After fixation, the cells were then incubated with stain solution (40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2) selleck containing 1 mg/ml X-gal at 37��C for overnight. Fluorescence-activated cell sorting Isolated PT were cultured overnight in PT medium supplemented with 10 mIU/ml bTSH. On the next day, PT were collected and incubated with anti-STRO-1 or anti-TPO (Clone MoAb47, Dako Cytomation) antibody. After labeling with secondary antibody conjugated with Alexa 488 and 7-AAD (BD Biosciences, Franklin Lakes, NJ), the cells were analyzed and sorted using a FACS Vantage SE (BD Biosciences). Real-time RT-PCR Total RNA was extracted using ISOGEN reagent (Nippon gene, Tokyo, Japan) and reverse transcribed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) with random hexamers.
The following PCR amplifications were performed using QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA) for TTF1 and SYBR Premix Ex Taq Perfect Real Time kit (TaKaRa Bio, Ohtsu, Japan) for other genes in a Thermal Cycler Dice Real Time System (TaKaRa Bio). The cycle threshold value, which was determined using second derivative, was used to calculate the normalized expression of the indicated genes using Q-Gene software [15], using ribosomal RNA 18S as a reference. The following primer pairs were used: 18S, 5��-GTAACCCGTTGAACCCCATT-3�� and 5��-CCATCCAATCGGTAGTAGCG-3��; TG, 5��-CTGGTGTGTCATGGACAGCGGAGAA-3�� and 5��-CCCGAGATTGTCTCACACAGGAT-3��; TSH-R 5��-CTATAGATGTGACTCTGCAGCAGCT-3�� and 5��-GAGGGCATCAGGGTCTATGTAAGT-3��; PAX8 5��-CCCCCTACTCCTCCTACAGC-3�� and 5��-ACTGTCCCCATGGCAACTAC-3��; TTF1 5��-CCATGAGGAACAGCGCCTC-3�� and 5��-CTCACG TCCCCCAGCGA-3��.
Microarray analysis Total RNAs were extracted from PT from three different samples (PT0808, PT0811 and PT0812) on the next day of initial plating and corresponding SAGM-grown lines at two weeks after plating using ISOGEN reagent (Nippon gene) and subjected to Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray analysis service (Bio Matrix Research, Nagareyama, Japan). The array contained 54,675 probes for mRNAs. GeneSpring Software (Agilent Technologies, Santa Clara, CA) was used for data analyses. The data were deposited in NCBI GEO site (accession number: “type”:”entrez-geo”,”attrs”:”text”:”GSE24553″,”term_id”:”24553″GSE24553).
Results Isolation of proliferative cells from normal human thyroid tissues In an attempt to isolate primitive human thyroid epithelial cells, we tried several commercially available media containing growth factors but not serum which is generally thought to AV-951 enforce differentiation program. Those were basically developed for growing human primary cells or tissue stem cells. Among them, we found that SAGM was the most suitable for our purpose, which was developed for growing human primary small airway epithelial cells.