H4435

For co-culture experiments, MSCs (2 × 10⁴/ml) were cultured in 24-well plates in 1 ml StemMACS+XF medium and treated daily with 2 µM PXS-5505 for 7 days. Monolayers were co-cultured with autologous or allogeneous CD34 + HSPCs (2 × 10⁴) in 1 ml StemSpan SFEM II medium with human HSPC expansion mix. Co-cultures were treated daily for 4 days with 5-AZA (100 nM), PXS-5505 (2 µM), P + A or vehicle (PBS). HSPCs were gently separated from co-culture and seeded into semi-solid MethoCult H4435 Enriched Methylcellulose-based medium with human recombinant cytokines (Stemcell Technologies, H4435) for CFU assays (3000 cells/35 mm dish, 1 ml). Colonies were counted manually with a Leica DMi1 inverted light microscope (Leica Microsystems) after 14-day incubation. CFU bulk cells were analyzed by flow cytometry. For transwell co-cultures, MSCs were treated daily with 2 µM PXS-5505 for 7 days in 24-well plates (Corning Falcon, 353504). The StemMACS+XF medium for MSC cultures was changed for StemSpan SFEM II medium with human HSPC expansion mix, and 1 × 10⁴ autologous CD34 + HSPCs were cultured in the 0.4 µm cell culture inserts (Corning Falcon, 353495) above MSC monolayers. The drugs were added daily for 4 days followed by CFU assays.

Methocult M3434 (murine) and H4435 (human) from STEMCELL Technologies were used for colony-forming assays. Methylcellulose was supplied with the indicated concentration of Epag or rTPO before cell seeding. Mouse bone marrow cells after red blood lysis were seeded at the concentration of 30,000 cells/mL. Bone marrow mononuclear cells from healthy donors and patients were seeded at the concentration of 100,000 cells/mL. CD34⁺ cells were infected with lentiviral TET2 targeting shRNA or nontargeting scr shRNA as described and characterized in our previous study (31 (link)). Two days after lentivirus infection, CD34⁺ cells were seeded at the concentration of 5000 cells/mL in Methocult in the presence of 5 μg/mL puromycin. Colonies were scored and cells were harvested for replating or flow cytometry analysis by FACSVerse (BD Biosciences) on days 10 to 14.

All human AML and cord blood samples were obtained with informed consent under local ethical approval (REC 07-MRE05-44). Primary AML cells or cord-blood-derived CD34⁺ cells were tested for colony-forming efficiency in H4435 semi-solid medium (Stem Cell Technologies) in the presence of the indicated concentration of MB3 or DMSO. Colonies were counted by microscopy 10–11 days (AML cells) or 12–14 days (CD34⁺ cells) after plating. See also Supplemental Information.