7s 100 light microscope

BMSCs at passage 3 were detached with trypsin EDTA (Hyclone; GE Healthcare) and plated at a low density ~1×10²/ml onto a fresh culture dish. The cells were cultured for 14 days at 37°C under humidified atmosphere of 5% CO₂ in DMEM (Hyclone; GE Healthcare) and half of the medium was renewed every 4 days. The cells were then fixed with 4% paraformaldehyde for 20 min and stained with Giemsa for 30 min at room temperature. The stained cells were observed under a Nikon 7S-100 light microscope (Nikon Corporation, Tokyo, Japan) and colonies including ≥50 cells were scored as a CFU-F.
Differentiation assay. BMSCs were harvested and seeded to 6-well culture dishes at a concentration of 2×10⁴/well with DMEM (Hyclone; GE Healthcare). When the culture reached 80% confluency ~5×10⁵/well, the medium was replaced with differentiation and induction medium from the StemPro Adipogenesis Differentiation kit, StemPro Osteogenesis Differentiation kit and the StemPro Chondrogenesis Differentiation kit (all kits from Invitrogen; Thermo Fisher Scientific, Inc.) for 2 weeks. The extent of adipogenesis, osteogenesis and chondrogenesis was observed by staining with oil red O, alizarin red S and alcian blue, respectively according to the manufacturer's protocol at room temperature (all dyes from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

An LTC-IC assay was conducted to detect the hematopoietic support ability of BMSCs. Firstly, BMSCs layers were seeded with 10 µl/ml mitomycin-c (Sigma-Aldrich) for 2 h at 37°C in humidified atmosphere of 5% CO₂. Then, HDs-HCs sorted using the CD34 MicroBead kit (Miltenyi Biotec Inc., Cambridge, MA, USA), were seeded onto the BMSC layers with MyeloCult H5100 medium (Stemcell Technologies, Inc., Vancouver, BC, Canada) for 5 weeks. Co-cultured CD34⁺ cells were then collected and cultured in MethoCult H4431 medium (Stemcell Technologies, Inc.) at a concentration of 1×10³/ml per dish for a further 14 days. Colony forming units of granulocyte/monocyte (CFU-GM) (≥40 cells), colony forming units of erythroid (CFU-E) (≥50 cells) and burst forming units of erythroid (BFU-E) (≥300 cells) were counted under the Nikon 7S-100 light microscope.