48 Article

Expression Analysis of Stemness Genes in a Rat Thyroid Cell Line FRTL5

Authors

A. Shimasue1, 2, N. Yamakawa1, 2, M. Watanabe2, Y. Hidaka1, Y. Iwatani2, T. Takano1

Affiliations

1

Key words ▶ FACS-mQ ● ▶ thyroglobulin ● ▶ TTF1 ● ▶ flow cytometry ● ▶ quantitative RT-PCR ●

Abstract

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1389924 Published online: October 14, 2014 Exp Clin Endocrinol Diabetes 2015; 123: 48–54 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence T. Takano Department of Laboratory Medicine Osaka University Graduate School of Medicine D2, 2-2 Yamadaoka Suita Osaka 565-0871 Japan Tel.:  + 81/6/6879 6633 Fax:  + 81/6/6879 6635 [email protected]. ac.jp

 Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan  Division of Health Sciences, Osaka University Graduate School of Medicine, Suita, Osaka, Japan

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Detection and analysis of a small subpopulation of cells such as stem cells or cancer stem cells are recognized to be a key technique in a recent regeneration and cancer science. However, in the thyroid, no marker that identifies stem cells has been established yet. We previously established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS), named mRNA quantification after FACS (FACS-mQ). By using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we analyzed the expression of stemness genes in a rat thyroid cell lines FRTL5 using FACS-mQ. 3

Introduction

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Recently, a minor population of cells in various tissues such as stem cells or cancer stem cells (CSCs) has been detected [1, 2], and the capacity of stem cells for tissue renewal and damage repair makes them attractive targets in the medical field [3]. Thus, many researchers are attempting to identify their biological characteristics. However, in the human thyroid gland, stem cells have not yet been identified and stem-like cells have not been detected clearly in thyroid-derived cell lines [4, 5]. Several marker genes, such as PROM1 [6], CD44 [7], and ALDH1 [8], have been reported to be associated with stem cells or CSCs in several tissues. However, in thyroid epithelial cells or thyroid-derived cell lines, genes which characterize stem cells have not been fully identified. In the rat thyroid cell line FRTL5, which is known to express thyroid-specific antigen, such as thyroglobulin (TG) and thyroid transcription factor 1 (TTF1, NKX2-1), a previous report failed to iden-

Shimasue A et al. Stemness Genes in FRTL5 …  Exp Clin Endocrinol Diabetes 2015; 123: 48–54

stemness genes, NANOG, ABCG2 and GATA4, were expressed in FRTL5. In FRTL5 cells, varied expression of thyroglobulin (TG) among cells was observed by flow cytometry. Cell populations with high or low TG expression were analyzed by FACS-mQ. The cell population with low TG expression showed increased expression of the stemness genes. Furthermore, Ki67-positive cells showed increased expression of TG, which suggested that cells with high TG proliferated rapidly. These results indicated that FRTL5 contains a cell population with high stemness gene expression and less differentiated features, resembling stem cells. These cells might regulate proliferation in FRTL5.

tify stem-like cells and failed to detect the expression of stemness genes [4]. Stem cells are usually separated by fluorescenceactivated cell sorting (FACS) using antibodies that specifically bind to cell-surface marker proteins or by the transfection of a plasmid carrying a cell-type-specific promoter and a reporter gene [9]. In this method, however, stem cells and CSCs do not possess an appropriated surface antigen and so cannot be sorted. We have previously established an in-tube immunohistochemical method named mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). In FACS-mQ, targeting any surface or intracellular antigens, a particular cell type can be stained with a fluorescent dye, allowing the stained cells to be sorting without causing RNA degradation [10–14]. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. Using this new technique, we found the existence of cell populations with a low and high TG expression in FRTL5 and the expression of several

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received 24.06.2014 first decision 07.08.2014 accepted 22.08.2014

2

stemness genes was examined in each cell population. Furthermore, we examined the relationship between the expression of these stemness genes and growth factors or cell proliferation. As far as we recognize, this is the first report on stemness gene expression in FRTL5.

