Superarray

To assess endogenous levels of osteoblastic mRNAs in osteogenically differentiating MSCs in contact with OBCs, MSCs and OBCs were co-cultured in a 1:4 ratio at 9,000-10,000 cells/cm². Before co-culture, trypsinized MSCs were stained with the cell tracker CM-DiI (Thermo Fisher, Waltham, MA; 10⁶ cells/mL + 8 μL CM-DiI, 37 °C, for five min, then 4 °C, for 15 min) to distinguish them from OBCs and to allow subsequent fluorescence-activated cell sorting (FACS). Three co-culture combinations were tested in both EM and OM: (1) DiI-labeled MSCs mixed with unlabeled OBCs, (2) DiI-labeled MSCs mixed with unlabeled MSCs, and (3) unlabeled, MSC-only cell populations, with the latter two conditions serving as controls. Samples were harvested pre-culture, and at culture days 6 and 12. MSC-only cultures were trypsinized, centrifuged, resuspended in 1 mL Trizol (Invitrogen), and stored at -80 °C; mixed cultures were trypsinized and frozen in dimethyl sulfoxide (DMSO) freeze medium (BioVeris Corporation, Gaithersburg, MD, USA) until the time course was complete. The DiI-positive cells were fractionated using a Becton-Dickinson three-laser Dako MoFlo FACS sorter, pelleted by centrifugation, resuspended in 1 mL Trizol, and stored at -80 °C. Osteogenesis was assessed by conventional real time RT-PCR and by PCR array analysis (SuperArray, Qiagen, Valencia, CA, USA; see below).

Total RNA was isolated from the hippocampi with RNeasy Lipid Tissue Mini Kit (Qiagen, cat# 74804). cDNA was synthesized from 0.5 to 1 μg of purified RNA by iScript Reverse Transcription Supermix (Bio-Rad, cat# 1708841) according to the manufacturer’s instructions. Quantitative PCR was performed in 20 μL reaction using IQ SYBR Green Master Mix and CFX96 Real-Time System standard protocol (Bio-Rad Laboratories, Hercules, CA). Specific validated primers for murine DCX, Ng2, Gro1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as human Gro1, fibroblast growth factor 2 (FGF2), and glial cell-derived neurotrophic factor (GDNF), were purchased (SuperArray, Qiagen, Germantown, MD). Triplicate PCR reactions yielded threshold cycle (Ct) average, with a coefficient of variance of < 0.05%, which were used to determine ΔCt values [ΔCt = Ct of the target gene minus Ct of the housekeeping GAPDH gene]. A comparative threshold cycle (C_T) method was used for relative gene expression quantification. All experiments included template-free (water) and reverse transcriptase-minus controls to ensure no contamination. Relative quantities of mRNA in experimental samples were determined, normalized to GAPDH, and expressed in arbitrary units as fold difference from control (control was taken as 1).