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Hematopoietic progenitor cells and interleukin-stimulated endothelium: expansion and differentiation of myeloid precursors.

Moldenhauer A, Genter G, Lun A, Bal G, Kiesewetter H, Salama A - BMC Immunol. (2008)

Bottom Line: In contrast, IL-1beta and IL-3 stimulation resulted in a 10- and 100-fold increase in cell numbers with more than 90% of these cells being CD33(+).Fewer 2-week cobblestones and greater amounts of 5-week cobblestones were observed with IL-6 and IL-3.IL-1beta and IL-3 stimulated endothelium induces proliferation and differentiation of myeloid precursors, while IL-6 treatment induced a benefit of HPC survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Transfusion Medicine, Charité - Universitätsmedizin Berlin, Germany. amolden@charite.de

ABSTRACT

Background: Cytokine-stimulated endothelial cells (EC) propagate hematopoietic progenitor cell (HPC) expansion. However, the effects on the functional capacities of cultured progenitors have not been evaluated. HPC were assessed by flow cytometry, colony and cobblestone assays and long-term cultures (LTC) after culturing in the supernatant of EC stimulated by IL-1beta, IL-3 or IL-6.

Results: EC incubation with IL-6 did not improve cell expansion in comparison to non-stimulated EC supernatant, while the HPCs' phenotype and functional capacities were retained. In contrast, IL-1beta and IL-3 stimulation resulted in a 10- and 100-fold increase in cell numbers with more than 90% of these cells being CD33(+). Plating efficiencies and LTC initiating cells were greatest in IL-6 supernatants, whereas the highest numbers of burst-forming units were observed using IL-3. IL-1beta supernatants diminished the number of 5-week cobblestone-areas, whereas the number of 2-week cobblestone areas remained equal to freshly isolated HPC. Fewer 2-week cobblestones and greater amounts of 5-week cobblestones were observed with IL-6 and IL-3. Expanded progenitors from all interleukin conditions were further matured into functional granulocytes.

Conclusion: IL-1beta and IL-3 stimulated endothelium induces proliferation and differentiation of myeloid precursors, while IL-6 treatment induced a benefit of HPC survival.

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Granulocytic functionality. Phagoburst results are shown in response to PMA, fMLP and E. coli of HPC expanded in IL1-stimulated EC and following further differentiation by G-CSF in comparison to granulocytes differentiated by cytokines alone. A) HPC differentiated following expansion in IL1-stimulated EC supernatant; B) HPC differentiated following expansion in IL1-stimulated EC supernatant and overnight storage in human serum prior to analysis; C) HPC differentiated in a cytokine combination of erythropoietin, SCF and G-CSF without endothelial supernatant. Shown is one representative result of eight independent experiments. Shaded histograms: sample fluorescence; white line: negative control. PMA: phorbol 12-myristate 13-acetate; E. coli: Escherichia coli; fMLP: N-formyl-methionyl-leucyl-phenylalanin.
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Figure 4: Granulocytic functionality. Phagoburst results are shown in response to PMA, fMLP and E. coli of HPC expanded in IL1-stimulated EC and following further differentiation by G-CSF in comparison to granulocytes differentiated by cytokines alone. A) HPC differentiated following expansion in IL1-stimulated EC supernatant; B) HPC differentiated following expansion in IL1-stimulated EC supernatant and overnight storage in human serum prior to analysis; C) HPC differentiated in a cytokine combination of erythropoietin, SCF and G-CSF without endothelial supernatant. Shown is one representative result of eight independent experiments. Shaded histograms: sample fluorescence; white line: negative control. PMA: phorbol 12-myristate 13-acetate; E. coli: Escherichia coli; fMLP: N-formyl-methionyl-leucyl-phenylalanin.

Mentions: Differentiated cells were analyzed for their granulocytic function. Cells which were harvested directly from G-CSF cultures had high spontaneous burst rates, which were even higher than after they had been exposed to Escherichia (E.) coli (Figure 4A). Yet, these cells responded two- and ten-fold better to N-formyl-methionyl-leucyl-phenylalanin (fMLP) and phorbol 12-myristate 13-acetate (PMA), respectively. When the differentiated cells were incubated overnight in human serum at 37°C, E. coli or PMA induced a ten-fold burst, whereas no effect was seen in response to fMLP (Figure 4B). Burst rates between cells, which had been stored overnight in human serum and those without serum incubation were significantly different (p ≤ 0.018). Oxygen radical formation was also significantly higher in granulocytes generated in stimulated EC supernatant than in granulocytes differentiated with cytokines alone (Figure 4C), but lower than in granulocytes from peripheral blood.


