Differentiation into osteocytes was achieved by adding 1-1000 nM dexamethasone, 0.25 mM ascorbic acid, and 1-10 mM beta-glycerophosphate to the medium. Differentiation of MSCs into osteoblasts

was achieved through morphological changes, Alzarin red staining of differentiated osteoblasts and RT-PCR gene expression of osteonectin in differentiated cells. Differentiation into chondrocyte was achieved by adding 500 ng/mL bone morphogenetic protein-2 (BMP-2; R&D Systems, USA) and 10 ng/ml transforming growth factor β3 (TGFβ3) (Peprotech, London) for 3 weeks[26]. In vitro differentiation into chondrocytes was confirmed by morphological changes, Alcian blue staining of differentiated chondrocytes and RT-PCR of Collagen II gene expression in cell homogenate. Total RNA was isolated from the differentiated MSCs using Trizol (Invitrogen, USA). RNA concentrations were measured by absorbance at 260 nm with a spectrophotometer, and 2 μg total RNA MK-8776 was used for reverse transcription using Superscript II reverse transcriptase (Invitrogen, USA). The cDNA was amplified using Taq Platinum (Invitrogen, USA). Osteonectin gene and collagen MEK162 price (II) primers used were designed according to the following oligonucleotide sequence: sense, 5′-GTCTTCTAGCTTCTGGCTCAGC-3′; antisense,5′-GGAGAGCTGCTTCTCCCC-3′

(uniGene Rn.133363) and sense, 5′-CCGTGCTTCTCAGAACATCA-3′; antisense, 5′-CTTGCCCCATTCATTTGTCT-3′ (UniGene Rn.107239). The RNA templates

were amplified at 33 to 45 cycles ioxilan of 94°C (30 sec), 58°C to 61°C (30 sec), 72°C (1 min), followed with 72°C for 10 min. PCR products were visualised with ethidium bromide on a 3% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as housekeeping gene to examine the extracted RNA integrity. CD29 gene expression was also detected by RT-PCR as a marker of MSCs [27]. Preparation of HCC Model Hepatocarcinogenesis was induced chemically in rats by injection of a single intraperitoneal dose of diethylnitrosamine at a dose of 200 mg/kg body weight followed by weekly subcutaneous injections of CCl4 at a dose of 3 mL/kg body weight for 6 weeks [28, 29]. At the planned time animals were sacrificed by cervical dislocations, blood samples and liver tissues were collected for assessment of the following: 1. Histopathological examination of liver tissues.   2. Gene expressions by qualitative and quantitative real time PCR for the following genes: β-catenin, PCNA, cyclin D and survivin genes   3. Alpha fetoprotein by ELISA (provided by Diagnostic Systems Laboratories, Inc., Webstar, Texas, USA.)   PCR detection of male-derived MSCs Genomic DNA was prepared from liver tissue homogenate of the rats in each group usingWizard® GenomicDNApurification kit (Promega, Madison, WI, USA). The presence or absence of the sex determination region on the Y chromosome male (sry) gene in recipient female rats was assessed by PCR.