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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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Cytospin preparations of freshly isolated HPC and following culture for two weeks in non-stimulated, BSA or IL-stimulated EC supernatant. Freshly isolated HPC (Post isolation) with a dense nucleus and small cytoplasmatic rim increased up to two-fold in size and gained cytoplasma in non-stimulated and BSA-stimulated supernatants. With IL-1β stimulated supernatant they developed into hypersegmented cells and also into monocytic cells in part, with an increase in cytoplasma content. More than 50% of the cells stimulated with IL-3 developed eosinophilic granula, whereas cells in IL-6 stimulated supernatant resembled freshly isolated cells. Cells cultured in IL-6, BSA- and non-stimulated supernatants were still positive for CD34 and CD133. Diffquik staining, size bar 1 μm. magnifications ×200. One representative result of twelve independent experiments.
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Figure 2: Cytospin preparations of freshly isolated HPC and following culture for two weeks in non-stimulated, BSA or IL-stimulated EC supernatant. Freshly isolated HPC (Post isolation) with a dense nucleus and small cytoplasmatic rim increased up to two-fold in size and gained cytoplasma in non-stimulated and BSA-stimulated supernatants. With IL-1β stimulated supernatant they developed into hypersegmented cells and also into monocytic cells in part, with an increase in cytoplasma content. More than 50% of the cells stimulated with IL-3 developed eosinophilic granula, whereas cells in IL-6 stimulated supernatant resembled freshly isolated cells. Cells cultured in IL-6, BSA- and non-stimulated supernatants were still positive for CD34 and CD133. Diffquik staining, size bar 1 μm. magnifications ×200. One representative result of twelve independent experiments.

Mentions: More than 93% of the freshly isolated cells were positive for CD34, CD33 and CD45. The latter two remained highly positive following a period of two weeks in all of the culture conditions analyzed. When cultured with IL-1β or IL-3-stimulated supernatant, expanded cells lost the CD34 antigen following a one week culture period (Table 1). In contrast, on average 34.8 ± 6.7% of the cells cultured in BSA, IL-6 or non-stimulated supernatant stained positive for CD133, and 17.7 ± 5.2% were still CD34 positive in IL-6 induced supernatant. Although the loss of CD34 antigen was paralleled by a loss of CD133, a subset of CD34(-) cells retained the CD133 glycoprotein (see additional file 1)). Following a two week culture period, half of the cells in the IL-1β stimulated EC supernatant were CD16(+), and 15–25% of the cells carried the monocytic marker CD14 (Figure 2). Other glycoproteins tested were CD15 and CD19, which were rarely present in freshly isolated CD34 cells and did not increase upon culturing.


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)

Cytospin preparations of freshly isolated HPC and following culture for two weeks in non-stimulated, BSA or IL-stimulated EC supernatant. Freshly isolated HPC (Post isolation) with a dense nucleus and small cytoplasmatic rim increased up to two-fold in size and gained cytoplasma in non-stimulated and BSA-stimulated supernatants. With IL-1β stimulated supernatant they developed into hypersegmented cells and also into monocytic cells in part, with an increase in cytoplasma content. More than 50% of the cells stimulated with IL-3 developed eosinophilic granula, whereas cells in IL-6 stimulated supernatant resembled freshly isolated cells. Cells cultured in IL-6, BSA- and non-stimulated supernatants were still positive for CD34 and CD133. Diffquik staining, size bar 1 μm. magnifications ×200. One representative result of twelve independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Cytospin preparations of freshly isolated HPC and following culture for two weeks in non-stimulated, BSA or IL-stimulated EC supernatant. Freshly isolated HPC (Post isolation) with a dense nucleus and small cytoplasmatic rim increased up to two-fold in size and gained cytoplasma in non-stimulated and BSA-stimulated supernatants. With IL-1β stimulated supernatant they developed into hypersegmented cells and also into monocytic cells in part, with an increase in cytoplasma content. More than 50% of the cells stimulated with IL-3 developed eosinophilic granula, whereas cells in IL-6 stimulated supernatant resembled freshly isolated cells. Cells cultured in IL-6, BSA- and non-stimulated supernatants were still positive for CD34 and CD133. Diffquik staining, size bar 1 μm. magnifications ×200. One representative result of twelve independent experiments.
Mentions: More than 93% of the freshly isolated cells were positive for CD34, CD33 and CD45. The latter two remained highly positive following a period of two weeks in all of the culture conditions analyzed. When cultured with IL-1β or IL-3-stimulated supernatant, expanded cells lost the CD34 antigen following a one week culture period (Table 1). In contrast, on average 34.8 ± 6.7% of the cells cultured in BSA, IL-6 or non-stimulated supernatant stained positive for CD133, and 17.7 ± 5.2% were still CD34 positive in IL-6 induced supernatant. Although the loss of CD34 antigen was paralleled by a loss of CD133, a subset of CD34(-) cells retained the CD133 glycoprotein (see additional file 1)). Following a two week culture period, half of the cells in the IL-1β stimulated EC supernatant were CD16(+), and 15–25% of the cells carried the monocytic marker CD14 (Figure 2). Other glycoproteins tested were CD15 and CD19, which were rarely present in freshly isolated CD34 cells and did not increase upon culturing.

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