Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Thyroid. 2013 Apr;23(4):385-91. doi: 10.1089/thy.2012.0644. Epub 2013 Mar 18.
The aim of this study was to assess the impact of transcriptional induction on thyroid follicular cell (TFC) differentiation from endodermally matured embryonic stem (ES) cells. The thyroid transcription factors-NKx2 homeobox 1 (NKx2-1, formerly called TTF-1) and Paired box gene 8 (Pax8)-are known to associate biochemically and synergistically in the activation of thyroid functional genes including the sodium/iodide symporter (NIS), thyrotropin (TSH) receptor (TSHR), thyroglobulin (Tg), and thyroid peroxidase (TPO) genes. In this study, we investigated the ability of ectopically expressed Pax8 and NKx2-1 to further the induction and differentiation of murine ES cells into potential TFCs.
ES cells were stably transfected with either the Pax8 gene, the NKx2-1 gene, or both genes to study the induction of NIS, TSHR, Tg, and TPO genes as assessed using both quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and protein expression. The derived cells were cultured with or without the presence of activin A to allow their differentiation into multipotent endodermal cells.
The four thyroid-specific genes NIS, TSHR, Tg, and TPO were all significantly activated by expressing both transcription factors within the same ES cell. In contrast, significant but much lower transcriptional activity of the TSHR, Tg, and TPO genes was detected in cells expressing just NKx2-1, and only the NIS and TSHR genes responded to Pax8 alone. No Tg protein expression could be detected prior to their development into endodermal derivatives. However, after further differentiation of postembryoid body ES cells with activin A and TSH into endodermal cell lines, those cells with dual transfection of Pax8 and NKx2-1 demonstrated greatly enhanced expression of the NIS, TSHR, Tg, and TPO genes to such a degree that it was similar to that found in control thyroid cells. Furthermore, these same cells formed three-dimensional neofollicles in vitro and expressed Tg protein, but these phenomena were absent from lines expressing only Pax8 or NKx2-1.
These findings provide further evidence that co-expression of Pax8 and NKx2-1 in murine ES cells may induce the differentiation of thyroid-specific gene expression within endodermally differentiated ES cells and commit them to form three-dimensional neofollicular structures.
本研究旨在评估转录诱导对从内胚层成熟的胚胎干细胞(ES 细胞)分化为甲状腺滤泡细胞(TFC)的影响。甲状腺转录因子-NKx2 同源盒 1(NKx2-1,以前称为 TTF-1)和配对盒基因 8(Pax8)在激活甲状腺功能基因方面已知具有生化和协同作用,包括钠/碘转运体(NIS)、促甲状腺激素(TSH)受体(TSHR)、甲状腺球蛋白(Tg)和甲状腺过氧化物酶(TPO)基因。在这项研究中,我们研究了异位表达的 Pax8 和 NKx2-1 进一步诱导和分化小鼠 ES 细胞为潜在 TFC 的能力。
将 Pax8 基因、NKx2-1 基因或这两个基因稳定转染 ES 细胞,以使用定量逆转录聚合酶链反应(qRT-PCR)和蛋白质表达来研究 NIS、TSHR、Tg 和 TPO 基因的诱导。衍生细胞在存在或不存在激活素 A 的情况下培养,以允许其分化为多能内胚层细胞。
四个甲状腺特异性基因 NIS、TSHR、Tg 和 TPO 均通过在同一 ES 细胞中表达两种转录因子而显著激活。相反,仅表达 NKx2-1 的细胞中 TSHR、Tg 和 TPO 基因的转录活性显著但低得多,而仅 Pax8 可单独响应 TSHR 基因。在发育为内胚层衍生物之前,无法检测到 Tg 蛋白表达。然而,在用激活素 A 和 TSH 将胚后体 ES 细胞进一步分化为内胚层细胞系后,那些双转染 Pax8 和 NKx2-1 的细胞表现出 NIS、TSHR、Tg 和 TPO 基因的表达显著增强,其程度与对照甲状腺细胞相似。此外,这些相同的细胞在体外形成了三维新滤泡,并表达 Tg 蛋白,但这些现象在仅表达 Pax8 或 NKx2-1 的细胞中不存在。
这些发现进一步证明,在小鼠 ES 细胞中共同表达 Pax8 和 NKx2-1 可能诱导内胚层分化的 ES 细胞中甲状腺特异性基因表达的分化,并促使它们形成三维新滤泡结构。