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Differentiating characterization of human umbilical cord blood ...

Differentiating characterization of human umbilical cord blood ...

571X.-Q. Kang et al. /

571X.-Q. Kang et al. / Cell Biology International 30 (2006) 569e5755 0 -CAAGCTGGCCTTCAGATTTC-3 0 regions.in PBS for 10 min. Cells were incubated with 3% hydrogen peroxide in methanolfor 10 min to block endogeneous peroxidase activity and then washedtwice in PBS for 5 min. Nonspecific binding sites were blocked with 10%normal goat serum. Cells were incubated overnight at 4 C with the primaryantibody (mouse anti-human monoclonal antibody of CK-18, 1:200; mouse3. Results3.1. Isolation and culture of MSCs from UCBanti-human monoclonal antibody of NF, 1:100; mouse anti-human monoclonal The cells isolated by gradient density centrifugationantibody of GFAP, 1:500). After being washed twice for 15 min in PBS, slides showed heterogeneity during the first 5 days. The adherentwere incubated (30 min at 37 C) with the secondary antibody (goat antimouseIgG marked by biotin), with streptoavidin marked by horseradish per-cells were observed on days 4 and 5. The spindle-shaped cellsappeared at the bottom of culture flasks. Many round cellsoxidase, and then developed color with DAB. PBS (0.01 M) was substitutedfor primary antibody as the negative control. The positive cells presented were suspended in the medium (Fig. 1A). Through continuouschange of medium, the suspended cells became fewerbrownish yellow.and fewer. When the medium was changed twice, the suspendedcells were completely removed from the medium.2.9. Total RNA isolation and reverse transcriptase-polymerasechain reaction (RT-PCR)The adhered cells were fibroblast-like and grew as a whirlpool.The sub-cultured cells were much purer and fibro-Total RNA was isolated from treated cells using Trizol (Gibco-BRL, MD,USA) according to the manufacturer’s protocol. Extracted RNA was solved in blast-like, the other cells were disappeared (Fig. 1B). Thediethylpyrocarbonate-treated water (DEPC water). The cDNA was synthesized primary culture cells reached confluence 10e12 days later,using RNA polymerase chain reaction kit (Takara Bio, Shiga, Japan) under the cells sub-cultured at a ratio of 1:3 reached confluenceconditions recommended by the manufacturer. Transcriptions of AFP, albumin8e10 days later. By this method, UCB-derived MSCs wereand CK-18 genes were examined by PCR using an LA-PCRÔ kit (TaKaRa,Dalian), a number of hepatocyte marker genes are listed in Table 1. PCR isolated from 10 samples out of 49, which were isolatedwas performed according to the manufacturer’s recommendation. RT-PCR within 6 h of collection. The rate of separation was 20.4%.was performed in 50 mL of mixture containing 1 mL of extracted RNA, 1 mLof 10 mmoL of both forward and backward primers (synthesized by Sangon,Shanghai, China), 5 mL of10 buffer (100 mM TriseHCl, pH 8.0, 500 mMMSCs could not be isolated from the other samples, whichwere isolated over 6 h after collection.KCl, 0.1% gelatin, 1% Triton X-100), 1 mL of 10 mM dNTP, 4 mL of25 mM MgCl 2 , 2.5 U of ribonuclease inhibitor, 200 U of M-MLV RT enzyme, 3.2. Differentiation of osteogenic, adipogenic and2 U of Taq polymerase, and DEPC water was added to the final volume ofneuron-like cells50 mL. The reaction mixture was kept at 42 C for 30 min to synthesizecDNA, and at 94 C for 3 min to denature DNA followed by 35 cycles of amplificationconsisting of 30 s of denaturing DNA at 94 C, 30 s of annealingDNA at 58 C and 30 s of extending DNA at 72 C. Amplified PCR productsOsteogenic differentiation was attained on day 24 followingtreatment. Under the influence of these reagents, approximately28.6% of the cells formed alkaline phosphatase-were separated on 1.2% agarose gel electrophoresis and the bands were identifiedby fluorescence.positive aggregates (Fig. 1C) while the controls did not,2.10. Periodic acid-Schiff treatment for glycogensuggesting that UCB-derived MSCs differentiated intoosteocytes. Under the influence of adipogenic differentiationmedium, UCB-derived MSCs formed oil red O positive cellsIn the hepatocyte differentiation group, on days 16, 20, 24 and 28, the mediumwas removed from the flasks, rinsed cells with PBS three times and fixed on day 8 after treatment, while the controls did not. If the lipidthem with 10% formaldehyde for 30 min. The cells were oxidized in 1% periodicdroplets appeared in the cytoplasm of adipocytes, oil red Oacid for 10 min and rinsed three times in dH 2 O. Afterwards, cells were would specifically stain them. On day 12 after treatment, ap-treated with Schiff’s reagent for 10 min, rinsed in dH 2 O for 10 min, stained proximately 52.8% of the cells induced adipocytes andwith hematoxylin for 2 min, differentiated by 1% alcohol/HCl, rinsed inreached confluence (Fig. 1D). Cells no longer changed theirdH 2 O again, and observed under a microscope.shapes after being treated with BME, DMSO and BHA for2.11. Statistical analysis7 h. The results showed cells expressed NF, but no GFAP(Fig. 1E).The data of AFP, albumin and urea were expressed as mean SD. The statisticalsoftware SPSS12.0 was used. The results were analyzed by t-test.3.3. Mesenchymal stem cells became small, round andP < 0.05 was considered statistically significant.epithelioid when cultured with FGF-4 and HGFTable 1When cells reached 70% confluence, they were treated withPrimers used for RT-PCRhepatocyte differentiation medium. The medium containedTarget SequenceSize of IMDM supplemented with 10% FBS, 20 ng/mL HGF, 10 ng/geneproductmL FGF-4, 100 U/mL penicillin and 100 U/mL streptomycin.(bp)Prior to the 8-day treatment they did not show any changeAFP 5 0 -GATGCACCTGACCCACTTTATAAA-3 0 395compared with the controls. After treatment, small, round cells5 0 -GAGATTGTCTGACCGATTCAGACT-3 0appeared in the treatment group, as well as epithelioid cells.Albumin 5 0 -CAACTATGTCCGTGAGCTTCCA-3 0 339 On day 28, approximately 63.6% of cells were small, round5 0 -GTGGTCGGTGCTGGTCTATATG-3 0or epithelioid. The control cells were still fibroblast-like,CK-18 5 0 -GAGATCGAGGCTCTCAAGGA-3 0 400 though increased in density. The cells overlapped in some

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