Materials and Methods

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Cell culture

The FRTL5 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and cultured in 10-cm culture dishes in Ham’s F12 medium (Gibco, Paisley, Scotland, UK), supplemented with 5 % newborn calf serum (Gibco); 2.5 mg/mL sodium bicarbonate (NaHCO3) (Wako, Osaka, Japan); 100 U/mL penicillin (Meiji, Tokyo, Japan); 100 μg/mL streptomycin (Meiji); 250 ng/mL fungizone (Gibco); and a 6-hormone preparation consisting of 100 μU/mL bovine thyroid stimulating hormone (TSH) (Sigma, St. Louis, MO, USA), 10 μg/mL bovine insulin (Sigma), 10 ng/mL somatostatin (Sigma), 360 pg/mL hydrocortisone (Sigma), 5 μg/mL transferrin (Gibco), and 100 ng/mL glycylL-histidyl-L-lysine acetate (Sigma) (6 H). FRTL-5 cells passaged less than 5 times were used in the following experiments. 8305C, 8505C, TCO1, cells were provided by the Human Science Research Resource Bank (Osaka, Japan) and were grown in RPMI 1640 medium (Gibco), supplemented with 10 % fetal bovine serum (Gibco); 2.5 mg/mL NaHCO3; 100 U/mL penicillin; 100 μg/ mL streptomycin; and 250 ng/mL fungizone. FTC133 cells were purchased from DS Pharma Biomedical (Osaka, Japan) and were grown in DMEM/F12 medium (Gibco), supplemented with 10 % fetal bovine serum; 2.5 mg/mL NaHCO3; 100 U/mL penicillin; 100 μg/mL streptomycin; and 250 ng/mL fungizone. All cells were maintained in a 5 % CO2-95 % air atmosphere at 37 °C, and the medium was changed every third day until the cells were subconfluent. In the analysis with growth factors, subconfluent FRTL5 cells were cultured under the conditions shown in ●  ▶  Table 1. G₀ consisted of Ham’s F-12 medium supplemented with 2.5 mg/mL NaHCO3, 100 U/mL penicillin, 100 μg/mL streptomycin and 250 ng/mL fungizone. These cells were used in both real-time quantitative reverse transcription-polymerase chain reactions (RT-PCR) and flow cytometry.

Flow cytometry and FACS-mQ

Flow cytometry and FACS-mQ were performed as previously reported with some modifications [10]. FRTL5 cells were dispersed with 6H medium supplemented with 1 g/L collagenase (Wako) and 1 g/L dispase II (Eidia, Tokyo, Japan), and other cells were dispersed with 0.25 % Tripsin-EDTA (Gibco). 10 ml tubes (Azone, Osaka, Japan) were used in all procedures. All media and buffers except the antibodies were mixed with 0.1 % diethylpyTable 1  Culture conditions with growth factors. Days

1–2

3–4

5

I II III IV V VI

6H 6H 6H 6H 6H 6H

6H G₀ G₀ G₀ G₀ G₀

6H G₀ G₀ + TSH (2.5 U/ml) G₀ + IGF1 *  (100 µg/ml) G₀ + TSH (2.5 U/ml) + IGF1 (100 µg/ml) 6H

 * Human recombinant insulin-like growth factor 1 (Sigma)

rocarbonate (DEPC) (Sigma), sorted overnight at room temperature, and then autoclaved. Then 106 cells were fixed with methanol (Wako) supplemented with 10 % polyethylene glycols (molecular weight 300 kD; Wako) (UM-Fix) for 15 min at 4 °C. UM-Fix was replaced by phosphate-buffered saline (PBS) (Wako) supplemented with 0.1 % Tween 20 (Sigma) (PBS-T) and kept in PBS containing 10 % dimethyl sulfoxide (DMSO) (Sigma) at  − 80 °C until use. On the day of in-tube immunocytochemistry, 1 × 10⁶ cells were thawed rapidly in a water bath at 37 °C and then permeabilized with 0.1 % TritonX-100 (Sigma) in PBS for 30 min at 4 °C. The cells were washed with PBS-T and blocked with 0.5 % blocking reagent (Roche Diagnostics, Tokyo, Japan) in PBS-T (blocking solution). The blocked cells were then incubated in 200 μl fluorescent-conjugated antibodies diluted in blocking buffer for 4 h at 4 °C. Control IgG1 (Dako Cytomation, Carpinteria, CA, USA) or monoclonal antibodies were labeled with fluorescein or R-phycoerythrin (R-PE) using a Fluorescein or R-PE Labeling KitNH₂ (Dojindo Molecular Technologies, Kumamoto, Japan), respectively. A mouse monoclonal anti-thyroglobulin antibody (DAK-Tg6; Dako Cytomation) conjugated with fluorescein and a mouse monoclonal anti-TTF1 antibody (AG7G3/1; Dako Cytomation) conjugated with R-PE was used at a concentration of 0.5 μg/ mL. An R-PE-labeled anti-Ki67 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used at a concentration of 5 μg/mL. After being washed twice with PBS-T, the cells were preserved in PBS-T containing 0.2 % paraformaldehyde (Wako) at 4 °C. The cells were analyzed and sorted using a FACSAria II cell sorter (Becton Dickinson, Mountain View, CA, USA). Data were analyzed using a DiVa software (Becton Dickinson). 10⁴ cells were collected and sorted in RNA later (Takara, Shiga, Japan).

