Characterization of murine pituitary-derived cell lines Tpit/F1, Tpit/E and TtT/GF

Abstract

The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.

Fig. 1.

Cell appearance and growth curves for TtT/GF, Tpit/F1 and Tpit/E cells. Morphology by light microscopy (left panels) and growth curves (right panels) for TtT/GF (A), Tpit/F1 (B) and Tpit/E cells (C) are indicated. Cell numbers of each line were counted during the period of 24 to 120 h or that of 24 to 168 h, and the doubling times of TtT/GF, Tpit/F1 and Tpit/E were calculated with the cell numbers for 48 to 72 h, 120 to 144 h and 120 to 144 h, respectively. The data are presented as the mean ± SD of duplicate counts in two independent experiments. Scale bar= 20 μm.

Fig. 2.

Real-time PCR of genes of interest expressing in Tpit/E, TpitF1 and TtT/GF cells. Quantitative real-time PCR was performed to estimate the mRNA level of the following genes: Sox2 (A), Sox9 (B), Sca1 (C), Cxadr (D), Prop1 (E), Prrx1 (F), Prrx2 (G), Pitx1 (H), Lhβ (I), Sf1 (J), E-cadherin (K), Vimentin (L), Snail2 (M) and S100β (N). Data were calculated by the comparative CT method to estimate the relative copy number contrasted to that of the TATA box binding protein gene (Tbp) used as an internal standard. The data are presented as the ± SD of duplicate PCRs in two independent experiments.

Fig. 3.

Immunocytochemistry for SOX2 and E-cadherin in Tpit/E, Tpit/F1 and TtT/GF cells. Immunostaining (red) for SOX2 (A) and E-cadherin (B) was performed and merged with an image of nuclear staining with DAPI (blue) (each right panel). Scale bar= 20 μm.

Fig. 4.

Real-time PCR of T-antigen in Tpit/E, TpitF1 and TtT/GF cells. Quantitative real-time PCR was performed to estimate the mRNA level of T-antigen in Tpit/E, TpitF1 and TtT/GF cells. Data were calculated as in Fig. 2.