpose of the Study:
- To investigate the reversibility of phorbol ester's direct inhibitory impact on CFC-GM.
- To determine if phorbol ester-treated accessory bone marrow cells can augment the in vitro recovery of cultured CFC-GM.
Main Methods:
- Human bone marrow cells were exposed to phorbol-12,13-dibutyrate (PDB) for 5 days in liquid cultures.
- Cultures were washed to remove PDB and continued for up to 8 weeks to assess cell counts and CFC-GM levels.
- Nonadherent and adherent bone marrow cell fractions were co-cultured to differentiate direct vs. indirect effects of PDB.
Main Results:
- PDB exposure led to a dose-dependent decrease in total nucleated cells and CFC-GM.
- PDB treatment resulted in an irreversible reduction in cell counts and CFC-GM in long-term cultures.
- Co-culture of control nonadherent cells with PDB-pretreated adherent cells showed increased nucleated cells, CFC-GM, and hematopoietic islands, linked to higher CSA levels.
Conclusions:
- Phorbol esters induce an irreversible decline in CFC-GM within long-term bone marrow cultures.
- PDB treatment enhances the capacity of adherent stromal cells to support normal CFC-GM growth in vitro.
- The study elucidates distinct direct and indirect mechanisms of phorbol ester action on hematopoiesis.
Abstract:
Phorbol esters have two opposing effects on bone marrow granulocyte/macrophage colony forming cells (CFC-GM): they reduce the number of CFC-GM which respond to colony-stimulating activity (CSA), and they also induce the release of CSA from accessory marrow cells. We questioned whether the direct inhibitory effect of phorbol esters on CFC-GM was reversible and whether phorbol ester-treated accessory bone marrow cells could increase the in vitro recovery of cultured CFC-GM. Normal human bone marrow cells were exposed to phorbol-12,13-dibutyrate (PDB) for 5 days in liquid cultures. Total nucleated cell counts decreased in a dose-dependent fashion to 42 +/- 6% of control at 5 X 10(-8) M PDB, and CFC-GM decreased to 20 +/- 8% of control. The cultures were then washed to remove PDB and continued for up to 8 weeks. Total cell counts and CFC-GM/ml remained reduced in PDB-pretreated cultures compared to control. To determine whether this was a direct effect of PDB on nonadherent, proliferating cells or whether PDB acted indirectly by affecting adherent, accessory cells, nonadherent bone marrow cells from control and PDB-preincubated cultures were co-cultured for 8 weeks with either control or PDB-preincubated adherent bone marrow cells. Nonadherent cells preincubated with PDB had a similar growth pattern whether or not the co-cultured adherent cells had been preincubated with PDB. In contrast, nonadherent cells preincubated in control medium and co-cultured with PDB-preincubated adherent cells displayed increased numbers of nucleated cells, CFC-GM and adherent hematopoietic islands compared to control. This was associated with increased amounts of CSA present in the culture supernatants. Thus PDB causes an irreversible decrease in CFC-GM in long term marrow cultures but enhances the ability of adherent stromal cells to support normal CFC-GM growth in culture.