The Human Thyroid-Derived CI-huThyrEC Cell Line Expresses the Thyrotropin (TSH) Receptor and Thyroglobulin but Lacks Other Essential Characteristics of Thyroid Follicular Cells

Abstract

Background: Thyroid hormone synthesis requires the normal function of thyroid follicular cells and adequate nutritional intake of iodine. For in vitro studies on thyroid cell pathophysiology, the immortalized FRTL5 rat thyroid cell line and a derivative thereof, the PCCL3 cell line, are widely used. However, a permanent human thyroid cell line is currently lacking. A recent report described a cell line obtained from human thyroid cells designated as Cl-huThyrEC. Methods: Four clones of Cl-huThyrEC cells were obtained and cultured in the presence of thyroid stimulating hormone (TSH). The expression of key genes defining the thyroid follicular cell phenotype was determined by reverse-transcription PCR (RT-PCR) in FRTL5, PCCL3, and Cl-huThyrEC cells. The latter were cultured as monolayers and as organoids in Matrigel. Iodide uptake was measured and compared among the cell lines. Results: Gene expression analysis reveals that Cl-huThyrEC cells express the thyroid-restricted transcription factors (PAX8, NKX2.1, FOXE1), the TSH receptor (TSHR), and thyroglobulin (TG), but they do not express the sodium-iodide symporter (NIS), thyroid peroxidase (TPO), and pendrin (SLC26A4). In functional studies, Cl-huThyrEC cells are unable to concentrate iodide. Conclusions: Despite the expression of certain key genes that are limited or restricted to thyroid follicular cells, Cl-huThyrEC cells lack some of the essential characteristics of thyroid follicular cells, in particular, NIS. Hence, their utility as a model system for thyroid follicular cells is limited.

Keywords: cell line; iodine; sodium iodide symporter; thyroid; thyroid peroxidase.

Figure 1

Microscopic appearance of the Cl-huThyrEC cells clones 1 to 4 (A–D) in the monolayer culture. Magnification 200×.

Figure 2

Relative expression of the thyroid-restricted transcription factors PAX8 (A), NKX2.1 (B), and FOXE1 (C) and key genes for thyroid hormone synthesis, TG (D), TSHR (E), NIS (F), TPO (G), and SLC26A4 (H), in the four CI-huThyrEC clones. For each condition, water was used to determine the background levels. One-way ANOVA with the Greenhouse–Geisser correction was calculated for the cDNA samples and compared to the negative control containing water. Asterisk: p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***. Results show the means of triplicates from one experiment performed independently three times.

Figure 3

Western blot analysis of FRTL5, PCCL3, and the four clones from Cl-huThyrEC cell lines. Membranes were incubated with antibodies against thyroglobulin (TG, (A)) and the sodium iodide symporter (NIS, (B)) and merged with molecular weight markers (kilo Dalton, kDa). The membranes were then incubated with an antibody against B-actin (arrow) as a loading control (C,D). Original images of (A–D) can be found in Supplementary Materials.

Figure 4

Microscopic appearance of Cl-huThyrEC cell clone 4 cultured in Matrigel. The cells self-organize and form organoid-like structures. Magnification 200×.

Figure 5

Relative expression of the thyroid-restricted transcription factors PAX8 (A), NKX2.1 (B), and FOXE1 (C) and key genes for thyroid hormone synthesis, TG (D), TSHR (E), NIS (F), TPO (G), and SLC26A4 (H), in CI-huThyrEC clone #4 cultured after organoid formation and cultured with 1 mU/mL (CI-huThyrEC-4 3D + 1 mU/mL TSH) or 2 mU/mL of TSH (CI-huThyrEC-4 3D + 2 mU/mL TSH). For each condition, water was used to determine the background levels. A one-way ANOVA with the Greenhouse–Geisser correction was calculated to compare the values obtained from cDNA samples to the negative control containing water. Asterisks: p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***. Results show the means of triplicates from one experiment performed independently three times.

Figure 6

(A,B) Relative expression of the thyroid-restricted genes Nis (A) and Slc26A4 (B) in rat PCCL3 and FRTL5 cells. As for the previous experiments, water was used to determine the background levels. A one-way ANOVA with the Greenhouse–Geisser correction was performed to compare the values obtained from cDNA samples to the negative control containing water. Asterisks: p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***. Results show the means of triplicates from one experiment performed independently three times. (C) Cumulative intracellular iodide levels in FRTL5 (black), PCCL3 (dotted), and clone 4 of CI-huThyrEC cells (grey) after adding 50 µM of NaI to the medium. NaI levels were measured in triplicates every 15 min via colorimetry using the Sandell–Kolthoff reaction. Bars indicate standard deviations (SD). (D) Counts per minute of intracellular ¹²⁵iodide after 2 min, 15 min, or 30 min of incubation with Na¹²⁵I in FRTL5 and huThyrEC-4 cells. A total of 100 µM of potassium perchlorate was added to the incubation buffer in some conditions in order to inhibit NIS transport. (E) Counts per minute of intracellular iodide 125 after 30 min of incubation in FRTL5 or huThyrEC-4 cells when incubated with 50 µM of cold iodide (+20 µCi/mmol of Na¹²⁵I) and 100 µM of KClO₄ or without NaI or KClO₄ to determine background levels. Asterisks: p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***. Results show the means of triplicates from one experiment. Bars indicate standard deviations (SD).