Thyroid follicle formation and thyroglobulin expression in multipotent endodermal stem cells.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA. risheng.ma@mssm.edu
Risheng
R
Ma
Rauf
R
Latif
Terry F
TF
Davies
D000818:Animals; D002454:Cell Differentiation; D004707:Endoderm / Q000166:cytology / Q000378:metabolism*; D007453:Iodide Peroxidase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D013954:Thyroglobulin / Q000235:genetics / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000099209:Sodium-Iodide Symporters
D007453:Iodide Peroxidase; D009687:Nuclear Proteins; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D011989:Receptors, Thyrotropin; D027981:Symporters; D013954:Thyroglobulin; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D000099209:Sodium-Iodide Symporters; C000614414:Nkx2-1 protein, mouse; C499496:Pax8 protein, mouse