Methocult h4434 medium

The differentiated cells from a coculture system were harvested by manual picking and treating with 2 mg/ml Collagenase IV incubation for 30 minutes, and then with 0.25% Trypsin‐EDTA (Life Technologies) for 20 minutes. The single cells were counted and seeded at 1–2 × 10⁵ cell/ml into Methocult H4434 medium (StemCell Technologies) specified for CFC assay according to the manufacturer's protocol. The cells were maintained at 37°C, 5% CO₂ and the pattern of colony forming units (CFUs) was examined at day 12–13. At day 16, burst‐forming unit erythroid colonies were identified and picked for CD235a (Glycophorin A [GPA]) positive cell selection by the MACS (Miltenyi Biotech, San Diego, CA, http://www.miltenyibiotec.com) according to the manufacturer protocols. The purified CD235a positive cells were used for determining hemoglobin chain expression pattern. The cell morphology was evaluated by being spun onto a glass slide using cytocentrifugation (Shandon Cytospin 4; Thermo Scientific, Waltham, MA, http://www.thermoscientific.com) at 1,000 rpm for 5 minutes and stained with Wright‐Giemsa staining solution (Sigma‐Aldrich).

Colony-forming assays were performed as described previously.^{16 (link),17 (link)} For CD34⁺ cells self-renewal assessment, CD34⁺ cells were seeded in serum-free HPGM supplemented with rhIL-3, rhIL-6 and rhSCF as mentioned above and cultured in the presence of IMiDs or DMSO. After 14 days in culture, the CD34⁺ cells of each group (vehicle, LEN and POM) were purified using the CD34⁺ cell isolation kit and were plated in MethoCult H4434 medium (StemCell Technologies) for 14 days (without vehicle, LEN or POM).

Clonogenic colony-forming capacities of healthy BM and mobilized PB (10,000–30,000 cells), AML BM mononuclear cells (10,000–30,000 cells), and total dissociated EB cell suspensions (20,000–30,000 cells) plated in Methocult H4434 medium (Stem Cell Technologies, Vancouver, Canada, http://www.stemcell.com) were monitored between days 7 and 16, and colonies were quantified based on morphology between 14 and 16 days. Individual colonies were isolated and assessed for single-cell morphology, and full wells were collected for fluorescence in situ hybridization (FISH) analysis. Depending on number of colonies generated in CFU assay, single-cell morphologies of at least three colonies were analyzed to confirm colony quantification criteria and evaluate the maturity of colonies. Briefly, colonies were isolated and resuspended in 100 µl PBS and spun onto microscope slides using the Shandon Cytospin 3 (Block Scientific, Inc., Bellport, NY, http://www.blockscientific.com). Morphological features were visualized by Giemsa-Wright staining performed using Shandon Kwik-Diff Stain Kit (Thermo Scientific, Waltham, MA, http://www.thermoscientific.com). Images were acquired using ScanScope CS digital slide scanner with Aperio ImageScope software (Leica Biosystems).