H5100

Human CD34+ cells were plated at a concentration of 500 cells/mL in semi-solid methylcellulose cultures for colony-forming cell (CFC) assay (H4435 Stem Cell Technologies). The type and number of CFC progenitors were scored 7 and 14 days later using an inverted microscope. Similarly, CML-derived CD34+ cells were subjected to 100 μM SR1 for 4, 7 and 14 days and assayed for CFC content in similar conditions as above. CML CD34+ cells subjected to SR1 treatment for 4 or 7 days were assayed for LTC-IC content as previously described [6 (link)]. Briefly, CD34+ were plated with a monolayer of MS5 stromal cells and cultured in long-term initiating medium (H5100 Stem cell Technologies) during 5 weeks in 96-well plates. After 5 weeks, all wells were sacrificed by trypsinization, washed and plated in methylcellulose cultures for colony-forming cell (CFC) assay and quantification of the number of LTC-IC as described previously [6 (link)].

AGM explants (AGM^ex) were performed as previously described (Medvinsky and Dzierzak, 1996 (link); Muller et al, 1994 (link)). Briefly, AGM regions were laid on a nylon membrane (Millipore) with metallic supports and cultured in MyeloCult M5300 or H5100 (Stem Cell Technologies) supplemented with 10 µM hydrocortisone (Sigma-Aldrich). After 3 days culture, explants were digested into single cells by 0.125% collagenase digestion (Sigma-Aldrich) for flow cytometry analysis or further culture. In some conditions, TNFSF14 or TNF was added into the explant cultures.
Flow-sorted ECs, pre-HSC I and pre-HSC II cells were cocultured with OP9-DL1 cells (stem cell factor, 100 ng/mL; IL-3, 100 ng/mL; and Flt3-ligand, 100 ng/mL; PeproTech) or OP9 cells (stem cell factor, 20 ng/mL; IL-7, 10 ng/mL; and Flt3-ligand, 10 ng/mL; PeproTech) for 3–10 days as previous report (Li et al, 2013 (link)). In some conditions, transwells were used to separate Mks and HECs. Cells were harvested by mechanical pipetting for flow cytometric analysis.

M2-10B4, a murine fibroblast cell line, was used as a feeder layer. At least 24 h before assay, M2-10B4 cells were radiated (80 Gy) and seeded in six-well plates as feeder cells (2.5 × 10⁵/well). The plates were coated with collagen solution (StemCell Technologies, Vancouver, Canada). The cells harvested from different coculture conditions at day 10 were resuspended with H5100 containing 10⁻⁶ M hydrocortisone (StemCell Technologies, Vancouver, Canada) and then seeded into the plate with the feeder layers. Half of the medium was replaced in weekly intervals. Both nonadherent and adherent cells were harvested at week 5 and reseeded in semisolid culture (H4434, StemCell Technologies, Vancouver, Canada) for CFC assay. After 16 days, colonies were scored under an inverted microscope. The LTC-IC number was calculated according to the manufacturer’s instructions. Each eight CFC colonies correspond to one LTC-IC, and the LTC-IC numbers per 5000 UCB-CD34+ cells before cultivation (day 0) and after harvesting (day 10) were calculated.

Human CD34⁺ cells were purified from umbilical cord blood using a Dynabeads 34 positive isolation kit (Miltenyl Biotec, Bergisch Gladbach, Germany). For coculture, MSCs from 2D or 3D culture were irradiated (1,500 cGy) 24 h before and subsequently cocultured with purified CD34⁺ cells for 5 days in long-term culture medium (H5100; STEMCELL Technologies, Vancouver, Canada) in the presence of a cytokine mixture (100 ng/mL human SCF; 100 ng/mL human Flt3L; and 20 ng/mL human IL-3, IL-6, and G-CSF; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel). For phenotypic analysis of ex vivo expanded hematopoietic cells, cocultured cells were stained with antibodies against CD45 (BD Pharmingen, San Jose, CA), CD34 (BD Pharmingen), and CD90 (BD Pharmingen) and then analyzed by flow cytometry after gating the CD45⁺ population, as previously described^{63 (link)}. Colony-forming assays were performed by plating the cocultured cells in semisolid medium (H4230; STEMCELL Technologies) for 14 days. The number and type of colony, such as colony-forming unit erythroid (CFU-E), burst-forming unit erythroid (BFU-E), colony-forming unit granulocyte–macrophage (CFU-GM), and colony-forming unit granulocyte–erythroid–macrophage–megakaryocyte (CFU-GEMM), were determined by microscopic examination of the colony.