Loss of Stem Cell Quiescence and Impaired Stem Cell Function of CD34⁺/CD38^low Cells One Year Following Autologous Stem Cell Transplantation

Abstract 1893

Autologous stem cell transplantation (ASCT) allows the application of high-dose chemotherapy and is included in the standard treatment regimens for multiple myeloma and relapsing lymphoma. The application of ASCT has considerably improved treatment outcome but in 30–50% of the patients the underlying malignant disorder relapses. In that case treatment options are limited, in part due to a diminished capacity of the transplanted bone marrow to tolerate chemotherapy. The transplanted bone marrow seems to be more vulnerable to chemotoxic stress. This is supported by our recent observations that progenitor cells from post-ASCT bone marrow demonstrate altered phenotypic and functional properties (Woolthuis et al. BMT 2011:110-115). In the present study, a more detailed examination of the hematopoietic stem cell (HSC) compartment post-ASCT was performed. The examined bone marrow cells were obtained from patients with normal peripheral blood cell counts one year after ASCT. First it was studied whether stem cell quiescence had been changed due to the ASCT procedure. Therefore the percentage of cells in the G0 phase was measured by staining the cells with Hoechst and Pyronin Y followed by flowcytometric analysis. Both the stem cell (CD34⁺/CD38^low) and progenitor (CD34⁺/CD38⁺) fractions were analyzed. Post-ASCT bone marrow cells (n=6) were compared with normal bone marrow cells (n=9) and mobilized peripheral blood stem cells (PBSC) (n=7). Interestingly, post-ASCT bone marrow contained a significantly lower percentage of quiescent cells in the CD34⁺/CD38^low fraction compared to normal bone marrow (mean percentage 23.6% (95%CI: 6.5–40.8) vs. 48.6% (95%CI: 31.4–65.9), p=0.045). In contrast no differences were observed in the CD34⁺/CD38⁺ fraction. To examine whether the loss of stem cell quiescence is associated with loss of stem cell function, stem cell frequency was analyzed in vitro by the long-term colony-initiating cell assay. These analyses revealed a strongly decreased stem cell frequency in post-ASCT CD34⁺ cells compared to normal CD34⁺ bone marrow cells (mean frequency 0.0016 (95%CI: 0.0003–0.0028) vs. 0.0206 (95%CI: 0.0162–0.0250), p=0.002). Importantly, CD34⁺ PBSC cells demonstrated a stem cell frequency comparable with the frequency observed in normal bone marrow, suggesting that the decrease in stem cell frequency observed in post-ASCT bone marrow can not simply be explained by the previous chemotherapy and the mobilization procedure. To obtain more insight into the mechanisms explaining our observations, levels of reactive oxygen species (ROS) were measured by flowcytometry. Comparable ROS levels were observed in post-ASCT and normal bone marrow CD34⁺/CD38^low cells while the CD34⁺/CD38⁺ post-ASCT cells demonstrated significantly higher ROS levels compared to normal bone marrow (p=0.01). In addition significant higher ROS levels were observed in CD34⁺/CD38^low PBSC compared to normal bone marrow (p=0.043). To define pathways associated with altered quiescence and/or ROS production, gene expression analysis was performed comparing CD34⁺ cells from post-ASCT bone marrow (n=6), normal bone marrow (n=31) and PBSC (n=5). Preliminary analysis reveals an upregulation in post-ASCT CD34⁺ cells of genes involved in cell cycle compared to normal bone marrow. In contrast, cell cycle genes appeared to be downregulated in CD34⁺ PBSC cells. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow post-ASCT is likely related to a loss of stem cell quiescence and enhanced ROS production by progenitor cells which will make the bone marrow cells more vulnerable for the effects of cytotoxic stress.