Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6 weeks. Four samples showed CD34+CD38− predominance, and four were predominantly CD34+CD38+. CD34+ CD38− predominant leukemia cells maintained the CD34+ CD38− phenotype and were viable for 6 weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34+ CD38+ predominant leukemic cells maintained the CD34+CD38+ phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4 weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34+ blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6 weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest MSCs provide pro-survival benefits to leukemic blasts through cell-cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

Background With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantitate immune-modulatory capacity of bone marrow derived mesenchymal stromal cells (BM-MSC). Methods Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third party BM-MSC. After stimulating with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression. Results The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16h) assay based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation (CV) between different responder T cells. Suppression by BM-MSC on different responders correlated with suppression by K299. We therefore used the K299 suppression as the reference to define suppression potency of BM-MSC in K299 Suppression Units (KSU). We found that inter-donor variability, passage number, method of manufacture, and exposure of BM-MSC to steroids or interferon gamma all affected BM-MSC potency of suppression. Conclusion This method provides a platform for standardizing suppressor function to facilitate comparison between laboratories and for use as a cell product release assay.

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