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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

Cumulative cell counts of proliferating progenitors in direct contact, non-contact and supernatant cultures. Cell counts were determined by demi-depopulation after 7, 14 and 21 days and summarized. Culture conditions were as follows: A) HPC in direct contact with IL1-β stimulated EC (Direct Contact, open squares), on a 0.4 μm microporous transmembrane above the IL-1β stimulated EC (Indirect Contact, open circles), in the supernatant of IL-1β stimulated EC (Supernatant, closed circles), B) in direct contact with IL-3 stimulated EC (Direct Contact), on a 0.4 μm microporous transmembrane above IL-3 stimulated EC (Indirect Contact) and in the supernatant of IL-3 stimulated EC (closed circles). Significant differences to contact cultures (*), to indirect contact cultures (#) and to bone marrow (§ were only found in IL-1β and IL-3 dependent conditions. C) No significant differences were determined among the IL-6 stimulated EC culture conditions or among bone marrow fibroblast cultures. The HPC cell count at the beginning was 5.5 × 104 per 3 ml. Each point represents the average of at least three independent measurements. Bone marrow (BM) fibroblast cocultures consisted of direct contact (open triangles), indirect contact (crosses) and supernatant cultures (closed triangles). Dotted lines: HPC cultured in endothelial supernatants, to which IL-1β, IL-3 or IL-6 was added.
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Figure 1: Cumulative cell counts of proliferating progenitors in direct contact, non-contact and supernatant cultures. Cell counts were determined by demi-depopulation after 7, 14 and 21 days and summarized. Culture conditions were as follows: A) HPC in direct contact with IL1-β stimulated EC (Direct Contact, open squares), on a 0.4 μm microporous transmembrane above the IL-1β stimulated EC (Indirect Contact, open circles), in the supernatant of IL-1β stimulated EC (Supernatant, closed circles), B) in direct contact with IL-3 stimulated EC (Direct Contact), on a 0.4 μm microporous transmembrane above IL-3 stimulated EC (Indirect Contact) and in the supernatant of IL-3 stimulated EC (closed circles). Significant differences to contact cultures (*), to indirect contact cultures (#) and to bone marrow (§ were only found in IL-1β and IL-3 dependent conditions. C) No significant differences were determined among the IL-6 stimulated EC culture conditions or among bone marrow fibroblast cultures. The HPC cell count at the beginning was 5.5 × 104 per 3 ml. Each point represents the average of at least three independent measurements. Bone marrow (BM) fibroblast cocultures consisted of direct contact (open triangles), indirect contact (crosses) and supernatant cultures (closed triangles). Dotted lines: HPC cultured in endothelial supernatants, to which IL-1β, IL-3 or IL-6 was added.

Mentions: Direct contact between IL-β or IL3 stimulated EC and HPC significantly reduced the cumulative cell output as compared to non-contact and supernatant cultures (Figure 1). Stimulated supernatants led to two to three times higher cumulative cell counts than non-contact cultures (IL-3: 14.1 × 106 versus 8.5 × 106; IL-1β: 9.3 × 106 versus 3.7 × 106), which were twice as high as in direct contact cultures (IL-3: 3.6 × 106 and IL-1β:1.9 × 106). Differences between IL-1β and IL-3 in cumulative cell numbers were not significant (p = 0.12). In IL-6 conditions, direct contact and supernatant conditions led to comparable cumulative cell counts (p > 0.13). Cell numbers in non-stimulated EC supernatant, to which single interleukins were added, had significantly lower cell counts in IL-1β and IL-3 conditions and lower cell numbers in IL-6 conditions, which was also the case, when HPC were cultured in endothelial plus stem cell medium including interleukins. IL-3 stimulated bone marrow fibroblasts led to significantly lower cumulative cell counts inducing on average a 15-fold cell expansion after two weeks. No significant differences were seen among different interleukins.


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)

Cumulative cell counts of proliferating progenitors in direct contact, non-contact and supernatant cultures. Cell counts were determined by demi-depopulation after 7, 14 and 21 days and summarized. Culture conditions were as follows: A) HPC in direct contact with IL1-β stimulated EC (Direct Contact, open squares), on a 0.4 μm microporous transmembrane above the IL-1β stimulated EC (Indirect Contact, open circles), in the supernatant of IL-1β stimulated EC (Supernatant, closed circles), B) in direct contact with IL-3 stimulated EC (Direct Contact), on a 0.4 μm microporous transmembrane above IL-3 stimulated EC (Indirect Contact) and in the supernatant of IL-3 stimulated EC (closed circles). Significant differences to contact cultures (*), to indirect contact cultures (#) and to bone marrow (§ were only found in IL-1β and IL-3 dependent conditions. C) No significant differences were determined among the IL-6 stimulated EC culture conditions or among bone marrow fibroblast cultures. The HPC cell count at the beginning was 5.5 × 104 per 3 ml. Each point represents the average of at least three independent measurements. Bone marrow (BM) fibroblast cocultures consisted of direct contact (open triangles), indirect contact (crosses) and supernatant cultures (closed triangles). Dotted lines: HPC cultured in endothelial supernatants, to which IL-1β, IL-3 or IL-6 was added.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Cumulative cell counts of proliferating progenitors in direct contact, non-contact and supernatant cultures. Cell counts were determined by demi-depopulation after 7, 14 and 21 days and summarized. Culture conditions were as follows: A) HPC in direct contact with IL1-β stimulated EC (Direct Contact, open squares), on a 0.4 μm microporous transmembrane above the IL-1β stimulated EC (Indirect Contact, open circles), in the supernatant of IL-1β stimulated EC (Supernatant, closed circles), B) in direct contact with IL-3 stimulated EC (Direct Contact), on a 0.4 μm microporous transmembrane above IL-3 stimulated EC (Indirect Contact) and in the supernatant of IL-3 stimulated EC (closed circles). Significant differences to contact cultures (*), to indirect contact cultures (#) and to bone marrow (§ were only found in IL-1β and IL-3 dependent conditions. C) No significant differences were determined among the IL-6 stimulated EC culture conditions or among bone marrow fibroblast cultures. The HPC cell count at the beginning was 5.5 × 104 per 3 ml. Each point represents the average of at least three independent measurements. Bone marrow (BM) fibroblast cocultures consisted of direct contact (open triangles), indirect contact (crosses) and supernatant cultures (closed triangles). Dotted lines: HPC cultured in endothelial supernatants, to which IL-1β, IL-3 or IL-6 was added.
Mentions: Direct contact between IL-β or IL3 stimulated EC and HPC significantly reduced the cumulative cell output as compared to non-contact and supernatant cultures (Figure 1). Stimulated supernatants led to two to three times higher cumulative cell counts than non-contact cultures (IL-3: 14.1 × 106 versus 8.5 × 106; IL-1β: 9.3 × 106 versus 3.7 × 106), which were twice as high as in direct contact cultures (IL-3: 3.6 × 106 and IL-1β:1.9 × 106). Differences between IL-1β and IL-3 in cumulative cell numbers were not significant (p = 0.12). In IL-6 conditions, direct contact and supernatant conditions led to comparable cumulative cell counts (p > 0.13). Cell numbers in non-stimulated EC supernatant, to which single interleukins were added, had significantly lower cell counts in IL-1β and IL-3 conditions and lower cell numbers in IL-6 conditions, which was also the case, when HPC were cultured in endothelial plus stem cell medium including interleukins. IL-3 stimulated bone marrow fibroblasts led to significantly lower cumulative cell counts inducing on average a 15-fold cell expansion after two weeks. No significant differences were seen among different interleukins.

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