Hematopoietic progenitor cells and interleukin-stimulated endothelium: expansion and differentiation of myeloid precursors.

Moldenhauer A, Genter G, Lun A, Bal G, Kiesewetter H, Salama A - BMC Immunol. (2008)

Granulocytic functionality. Phagoburst results are shown in response to PMA, fMLP and E. coli of HPC expanded in IL1-stimulated EC and following further differentiation by G-CSF in comparison to granulocytes differentiated by cytokines alone. A) HPC differentiated following expansion in IL1-stimulated EC supernatant; B) HPC differentiated following expansion in IL1-stimulated EC supernatant and overnight storage in human serum prior to analysis; C) HPC differentiated in a cytokine combination of erythropoietin, SCF and G-CSF without endothelial supernatant. Shown is one representative result of eight independent experiments. Shaded histograms: sample fluorescence; white line: negative control. PMA: phorbol 12-myristate 13-acetate; E. coli: Escherichia coli; fMLP: N-formyl-methionyl-leucyl-phenylalanin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570655&req=5

Figure 4: Granulocytic functionality. Phagoburst results are shown in response to PMA, fMLP and E. coli of HPC expanded in IL1-stimulated EC and following further differentiation by G-CSF in comparison to granulocytes differentiated by cytokines alone. A) HPC differentiated following expansion in IL1-stimulated EC supernatant; B) HPC differentiated following expansion in IL1-stimulated EC supernatant and overnight storage in human serum prior to analysis; C) HPC differentiated in a cytokine combination of erythropoietin, SCF and G-CSF without endothelial supernatant. Shown is one representative result of eight independent experiments. Shaded histograms: sample fluorescence; white line: negative control. PMA: phorbol 12-myristate 13-acetate; E. coli: Escherichia coli; fMLP: N-formyl-methionyl-leucyl-phenylalanin.
Mentions: Differentiated cells were analyzed for their granulocytic function. Cells which were harvested directly from G-CSF cultures had high spontaneous burst rates, which were even higher than after they had been exposed to Escherichia (E.) coli (Figure 4A). Yet, these cells responded two- and ten-fold better to N-formyl-methionyl-leucyl-phenylalanin (fMLP) and phorbol 12-myristate 13-acetate (PMA), respectively. When the differentiated cells were incubated overnight in human serum at 37°C, E. coli or PMA induced a ten-fold burst, whereas no effect was seen in response to fMLP (Figure 4B). Burst rates between cells, which had been stored overnight in human serum and those without serum incubation were significantly different (p ≤ 0.018). Oxygen radical formation was also significantly higher in granulocytes generated in stimulated EC supernatant than in granulocytes differentiated with cytokines alone (Figure 4C), but lower than in granulocytes from peripheral blood.

Bottom Line: In contrast, IL-1beta and IL-3 stimulation resulted in a 10- and 100-fold increase in cell numbers with more than 90% of these cells being CD33(+).Fewer 2-week cobblestones and greater amounts of 5-week cobblestones were observed with IL-6 and IL-3.IL-1beta and IL-3 stimulated endothelium induces proliferation and differentiation of myeloid precursors, while IL-6 treatment induced a benefit of HPC survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Transfusion Medicine, Charité - Universitätsmedizin Berlin, Germany. amolden@charite.de

ABSTRACT

Background: Cytokine-stimulated endothelial cells (EC) propagate hematopoietic progenitor cell (HPC) expansion. However, the effects on the functional capacities of cultured progenitors have not been evaluated. HPC were assessed by flow cytometry, colony and cobblestone assays and long-term cultures (LTC) after culturing in the supernatant of EC stimulated by IL-1beta, IL-3 or IL-6.

Results: EC incubation with IL-6 did not improve cell expansion in comparison to non-stimulated EC supernatant, while the HPCs' phenotype and functional capacities were retained. In contrast, IL-1beta and IL-3 stimulation resulted in a 10- and 100-fold increase in cell numbers with more than 90% of these cells being CD33(+). Plating efficiencies and LTC initiating cells were greatest in IL-6 supernatants, whereas the highest numbers of burst-forming units were observed using IL-3. IL-1beta supernatants diminished the number of 5-week cobblestone-areas, whereas the number of 2-week cobblestone areas remained equal to freshly isolated HPC. Fewer 2-week cobblestones and greater amounts of 5-week cobblestones were observed with IL-6 and IL-3. Expanded progenitors from all interleukin conditions were further matured into functional granulocytes.

Conclusion: IL-1beta and IL-3 stimulated endothelium induces proliferation and differentiation of myeloid precursors, while IL-6 treatment induced a benefit of HPC survival.

Show MeSH
Related in: MedlinePlus