Ex Vivo Assays to Study Self-Renewal, Long-Term Expansion, and Leukemic Transformation of Genetically Modified Human Hematopoietic and Patient-Derived Leukemic Stem Cells

Abstract

With the emergence of the concept of the leukemic stem cell (LSC), assays to study them remain pivotal in understanding (leukemic) stem cell biology. Although the in vivo NOD-SCID or NSG xenotransplantation model is currently still the favored assay of choice in most cases, this system has some limitations as well such as its cost-effectiveness, duration, and lack of engraftability of cells from some acute myeloid leukemia (AML) patients. Here, we describe in vitro assays in which long-term expansion and self-renewal of LSCs isolated from AML patients can be evaluated. We have optimized lentiviral transduction procedures in order to stably express genes of interest or stably downmodulate genes using RNAi in primary AML cells, and these approaches are described in detail here. Also, we describe bone marrow stromal coculture systems in which cobblestone area-forming cell activity, self-renewal, long-term expansion, and in vitro myeloid or lymphoid transformation can be evaluated in human CD34(+) cells of fetal or adult origin that are engineered to express oncogenes. Together, these tools should allow a further molecular elucidation of derailed signal transduction in LSCs.

The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

“…Cell lines were all tested mycoplasma free using a PCR-based assay. Primary AMLs were cultured in Gartner’s medium supplemented with G-CSF, N-plate, and IL3 (all 20 ng/ml), as described before ( Sontakke et al., 2014 ; van Gosliga et al., 2007 ). CB MLL-AF9 liquid and MS5 co-cultures under myeloid or lymphoid permissive conditions were performed as described previously ( Horton et al., 2013 ; Schuringa et al., 2004 ).…”

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms

“…Similarly, CD34 + hematopoietic progenitor cells transduced with shRNA against DDX41 showed significantly enhanced colony formation (Figure 4C). When serial replating assays were used to assess the effects of DDX41 on retention of clonogenic capacity (Sontakke et al, 2014;He et al, 2011), knockdown cells showed significantly increased replating efficiency consistent with retained clonogenic properties (Figure 4C). Anti-proliferative properties of DDX41 were also suggested by the results of cultures performed in the presence of various growth conditions.…”

Establishing human leukemia xenograft mouse models by implanting human bone marrow–like scaffold-based niches

“…In all cases, CD34 1 -sorted AML cells were grown with or without a cocktail of human cytokines, IL-3, G-CSF, and thrombopoietin, as previously described. [21][22][23] No long-term cultures could be established without stroma, although some expansion was observed for #2 in liquid culture conditions with cytokines, but within 20 days, cells differentiated and stopped expanding (supplemental Figure 5A). Human MSCs clearly were superior in supporting longterm expansion of both AML samples even in the absence of additional cytokines (supplemental Figure 5A).…”