Hbmscs

Mouse BMSCs were isolated from 8-weeks-old male C57BL/6 J mice as described previously described [18 (link)]. Bone marrow was flushed out from mouse tibia and femur into Eppendorf tubes, centrifuged for 1 min at 400 g to collect the marrow cells. Cell were then filtrated through a 70-μm nylon mesh filter and cultured in 175 cm2 flasks in basal culture media (BCM) consists of RPMI-1640 medium supplemented with 12% FBS (Gibco Invitrogen, USA), 12 μM L-glutamine (Invitrogen) and 1% penicillin/streptomycin (P/S) (Gibco Invitrogen, USA). After 24 h, non-adherent cells were removed from the cultured medium, washed with PBS, and cultured in 30 ml of fresh medium in 60 cm2. Medium was changed every 3–4 days and cells were washed, trypsinized and regularly sub-cultured.
Human BMSC cells (hBMSCs) were purchased from Cell Applications Inc. (San Diego, CA). Cells were cultured according to the manufacturer’s instruction, in Dulbecco’s modified Eagle medium (DMEM)/low glucose (Sigma-Aldrich) containing 10% FBS Gibco Invitrogen, USA) and 1% penicillin/streptomycin. Non-adherent cells were removed after 2 days in culture and medium was changed every 2–3 days.

Karyotype analysis was performed by Q-banding using conventional methods (Nihon Gene Research Laboratories Inc., Sendai, Japan). The hBMSCs obtained by purchasing from Cell Applications Inc. (San Diego, CA) were prepared as mentioned above. The cells were cultured with autologous PL supplementation and passed twice before the analysis. The cells were divided into two groups about pretreatment; thus, one was treated with the synchronous culture using thymidine, while the other was not treated with it. The cells in both groups were incubated in quinacrine mustard dihydrochloride for 10 min and then immersed in PBS. They were stained with Hoechst 33258 for 10 min and immersed in distilled water. They were mounted on the slides with an antifade reagent.