**2.1. Patients and donor characteristics**

BM samples from 116 MM patients were studied: 69 patients with MM at the time of diagnosis and 47 at relapse. All patients had indications for treatment. The main clinical characteristics of patients are shown in **Table 1**. Twelve bone marrow aspirates were received from healthy donors (HDs) as control. Written informed consent was obtained from all patients and donors in accordance with the Declaration of Helsinki and the ethical guidelines of the Charité University School of Medicine, which approved this study (Votum No.: EA4/131/13).

**2.3. Co-culture and transwell culture experiments**

For transwell cultures (0.4 μM pore size, Corning), 2 × 104

**2.4. Detection of SAβGalA and cell cycle analysis**

Data were studied using a logarithmic scale.

analyzed with the Rotor Gene 6000 software.

**2.6. Quantitative methylation-specific PCR (qMSP)**

**2.5. Quantitative real-time PCR (qPCR)**

lower chamber of a 12-well plate and incubated for 4 h. Then, 2 × 104

KMS12-PE cells served as negative control for transwell cultures and co-cultures.

KMS12-PE cells received from DSMZ (ACC606) were cultured in enriched RPMI media. For cocultures, MM-BMMSCs were seeded in a six-well plate and incubated for 4 h. Then, KMS12-PE myeloma cells were added followed by incubation for 72 h. After incubation, KMS12-PE cells were removed. The absence of CD138+ cells was confirmed using microscopy and checked with FACS analysis. MM-BMMSCs were washed twice with PBS and applied for future analysis. Co-cultured KMS12-PE myeloma cells were suspended in TRIzol for future analysis.

cells were added to the upper chamber. Incubation was performed for 72 h. Cultures without

SAβGalA was measured using the median fluorescence intensity (MFI) as previously reported [45]. Co-cultures of HD-BMMSC and HS-5 stromal cells (CRL-11882) were used as controls. In addition, β-galactosidase activity was analyzed using the "Senescence Cells Histochemical Staining Kit" (Sigma-Aldrich) as recommended by the manufacturer. Cell cycle analysis was performed using the "Cell Cycle Assay Kit" (Abcam) as recommended in the prescription.

Total RNA was extracted using TRIzol as described previously [46]. RNA was treated with DNase (Ambion) and poly(A)-polymerase (NEB) according to the manufacturer's instructions. About 800 ng of RNA was used for cDNA synthesis with a Transcriptor First Strand cDNA Synthesis Kit (Roche) and 2.5 μl of poly(T)VN adaptor primer (10 pmol) in a 20 μl reaction.

qPCR was performed with the FastStart Universal SYBR Green Master Mix (Roche). Primers were designed for each mRNA target using Primer3, OligoCalc, and OligoIDT. MiRNA detection was conducted using a specific miRNA primer and a universal reverse primer complementary to the adaptor sequence [47]. GAP-DH (for mRNA) and 5.8S rRNA (for miRNA) were chosen as housekeeping genes. QPCR was carried out with the Rotor Gene 6000 Real-Time PCR cycler. Cycling condition comprised 10 min at 95°C, 45 cycles of 15 s at 95°C and 60 s at 59°C, followed by a melting curve analysis from 60 to 98°C, rising by 1°/s. Efficiencies of qPCR were determined using linear regression analysis [48, 49] using LinRegPCR software, and relative quantifications were estimated with the Pfaffl method [50]. Received data were

DNA isolation was performed using Puregene reagents (Qiagen) according to the manufacturer's instructions. Genomic DNA was subjected to bisulfite treatment with the EpiTect Fast Bisulfite Conversion Kit (Qiagen) as recommended in the manual. Primers were used as

MM-BMMSCs were seeded in the

http://dx.doi.org/10.5772/intechopen.77179

Molecular Aberrations in Bone Marrow Stromal Cells in Multiple Myeloma

KMS12-PE myeloma

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