Recombinant human basic fgf

hESCs were used according to the hESC research guidelines of the Japanese government. For the experiments shown, we used the VA22-N37 cell line^{11 (link)}; however, for reproducibility, the results were confirmed using KhES-1 (KUIMSe001-A) and KhES-3 (KUIMSe003-A) cells (Fig. S4). VA22-N37 is a RAX::Venus reporter hESC line^{11 (link)} established based on KhES-1 (KUIMSe001-A) and they are biological replicates. Undifferentiated hESCs were maintained on a feeder layer of mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C treatment in DMEM/F12 (D6421/Sigma) supplemented with 20% (vol/vol) KSR (lot No. 1517496/Invitrogen), 0.1 mM non-essential amino acids (11140-050/Invitrogen), 2 mM L-glutamine, 5 ng/mL recombinant human basic FGF (068-04544/Wako), and 0.1 mM 2-mercaptoethanol under an atmosphere of 2% CO₂. For passaging, hESC-colonies were detached and recovered en bloc from culture dishes by treating them with 0.25% (wt/vol) trypsin and 0.1 mg/mL collagenase IV in PBS containing 20% (vol/vol) KSR and 1 mM CaCl₂ at 37 °C for 8 min. The detached hESC clumps were broken into smaller pieces by gentle pipetting. The passages were performed at a 1:5 split ratio. When we performed SFEBq culture, small broken pieces of hESC clumps were incubated on gelatin-coated plate for 90 minutes to eliminate MEFs (Fig. S5).

HEK293T cells were obtained from the RIKEN Cell Bank (Saitama, Japan) and cultured in DMEM (FUJIFILM Wako Pure Chemical) supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO₂ (42 (link)). The patient-derived OS cell line 143B was obtained from the ATCC (Manassas, USA) and cultured in adherent medium containing DMEM supplemented with 10% FBS, 110 μg/mL sodium pyruvate (FUJIFILM Wako Pure Chemical), and 1% penicillin/streptomycin. Both cell types were cultured in tissue culture dishes (SARSTEDT) to ensure optimal adherence and expansion. To enrich stem-like cells, 143B cells were harvested using trypsin (BD Bioscience) and EDTA (FUJIFILM Wako Pure Chemical), then cultured in osteosphere medium containing DMEM/F12 (FUJIFILM Wako Pure Chemical) supplemented with 20 ng/mL recombinant human EGF (FUJIFILM Wako Pure Chemical), 20 ng/mL recombinant human basic FGF (FUJIFILM Wako Pure Chemical), B27 supplement without vitamin A (Gibco), GlutaMAX (Thermo Fisher Scientific), and 1% penicillin/streptomycin. Under these conditions, the cells were incubated in Ultra-Low Attachment Surface culture dishes (Corning). To assess the differentiation potential of OSCs, the cells were transferred from osteosphere to adherent medium, and from Ultra-Low Attachment Surface to tissue culture dishes, to promote adherence and differentiation.

We used chicken B cell lymphoma cell line DT40. In addition, we used two DT40 cell lines containing one human chromosome 4 (DT40 + hCh4) or one mouse chromosome 11 (DT40 + mCh11), which were created via microcell-mediated cell fusion (Oshimura et al. 2015 (link)). Chicken DT40 cells were cultured in RPMI medium 1640 (Gibco^®, Thermo Fisher Scientific, Waltham, MA, USA), 10% foetal bovine serum (FBS, Corning, NY, USA), 0.1% chicken serum, 0.1 mM β-mercaptoethanol (2ME, Sigma-Aldrich, St. Louis, MO, USA). Cells were grown under 5% CO₂ at 39˚C. Mouse J1 ESCs were cultured on the feeder cells in DMEM F12 (Wako, Osaka, Japan) supplemented with 10% FBS, 0.1 mM nonessential amino acid and sodium pyruvate (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), penicillin–streptomycin and 2 mM l-glutamine (Gibco^®), 0.1 mM 2ME, and 10³ U/ml ESGRO^® LIF (Merck, Kenilworth, NJ, USA). Feeder cells were mitomycin C-treated mouse embryonic fibroblasts (MEF) derived from embryonic day 12.5. Human female iPSCs (KAC, Kyoto, Japan) were cultured on feeder cells in DMEM F12 HAM (Wako) supplemented with 20% KnockOut Serum Replacement (KSR™, Thermo Fisher Scientific), 0.1 mM nonessential amino acid, 2 mM L-glutamine, 1 × penicillin–streptomycin, 0.1 mM 2ME, and 5 ng/ml recombinant human basic FGF (Wako). Mouse and human cells were grown under 5% CO₂ at 37 °C.