Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells - Implications for myeloma bone disease

In this study we analyzed the myeloma cell contact-mediated changes on the transcriptome of skeletal precursor cells. Therefore, human mesenchymal stem cells (MSC) and osteogenic precursor cells (OPC) were co-cultured with the representative myeloma cell line INA-6 for 24 h. Afterwards, MSC and OPC were separated from INA-6 cells by fluorescence activated cell sorting. Total RNA of MSC and OPC fractions was used for whole genome array analysis. Primary MSC from the cancellous bone from the acetabulum received from donors after total hip arthroplasty were isolated as described by Noth et al., 2002 (PMID:12382974) and expanded in DMEM/Ham´s F12 medium including 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml L-Ascorbic acid 2-phosphate. MSC were passaged at least once before they were used for experiments. Osteogenic precursor cells (OPC) were generated by incubating MSC with DMEM High Glucose medium for two weeks supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mmol/l beta-glycerophosphate, 100 nmol/l dexamethasone, and 50 µg/ml L-Ascorbic acid-2-phosphate. The myeloma cell medium consisted of RPMI 1640 medium, 20% FCS, 100 µg/ml gentamicin, 2 mmol/l L-glutamine, and 1 mmol/l sodium pyruvate. One day before co-culturing, confluent MSC and OPC monolayers were adapted to cell culturing conditions by incubating them with a 1:1 (v/v) mixture of MSC/myeloma cell medium and OPC/myeloma cell medium and INA-6 cells were stained with 5 µmol/l Cell Tracker® Green 5-chloromethylfluorescein diactetate. Afterwards, INA-6 cells were washed with PBS and used for co-culture. After 24 h, cells were trypsinized and separated by fluorescence activated cell sorting (BD FACS AriaTM III cell sorter). The control cells were treated accordingly. Cell pellets of MSC and OPC fractions were lysed in RA1 buffer containing 1% 2-mercaptoethanol and stored at -80°C until total RNA extraction. Total RNA was isolated with the NucleoSpin® RNA II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer´s instructions. The whole genome array analysis was performed using the Affymetrix HG-U133 Plus 2.0 GeneChips (Affymetrix, High Wycombe, United Kingdom). Each 5 control specimens and 5 co-cultured samples of both MSC and OPC were used for hybridization.