Extraction of RNA from the cells and RT-PCR

In RT-PCR analysis and quantitative RT-PCR analysis without FACS-mQ, RNAs were extracted from dispersed cells as previously described [15] and 1 µg of total RNA was used for the RT reaction. In FACS-mQ analysis, total RNA was extracted using NucleoSpin FFPE RNA (Takara) according to the protocols recommended by the manufacturer, and the whole sample was used for the RT reaction. The extracted RNA was reverse transcribed in an RT mixture, which contained 4 µl of 5 ×  first strand buffer (Invitrogen, Tokyo, Japan), 10 mmol/L dithiothreitol (Invitrogen), 0.5 mmol/L deoxynucleotide triphosphatase (dNTP) (Takara), 200U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitogen), 2 U/µL RNase inhibitor (Takara), and 2.5 µmol/L random hexamer (Takara) in a total volume of 20 µL. The RT reaction was carried out for 10 min at 25 °C, 50 min at 42 °C, and 15 min at 70 °C. Primers used this study are listed in ●  ▶  Table 2. All primers were purchased from Greiner Japan (Tokyo, Japan). In RT-PCR analyses, cDNAs were amplified with Ex-Taq polymerase (Takara) according to the protocols recommended by the manufacturer. The representative PCR conditions were as follows: 94 °C for 2 min, and 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. In gel electrophoresis, 5 µL PCR product was loaded on a 2 % agarose gel and, after electrophoresis, the gel was stained with SYBR Green I (Takara). The PCR products were extracted from the gel using a QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan). The sequences of the PCR products were confirmed by direct sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Shimasue A et al. Stemness Genes in FRTL5 …  Exp Clin Endocrinol Diabetes 2015; 123: 48–54

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Article 49

50 Article Real-time quantitative PCR for rat genes was performed using SYBR Green Master Mix and an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer’s recommendations. One microliter of cDNA was used in the subsequent assays. Primers used this study are listed in ●  ▶  Table 2. All primers were purchased from Greiner Japan (Tokyo, Japan). The conditions for PCR were as follows: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A Table 2  Primers used in this study. Primers

NANOG

5′-CCTTGCCGTTGGGCTGACATGAG-3′ 5′-AGGCAGTCTCTGTGCAGAGCAT-3′ 5′-GCTGCCTCTTTAAGACTAGGGCTGG-3′ 5′-GGCGAAGTGCAATTGGGA-3′ 5′-CCTGCAGCTCAGCCTTAAGA-3′ 5′-GCACTGCAGAAACATGTTCT-3′ 5′-GCTCAGTACCAACCTGACG-3′ 5′-TCCAGATCGGTTCTGTCG-3′ 5′-GTCGGTGTGCGAGTCAGG-3′ 5′-AAACTTAGCACATCTCCCTCTGCGA-3′ 5′-ATCCAGTGCTGTCGGCTCTGAAGC-3′ 5′-GGGGTGCTGATTACGCGGTGAT-3′ 5′-GTGAGCACCTGCTGCTTGGAGC-3′ 5′-TGATGGCTTTGAGGCAGGCGTA-3′ 5′-ATGGCCAGTACCTACGTGAA-3′ 5′-CCTTTGCCCTGTTGATAAGCC-3′ 5′-AGTTGGACTAAATGCCTAGT-3′ 5′-ATATACACAGGCTTGTCCA-3′ 5′-CTGACAGGATGCAGAAGGA-3 5′-TGATCCACATCTGCTGGAA-3′

SOX2 POU5F1 PROM1 ABCG2 GATA4 HNF4A TG NKX2–1 ACTB

a

Statistical analysis

Data were analyzed with StatMate software (Atoms, Tokyo, Japan). Statistical analysis of differences between the groups was carried out using the Mann-Whitney U test. P