C 28010

Normal human osteoblasts (CC-2538, LONZA, Basel, Switzerland) were maintained in the OGM Bullet Kit (CC-3207, LONZA). Human mesenchymal stem cells from umbilical cord matrix (C-12971, Promo Cell, Heidelberg, Germany) were maintained in mesenchymal stem cell growth medium (C-28010, Promo Cell). Normal human dermal fibroblasts (KF-4109, KURABO, Osaka, Japan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (044-29765, Wako Pure Chemical Industries, Osaka, Japan) containing 10% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan) and 1% penicillin-streptomycin (168-23191, Wako Pure Chemical Industries). The cells were maintained at 37 °C with 5% CO₂ and were used within 4 to 6 passages.

At passage 3, HMSCs were trypsinized, washed, and resuspended in mesenchymal stem cell medium (PromoCell, C-28010) at a concentration of 1 × 10⁵ cells/mL. HMSCs were fixed with paraformaldehyde at 4°C for 15 min and washed with distilled water before permeabilization in 0.5% Triton-X100. Non-specific binding was blocked using blocking solution (PBS containing 1% bovine serum albumin (BSA)) for 1 hour at room temperature. HMSCs were then incubated 2 hours at room temperature with primary antibodies from rabbit against antigrowth associated protein-43 (GAP-43, 1 : 200) (Chemicon, AB5220) and against antiglial fibrillary acidic protein (GFAP, 1 : 500) (Chemicon, AB5804) and from mouse against antineuronal nuclei (NeuN, 1 : 100) (Chemicon, MAB377). After washing, HMSCs were incubated 15 minutes with goat anti-rat IgG (Millipore, AP136P) and goat anti-rabbit IgG (Millipore, 12-348MN) secondary antibodies. After several washes in PBS, HMSCs were incubated with horseradish peroxidase (HRP-) coupled streptavidin for 10 min. DAB (diaminobenzidine) served as chromogen (Figure 2).

All hMSCs were thawed in Reference Medium; for commercial hMSCs, MSC basal medium (ref. PT3238, Lonza) or MSC cell growth medium (ref. C-28010, PromoCell) were used depending on the hMSCs commercial supplier. Non-commercial hMSCs were thawed in FBS-Medium, composed of DMEM low glucose (ref. 21969, Gibco-Life Technologies) supplemented with 10% MSC-qualified FBS (ref. 10500, Gibco-Life Technologies), 1% L-glutamine (ref. 25030, Gibco-Life Technologies) and 1% penicillin/streptomycin (ref. 15140, Gibco-Life Technologies). hMSCs were cultured at 37 °C and 8% CO₂. Medium was replaced every 3–4 days until arriving at 80% confluence, when cells were detached using xeno-free trypsin (Tryple Express, ref. 12604, Gibco-Life Technologies). hMSCs were split and seeded at a cell density of 5000–6000 cells/cm² using the different media (Reference Medium, XF-Medium lot 1 and lot 2, PL-Medium lot 1 and lot 2). After each passage, population doubling time (PDT) was calculated as described elsewhere [29 (link)]. Statistical analysis was performed using GraphPad Prism Version 5.01 (GraphPad Software, San Diego, CA, USA) and applying the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison test.