LTC-IC and CFU assays

Long-term culture-initiating cells (LTC-ICs) assay were following the protocol described previously²⁵. The murine stromal cell lines (M2-10B4 and SL/SL mixed at 1:1) and irradiated with 8000 cGy were plated in 96-well collagen-coated microtiter plates (5000 cells/well of each cell line) and cultured in long-term culture medium (MyeloCult H5100; Stem Cell Technologies) supplemented with hydrocortisone 21–hemi-succinate (10⁻⁶ M). Limiting dilution analysis (LDA) for quantitating LTC-ICs present in the Lin−CD34+CD38−CD45RA−CD90+CD49f+ cell subset 5 days after expansion in C₃-cytokines plus Scriptaid or the vehicle control. Cells were plated at 50 to 150 cells per well (10–100 for unexpanded condition) by flow sorting⁴³. Co-cultures were maintained at 37 °C in high humidity and with 50% medium exchange every week. After 6 weeks, all cells were plated in methylcellulose cultures supplemented with complete methylcellulose-based medium (MyeloCult H4435; Stem Cell Technologies)^25,42,43. Long-term culture colony-forming cells (LTC-CFCs; readout of LTC-IC assay) were scored after an additional 14–16 days of culture^25,43. Limiting Dilution Assay (LDA) analysis was performed on ELDA (http://bioinf.wehi.edu.au/software/elda/) based on the number of negative wells (showing no CFCs) in each condition with a confidence interval of 0.95.