Abstract 3408

The CXCR4-SDF1α axis plays an important role in maintaining the stemness of human hematopoietic stem cells (HSC). In the present study we established a surrogate model for the bone marrow niche by culturing HSC on a feeder-layer of human mesenchymal stromal cells (MSC) and investigated the proliferation and differentiation behaviour upon perturbation by Plerixafor.

HSC (CD34+ cells) were isolated from umbilical cord blood by fluorescent activated cell sorting (FACS). MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were stained with carboxyfluorescein succinimidyl ester (CFSE) and cultured on MSC feeder-layer for 6 days. For evaluating the influence of the culture medium on MSC cultures, three different media conditions (M1-M3) were used. Medium 1 (M1) contained 2% fetal calf serum (FCS), medium 2 (M2) contained 10% FCS and medium 3 (M3) contained GMP-grade human platelet lysate (hPL). Proliferation of HSC was calculated by analyzing the distribution of the CFSE dye (measured at day 1 and day 6). Plerixafor was added in concentrations of 0.1, 1.0 and 10.0 μM. On day 6, HSCs were harvested and analyzed by flow cytometry for CD34, CD38 and CXCR4 expressions in relationship to the cell division rate.

When co-cultured with MSC, the division kinetics of HSC was increased, while the proportion of CD34+ cells remained significantly higher compared to HSC without MSC. Accordingly, more CD38 HSC were found after 6 days upon co-culture with MSC. All three MSC preparations supported self-renewing proliferation of HSC, whereas MSC M1 induced the strongest effect. This underlines that co-culture with MSC has a significant supportive function for hematopoiesis. The additional exposure to Plerixafor in the co-culture system partially reversed this effect in a dose-dependent manner: division rate of HSC and the proportion of CD34+ and CD38 cells were reduced with higher concentrations of Plerixafor. The reduction of self-renewing proliferation by Plerixafor was not observed in controls consisting of HSC without MSC. Plerixafor also rendered the CXCR4 receptors undetectable on the surface of CD34+ cells for up to 6 days, most probably due to a persisting blockade of the antibody-binding site.

Human HSC co-cultured with MSC showed an increased cell division rate and produced a higher proportion of CD34+/CD38 cells. Different MSC culture media were systematically analysed in this setting and subtle differences in the supportive function could be observed. The addition of Plerixafor neutralized the effects of MSC, leading to an earlier loss of “stemness” and to lineage-commitment of HSC, thus providing evidence for the role of the CXCR4/SDF1α axis in terms of supportive function of MSC for self-renewal of HSC.


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Author notes


Asterisk with author names denotes non-ASH members.