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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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Related in: MedlinePlus

Flow cytometry of expanded cells before and after culturing for a subsequent week in G-CSF. Expression of CD16 and CD66 was up-regulated in HPC expanded in IL-3 and IL-6 stimulated EC cultures (p < 0.05), while in IL-1β cultures, no further up-regulation was observed. Increase of granulocytic glycoproteins occurred in parallel to the development of granulocytic morphology. Pictures were taken from one representative result of six independent experiments. A) forward scatter – side scatter, IgG control; B) CD16 and CD66 expression before culturing with G-CSF; C) CD16 and CD66 expression and cell morphology after culturing with G-CSF.
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Figure 3: Flow cytometry of expanded cells before and after culturing for a subsequent week in G-CSF. Expression of CD16 and CD66 was up-regulated in HPC expanded in IL-3 and IL-6 stimulated EC cultures (p < 0.05), while in IL-1β cultures, no further up-regulation was observed. Increase of granulocytic glycoproteins occurred in parallel to the development of granulocytic morphology. Pictures were taken from one representative result of six independent experiments. A) forward scatter – side scatter, IgG control; B) CD16 and CD66 expression before culturing with G-CSF; C) CD16 and CD66 expression and cell morphology after culturing with G-CSF.

Mentions: Extension of the cell culture for an additional week with G-CSF induced the up-regulation of the granulocytic markers CD16 and CD66 in all three interleukin conditions (Figure 3). Prior to G-CSF addition, only cells cultured in IL-1β-stimulated endothelial supernatant already had a high frequency of CD16 and CD66 positive cells, which was further increased following the addition of G-CSF. Thereafter, the cells also became highly positive for CD15, CD11b and CD11c. Control granulocytes differentiated in stem cell medium plus cytokines in the absence of endothelial supernatant developed an equivalent morphology and immunephenotype. There were no differences between the burst activities of G-CSF matured granulocytes from different interleukin conditions (p > 0.05, Table 4).


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)

Flow cytometry of expanded cells before and after culturing for a subsequent week in G-CSF. Expression of CD16 and CD66 was up-regulated in HPC expanded in IL-3 and IL-6 stimulated EC cultures (p < 0.05), while in IL-1β cultures, no further up-regulation was observed. Increase of granulocytic glycoproteins occurred in parallel to the development of granulocytic morphology. Pictures were taken from one representative result of six independent experiments. A) forward scatter – side scatter, IgG control; B) CD16 and CD66 expression before culturing with G-CSF; C) CD16 and CD66 expression and cell morphology after culturing with G-CSF.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Flow cytometry of expanded cells before and after culturing for a subsequent week in G-CSF. Expression of CD16 and CD66 was up-regulated in HPC expanded in IL-3 and IL-6 stimulated EC cultures (p < 0.05), while in IL-1β cultures, no further up-regulation was observed. Increase of granulocytic glycoproteins occurred in parallel to the development of granulocytic morphology. Pictures were taken from one representative result of six independent experiments. A) forward scatter – side scatter, IgG control; B) CD16 and CD66 expression before culturing with G-CSF; C) CD16 and CD66 expression and cell morphology after culturing with G-CSF.
Mentions: Extension of the cell culture for an additional week with G-CSF induced the up-regulation of the granulocytic markers CD16 and CD66 in all three interleukin conditions (Figure 3). Prior to G-CSF addition, only cells cultured in IL-1β-stimulated endothelial supernatant already had a high frequency of CD16 and CD66 positive cells, which was further increased following the addition of G-CSF. Thereafter, the cells also became highly positive for CD15, CD11b and CD11c. Control granulocytes differentiated in stem cell medium plus cytokines in the absence of endothelial supernatant developed an equivalent morphology and immunephenotype. There were no differences between the burst activities of G-CSF matured granulocytes from different interleukin conditions (p > 0.05, Table 4).

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