Methylcellulose medium

To evaluate the hematological toxicity of TC11, 4×10⁴ cells/mL of bone marrow cells from 13-wk-old male ICR mice were cultured in methylcellulose medium (Stem Cell Technologies, Vancouver, BC) containing FBS, 2-mercaptoethanol, 20 ng/mL mouse stem cell factor (mSCF), 20 ng/mL mouse interleukin 3 (mIL-3), 10 ng/mL mouse interleukin-6 (mIL-6), and 1 U/mL human erythropoietin (hEPO) (kindly provided by Kyowa Hakko Kirin Co., Tokyo) in the presence or absence of TC11. On day 14, various types of colony-forming cells were counted.

Parental NCI-H460-Luc2 cells (Caliper Life Sciences, Hopkinton, MA) and CDDP-R H460-Luc2 cells were cultured in RPMI 1640 (American Type Culture Collection, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 1% penicillin-streptomycin (Invitrogen) at 37°C and humidified 5% CO₂. Cisplatin (CDDP), basic fibroblast growth factor (bFGF), and insulin were obtained from Sigma-Aldrich (St Louis, MO). Epidermal growth factor (EGF) was obtained from BD Biosciences (San Jose, CA). Methylcellulose medium was purchased from STEMCELL Technologies (Vancouver, BC, Canada). BEZ-235 was obtained from Novartis Pharmaceuticals (Basel, Switzerland).

Purified umbilical cord blood CD34⁺ cells were co-cultured with hESC-MSCs or hBM-MSCs for 2 weeks [15 (link)]. To estimate the effects of MSCs on CFU growth in vitro, the co-cultured UCB-CD34⁺ cells were collected and seeded on a sterile 24-well plate and incubated in methylcellulose medium (Stem Cell). Then, numbers of colonies were calculated. Each group’s experiments were implemented in triplicates.

Colony formation assays were performed in accordance to a modified version of a previously reported protocol 16 (link). Donor murine BM cells were isolated from 6-week old B6.SJL (CD45.1) after injecting 5-fluorouracil (5-FU; 150mg/kg) for 5 days, and hematopoietic progenitor cells were isolated via a Mouse Lineage Cell Depletion Kit (Miltenyi Biotec Inc., Auburn, USA). These progenitor cells then underwent infection with the indicated viruses using a spinoculation protocol with the assistance of polybrene 36 (link)-38 (link). Following transduction, cells were incubated overnight at 37^oC in fresh media, and this was then repeated the following day. Thereafter, 2 × 10⁴ cells were plated in methylcellulose medium (Stem Cell Technologies Inc, Vancouver, Canada) containing 10 ng/ml IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), 30 ng/ml SCF, and 2 μg/ml puromycin and/or 1 mg/ml G418, as appropriate. After incubating for 7 days, colony formation in each of the experimental groups was assessed, and these colonies were then replated.

Mononuclear cells (MNCs) were prepared from bone marrow or spleen tissue using Histopaque (Sigma), and 1.0×10⁵ MNCs were cultured in a 3.5-cm dish containing methylcellulose medium (Stem Cell Technologies) supplemented with CID or rHuEPO. After 3 days of culture, the cells were stained with benzidine (Wako), and positive colonies were counted as colony-forming unit-erythroid (CFU-E). To count erythroid burst-forming unit erythroid (BFU-E)–derived colonies, the cells were cultured with 2.0 U/mL rHuEPO and 100 ng/mL stem cell factor (SCF, R&D Systems) for 7 days [23 (link)].