Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells

Abstract

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 6weeks. 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 6weeks 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 4weeks. 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 6weeks 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 that 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.

Figure 1. Numbers of viable AML blasts generated during culture

(A) Trend of absolute numbers of viable LSC-like leukemic blasts excluding UPN1 (separately shown in Supplementary Figure 2). Better viability of leukemic blasts was observed when co-cultured with MSCs, MSCs supplemented with cytokines (MSCs + Cyto) (IL-3, Flt3-ligand, and SCF), cytokines alone (Cyto) in contrast to medium only (Medium). (B) Trend of absolute numbers of viable non-LSC-like leukemic blasts. (C) Microscopic appearance of leukemic blasts co-cultured with MSCs. Representative images for UPN3 (CD34⁺ CD38⁻ predominant leukemia) indicating preserved morphology of leukemic blasts cultured for 2 weeks in the presence (middle panel) or absence (right panel) of MSCs.

Figure 2. Phenotype of AML blasts during culture

(A) Percentage of CD34⁺CD38⁻ cells in CD34⁺ CD38⁻ predominant leukemic samples during culture with MSCs, MSCs with cytokines (MSCs + Cyto), cytokines alone (Cyto), or medium alone (Medium). CD34⁺ CD38⁻ predominant leukemias (average %CD34⁺CD38⁻ 72.5%; range 55.6-90.0% at day 0) maintained a higher proportion of CD34⁺CD38⁻ cells when co-cultured with MSCs compared to other culture conditions. (B) Percentage of CD34⁺CD38⁺ cells in CD34⁺ CD38⁺ predominant (average %CD34⁺CD38⁺ 55.6%; range 30.3- 89.9% at day 0) leukemic samples during culture. CD34⁺ CD38⁺ predominant leukemias maintained a higher proportion of CD34⁺CD38⁺ cells when co-cultured with MSCs. (C) Trend of absolute numbers of viable CD34⁺CD38⁻ cells in CD34⁺ CD38⁻ predominant leukemic blasts excluding UPN1 (separately shown in Supplementary Figure 2). (D) Trend of absolute numbers of viable CD34⁺CD38⁺ cells in CD34⁺ CD38⁺ predominant leukemic blasts. (E) Representative immunophenotype data of CD45⁺ leukemic blasts from patient UPN4 (CD34⁺ CD38⁻ predominant leukemia) and UPN6 (CD34⁺ CD38⁺ predominant leukemia) during culture. Co-culture with MSCs favored persistence of the original phenotype compared to other culture conditions.

Figure 3. Cell cycle analysis of AML blasts during culture

(A) Representative cell cycle analysis of UPN2 (CD34⁺ CD38⁻ predominant leukemia) 1 week after culture with MSCs, MSCs with cytokines (MSCs + Cyto), or cytokines alone (Cyto). (B) Summary of cell cycle analysis before co-culture and 1 week after co-culture (n=8). A higher proportion of cells remained in G0 when co-cultured with MSCs compared to other culture conditions using cytokines. (C) Trend of absolute numbers of CD34⁺ CD38⁻ predominant leukemic cells in the G0 phase of the cell cycle. (D) Trend of absolute numbers of CD34⁺ CD38⁺ predominant leukemic cells in the G0 phase of the cell cycle.

Figure 4. Transplantation of AML blasts in NSG mice

(A) Summary (post MSCs, n=5 mice; uncultured, n=2 mice) and (B) representative experiment of human cell engraftment in the bone marrow of NSG mice 8 weeks after transplantation. Both leukemic blasts harvested 4 and 6 weeks after MSC co-culture, respectively, maintained their ability to engraft compared to uncultured blasts derived from the original leukemic samples. The engrafted cells retained the original phenotypes regarding to CD34 and CD38 expression.

Figure 5. Cytogenetic analysis of leukemic blasts

(A) Representative images of spectral karyotype analysis (SKY) and (B) cytogenetic abnormalities from clones found in leukemic blasts. For both UPN1 blasts (CD34⁺ CD38⁻ predominant leukemia) and UPN6 blasts (CD34⁺ CD38⁺ predominant leukemia), the original chromosomal abnormalities (upper panels) persisted in CD45⁺ blasts sorted 6 weeks after MSC co-culture (middle panels) as well as in CD45⁺ blasts sorted from the bone marrow of NSG mice 8 weeks after transplantation (lower panels). Additional chromosomal aberrations were also detected.

Figure 6. Chemosensitivity of leukemic blasts to cytarabine

Cells from six primary leukemic samples were cultured for 48 hours with or without MSCs in the presence of different doses of cytarabine. Data are represented as a relative percentage of viability compared MSC co-cultured blasts. The viability of leukemic blasts was significantly higher when co-cultured with MSCs.