Derivation of hAFS cells with the starter cell method
Five milliliters of fifteen independent amniotic fluid samples were obtained from 16-20 week pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis. The study protocol was approved by the Ethics Committee of Siriraj hospital, Mahidol University, Thailand and each participant received an informed consent document. Cells were immediately isolated from amniotic fluid and cell debris by centrifugation at 2,100 rpm for 5 min. The pellet cells were resuspended with 5 ml of Chang medium (Irvine Scientific, CA, USA), which has been used in routine amniocentesis culture. A resuspension was plated on a 100 mm2 tissue culture dish (Nunc, NY) and incubated at 37°C in humidified 5% CO2, 5% O2 for 3-4 days. To identify the starter cells, the distinct fibroblast-like cells in the primary culture dish were observed under inverted microscope. The individual single hAFS cells were cultured continuously as starter cells to form hAFS cell colonies. To keep each colony at the proper distance, only one starter cell was allowed to exist under a microscopic field using 10× magnifications. Unneeded starter cells were mechanical removed by pipette tip. For a colony forming from a starter cell, only the starter cell was continued in culture. The medium and non-adherent cells were removed from the primary culture dish. Five milliliters of hAFS cell medium containing α-MEM medium (Gibco, Invitrogen, CA) supplemented with 15% ES-FBS (PAA, Pasching, Austria), 1% glutamine (Sigma, MO), 1% penicillin/streptomycin (Biochrom, Berlin, Germany), 18% Chang B and 2% Chang C (Irvine Scientific, CA) and 10 ng/ml bFGF (Chemicon, Millipore, MA) was added to the culture dish. The culture was maintained in the primary culture dish for 48 h. The cells in each colony were counted in order to predict and select a clone for hAFS cell expansion. Next, each hAFS cell colony was mechanically picked up under inverted microscope using fine-tipped pipettes. The cells in each hAFS cell colony were re-seeded into a well of a 24-well plate. The culture was performed at 37°C under humidified 5% CO2, 5% O2. When a clonal hAFS reached confluence at 70% of the culturing area, subculturing was performed by trypsinization and re-plating into a 25 cm2 tissue culture flask. The medium was changed every other day. The hAFS cells were allowed to expand to 70% confluence and then routinely subcultured with a dilution of 1:3.
Immunofluorescent staining of hAFS cells
hAFS cell markers were characterized using fluorescence microscope detection and flow cytometry analysis. For immunofluorescence, hAFS cells at 70% confluence were fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany). Cells were stained overnight with primary antibody against Oct-4a (Santa Cruz Biotechnology, CA) and SSEA-4 (Chemicon, Millipore, MA). The cells were then washed twice with PBS-Tween (PBS+0.5% Tween-20; UBS, OH) before staining with a 1:200 dilution of rabbit anti-human IgG secondary antibody (Chemicon, Millipore, MA). The specificity of each reaction was visualized by inverted fluorescent microscopy. For flow cytometry analysis, hAFS cells were harvested and stained with FITC or PE-conjugated antibodies against CD29, CD44, CD90, CD105, CD133 (E-bioscience, CA), CD34, CD45, CD73, SSEA-4 (Beckton Dickinson, NJ) and Oct-4a. The cells were fixed with 1% paraformaldehyde. The analysis was performed using a Beckton Dickinson flow cytometer (Beckton Dickinson, NJ).
Total RNA was extracted from hAFS cells by Phenol-Chloroform and used as a template for reverse transcription. The cDNA was made by using RevertAid First Strand cDNA Synthesis Kit (Fermentas, EU). The primers used in PCR are as follows:
Oct-4 (247 bp) sense, 5'-CGTGAAGCTGGAGAAGGAGAAGCTG-3', and antisense, 5'-CAAGGGCCGCAGCTTACACATGTTC-3';
HLA-ABC (394 bp) sense, 5'-GTATTTCTTCACATCCGTGTCCCG-3', and antisense, 5'-GTCCGCCGCGGTCCAAGAGCGCAG-3';
HLA-DR (220 bp) sense, 5'-CTGATGAGCGCTCAGGAATCATGG-3', and antisense, 5'-GACTTACTTCAGTTTGTGGTGAGGGAAG-3';
Nestin (395 bp) sense, 5'-CCAGAAACTCAAGCACCAC-3', and antisense, 5'-TTTTCCACTCCAGCCATCC-3';
Nanog (161 bp) sense, 5'-AGTCCCAAAGGCAAACAACCCACTTC-3', and antisense, 5'-
Sox2 (449 bp) sense, 5'-CCCCCGGCGGCAATAGCA-3', and antisense, 5'-TCGGCGCCGGGAGATACAT-3';
and beta-Actin (107 bp) sense, 5'-ATGTGGCCGAGGACTTTGATT-3', and antisense, 5'-AGTGGGGTGGCTTTTAGGATG-3'.
The cDNA amplification for Oct-4 and HLA-ABC were performed by the following PCR conditions: initial denaturing at 95°C for 5 min and 35 cycles of 94°C for 45 sec, 57°C for 1 min, 70°C for 1 min and extension at 70°C for 10 min. For HLA-DR, Nestin, Nanog, Sox2 and β-actin, the PCR conditions were as follows: 95°C for 5 min and 35 cycles of 94°C for 45 sec, 52°C for 1 min, 70°C for 1 min and extension at 70°C for 10 min.
To investigate the differentiation capacity, hAFS cells were in vitro differentiated into three different cell specific lineages, including adipogenic, osteogenic and neurogenic lineages. This hAFS cells were cultured in hAFS medium until 70% confluence and then shifted to a specific induction medium under the same condition of 5% CO2, 5% O2 at 37°C. The medium was changed twice a week. For differentiation to osteogenic and adipogenic lineages, the obtained cells were cultured in the appropriate medium. The osteogenic medium contains alpha-MEM (Gibco) supplemented with 10% ES-FBS (PAA), 0.1 μM dexamethasone (Sigma), 10 mM glycerol-2-phosphate (Sigma) and 50 μM ascorbic acid (Sigma). The adipogenic medium contains alpha-MEM (Gibco) supplemented with 10% ES-FBS (PAA), 1 μM dexamethasone (Sigma), 5 μg/ml insulin (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), and 60 μM indomethacin (Sigma). For neural differentiation, hAFS cells were cultured in neurogenic inducing medium containing alpha-MEM (Gibco) supplemented with 20% ES-FBS (PAA), 1 μM beta-mercaptoethanol (Sigma), and 5 ng/ml basic FGF (Invitrogen) for 24 h and then shifted to serum-depleted medium (alpha-MEM, 10 uM beta-mercaptoethanol) for 5 h.
Immunochemical staining of differentiated cells
The osteogenesis was assessed by alkaline phosphatase enzyme activity. Calcium accumulation was examined using von Kossa staining. The Oil Red O staining was used for detection of intracellular lipid droplet formation for evaluating adipogenesis. For evaluation of neural differentiation, the neuron specific class III β-tubulin (TuJ-1) was used.