In this study we demonstrated that unrelated

In this study, we demonstrated that unrelated allogeneic human MSCs supported a LSC phenotype in a largely quiescent state in a long-term vitro culture system. We then confirmed the persistence of LIC after 6weeks co-culture with MSCs using the NSG mouse xenotransplant model. Our results also revealed that MSCs, in contrast to cocktails of stem cell growth factors or medium alone, support survival of quiescent LSCs with minimal outgrowth of more differentiated leukemic populations. Of interest, for one of 8 leukemic samples studied, LSCs were maintained in culture for up to 6weeks even without MSC support. Variability between leukemic samples has been described before (Klco et al., 2013). While the mechanisms underlying the atypical in vitro growth pattern of leukemic blasts in this patient remain unclear, we have observed increased telomere length in this sample compared to leukemic s ibuprofen cost derived from age-matched patients (unpublished data). The maintenance of viable leukemic blasts in a stromal co-culture system is consistent with the previous findings observed by van Gosliga et al (van Gosliga et al., 2007) using murine MS5 stromal cell lines and by Klco et al (Klco et al., 2013) using human HS27 stromal cell lines to expand primary AML blasts. However, both groups combined MSCs with growth factors (SCF, IL-3, Flt-3 ligand +/− TPO), and one study demonstrated loss of the CD34+ cell population after long-term co-culture (van Gosliga et al., 2007). While the distinct stromal cells utilized in each study may account for the differences observed, our data suggest that addition of growth factors during co-culture results in depletion of LSCs by promoting their differentiation in vitro. We also demonstrated that the protective effect on LSCs requires direct cell-to-cell contact since a transwell culture system failed to replicate the findings in vitro. The nature of the interactions between MSCs and leukemic blasts that favor survival, quiescence and maintenance of the original phenotype remains to be elucidated. Despite the prolonged culture (and in keeping with the preservation of quiescence) we found that the clonality of the original leukemic population was largely preserved by MSCs. This ability of MSCs to preserve the original leukemic phenotype was observed more significantly in CD34+ CD38− predominant leukemias than CD34+ CD38+ predominant leukemias. The non-LSC-like leukemias had two logs fewer leukemia initiating cells than the LSC samples, thus the preferential pro-survival effects of MSCs in vitro may be largely restricted to preserving leukemic stem cells. Further studies to identify the mechanisms of LSC support by MSCs should shed light on the role of MSCs in the marrow milieu. While remission induction has continued to improve in patients with AML, disease relapse remains a major limitation for cure. We showed that leukemic blasts maintained on MSCs were more resistant to cytotoxicity from cytarabine, a widely used nucleoside analogue to induce remission in AML. Currently allogeneic stem cell transplantation represents one of the most reliable strategies for eliminating residual disease through a graft-versus-leukemia (GVL) effect. Leukemic cells capable of initiating leukemia in a xenograft model are currently the best descriptor of the cell type responsible for leukemic relapse after chemotherapy. Our in vitro model could be used to study interactions of alloreactive lymphocytes with GVL-like effects on LSCs as well as to explore new agents with specificity for quiescent LSCs. While we show differences in the supportive capacity of MSCs for CD34+ CD38− predominant and CD34+ CD38+ predominant leukemias, additional studies will be needed to clarify the nature of MSC interactions with specific AML subtypes, and to correlate LSC maintenance in vitro with relapse probability in vivo.
Conclusion The following are the supplementary data related to this article.
Acknowledgments
Introduction