Dulbecco s modified eagle s medium dmem lg

This study was approved by the Institutional Review Board (IRB) of Yonsei University College of Medicine (IRB No: 4-2017-0232) and informed consent was obtained from all participants for human bone marrow samples used in this study. Bone marrow aspirates were obtained from the posterior iliac crests of seven adult donors (55–65 years old) and isolated as previously described^{52 (link)}. hBMSCs were maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG; Gibco) supplemented with 10% FBS (Gibco) and 1% antibiotic/antimycotic solution (Gibco) and were used within five passages. RAW264.7 cells (Korean Cell Line Bank, Seoul, South Korea) were maintained in high-glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% antibiotic/antimycotic solution (Gibco). mBMMs were cultured in α-minimum essential medium (Gibco) containing 10% FBS and 1% antibiotic-antimycotic solution. Polaprezinc (CAS 107667-60-7, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was dissolved in 1 M hydrogen chloride and used at a final concentration of 50 μM. ZnSO₄ was purchased from Sigma-Aldrich.

This study was approved by the Institutional Review Board of Yonsei University College of Medicine (IRB no. 4-2017-0232). Human bone marrow aspirates were obtained from the point 3 cm posterior to the anterior superior iliac spine of 10 healthy adult donors with a mean age of 35 years (range: 30–40 years). hBMSCs were maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG; Gibco BRL, Rockville, MD, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic-antimycotic solution (Gibco). Human embryonic kidney 293 (HEK293) and 293FT cells were obtained from American Type Culture Collection (Manassas, VA, USA). These cells were maintained in high-glucose DMEM (DMEM-HG; Gibco) containing 10% FBS and 1% antibiotic-antimycotic solution (Gibco). DKK1 (R&D Systems) was used at a concentration of 0.1 mg/mL in hBMSCs^{53 (link)}. Wnt3a (cat. no. 5036-WN; R&D Systems) was used at a concentration of 10 ng/mL in hBMSCs^{54 (link),55 (link)}.

Human bone marrow from four healthy donors were collected in an aseptic condition from Ramathibodi Hospital with informed consent as part of the study protocol that was approved by the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (MURA2017/603). Fresh human bone marrow was diluted with 1× PBS (ratio 1:1) and was gently layered on Histopaque 1.077 at a dilution of 1:1 in 15 ml centrifuge tube that is subsequently centrifuged at 400 × g for 30 min at 25°C. The PBMC layer was transferred to a new centrifuge tube and washed twice with 1× PBS. Cell pellet was resuspended with low-glucose Dulbecco’s Modified Eagle’s medium (DMEM-LG; GIBCO, United States) supplemented with 10% Fetal Bovine Serum (FBS; Merck Millipore, United States), 1% GlutaMAX (GIBCO, United States), and 1% Penicillin and Streptomycin (GIBCO, United States) and incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. All experiments in this study were performed by MSCs passage 3–6.