Abstract
The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500μU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.
Highlights
The TSH receptor (TSHR) is the major trophic activator of the thyroid gland and controls thyroid hormone synthesis and secretion
We have previously described the transformation of transfected WT mES cells into thyroid progenitor cells and their expression of thyroid specific genes [7] and these cells were used for the SCID experiment
The TSH receptor knockout (TSHR-KO) heterozygous cells were purified using GFP and mCherry expression as markers. By gating on these double positive cells, we obtained nearly 92% purity of these cells (Figures 3A–E). These purified and fully differentiated thyroid follicular cells (TFCs) were used for the longer-term implantation experiments in TSHR-KO mice
Summary
The TSH receptor (TSHR) is the major trophic activator of the thyroid gland and controls thyroid hormone synthesis and secretion. The TSHR-KO mice have a small thyroid gland indicating that development of the gland is not totally dependent on TSH, but the mouse is markedly hypothyroid with only rudimentary thyroid follicles (Figure 1), low thyroxine levels and very high serum TSH. These mice remain stunted unless weaned at 3 weeks and put on a thyroid replacement diet. Rendering such hypothyroid mice to be consistently euthyroid has not, been easy with the variable ability and availability of thyroid hormone replacement strategies which usually require a special, and sometimes unreliable diet, to be commissioned
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
