Stem cell antigen 1-positive mesenchymal cells are the origin of follicular cells during thyroid regeneration

Abstract

Many tissues are thought to contain adult stem/progenitor cells that are responsible for repair of the tissue where they reside upon damage and/or carcinogenesis, conditions when cellular homeostasis becomes uncontrolled. While the presence of stem/progenitor cells of the thyroid has been suggested, how these cells contribute to thyroid regeneration remains unclear. Here we show the origin of thyroid follicular cells and the process of their maturation to become follicular cells during regeneration. By using β-galactosidase (β-gal) reporter mice in conjunction with partial thyroidectomy as a model for thyroid regeneration, and bromodeoxyuridine (BrdU) long label-retaining cell analysis, we demonstrated that stem cell antigen 1 (Sca1) and BrdU-positive, but β-gal and NKX2-1 negative cells were found in the non-follicular mesenchymal area 7 days after partial thyroidectomy. They temporarily co-expressed cytokeratin 14, and were observed in part of follicles by day 35 post-partial thyroidectomy. Sca1, BrdU, β-gal, and NKX2-1-positive cells were found 120 days post-partial thyroidectomy. These results suggested that Sca1 and BrdU positive cells may participate in the formation of new thyroid follicles after partial thyroidectomy. The process of thyroid follicular cell regeneration was recapitulated in ex vivo thyroid slice collagen gel culture studies. These studies will facilitate research on thyroid stem/progenitor cells and their roles in thyroid diseases, particularly thyroid carcinomas.

Figure 1. Immunohistochemistry and immunofluorescence of Sca1, BrdU, β-gal, and NKX2-1.

(A) Sca1 immunohistochemistry of normal thyroid. The image on the right shows the magnified image boxed on the left. Arrow indicates positive staining (brown in color) in the vascular endothelial cells. Scale bar: 200 µm. (B) Immunofluorescence for Sca1, BrdU, DAPI, β-gal, and NKX2-1 in 7 days post-PTx thyroid. Arrow indicates a positive cell for Sca1 and BrdU, but negative for β-gal, and NKX2-1, located in the mesenchymal area. Merged image is shown on the lower right. Scale bar: 20 µm.

Figure 2. Co-expression of Sca1 and Krt14, CD34, or CD133 in the thyroid 14 days post-PTx by immunofluorescence.

(A, B) Sca1(+) cells located in the follicles (A) or in the mesenchymal areas (B) co-express Krt14. Arrow indicates cells co-expressing Sca1, Krt14, and/or BrdU. (C) Sca1(+);BrdU(+) cells (shown by an arrow) do not co-express either CD34 or CD133. Merged image is shown on the lower right. Scale bar: 20 µm.

Figure 3. Thyroid of 35 days post-PTx.

(A) H&E staining of affected areas after PTx. Left: the asterisk shows the representative follicle under repair with no colloid and in irregular shape. The dotted line demarcates the affected areas (above the line), where many irregular follicles with low or no colloid and fibrosis (arrows) are seen. Right: High magnification image of the follicle with the asterisk in A, in which many ciliated intrafollicular cells are seen (representatives shown by arrows). Scale bar: 100 µm (left), 20 µm (right). (B) Immunohistochemistry for Sca1 and β-gal shown in brown. Serial sections to that used in A were used for immunostaining. The asterisk is used to orient the sections. Scale bar: 100 µm. (C) Number of ciliated cells per 1000 intrafollicular cells in the affected area on various days post-PTx. Significant differences were found between control (no surgery), 7 days, and 14 days post-PTx thyroids vs. 35 and 75 days post-PTx thyroids. No statistically significant differences were found among control, 7 days, and 14 days post-PTx thyroids, and between 35 vs. 75 days post-PTx thyroids.

Figure 4. Immunofluorescence of BrdU, Sca1, NKX2-1, and β-gal in 35 and 120 days post-PTx thyroids.

(A) Immunofluorescence in 35 days post-PTx thyroid. Sca1(+);BrdU(+), but β-gal(−);NKX2-1(−) cell is located in an irregular shaped follicle (shown by an arrow). (B) Immunofluorescence in 120 days post-PTx thyroid. Sca1(+);BrdU(+) cell also expresses β-gal and NKX2-1 (shown by an arrow). Arrowhead in the Sca1 panel in B indicates cilia that are strongly positive for Sca1 due to the cell having these cilia is Sca1(+). Merged image is shown on the lower right. Scale bar: 20 µm. (C) Number of Sca1(+);β-gal(−) cells in 14 and 35 days post-PTx thyroids. The number shown is based on 1000 intrafollicular cells counted. N = 7–10.

Figure 5. Ex vivo three-dimensional primary thyroid slice culture.

(A) Thyroid slices were treated with BrdU two hours before harvest at 1, 3, and 7 days after the start of culture. Sections were immunostained for Sca1, BrdU, and NKX2-1 on indicated days. Serial sections were used. Red arrow shows representative Sca1, BrdU, and/or NKX2-1 positive cells. The dotted line in the panel of day 7 BrdU was arbitrarily drawn to separate the central and the peripheral area. They were used to count cell numbers shown in B. Scale bar: 20 µm (Day 1, 3, and 7 upper panel), 50 µm (Day 7 lower panel). (B) Number of Sca1(+) and BrdU(+) cells in primary thyroid slice cultures. Cell numbers were determined based on those obtained after counting 300 intrafollicular (Follicle) or mesenchymal cells (Mesen) in the central (C) or the peripheral (P) area using 10 sections prepared from a culture dish on day 3 and 7. Each dot indicates cell numbers from one culture dish.

Figure 6. Model depicting two independent regenerative pathways that may operate in thyroid after damage caused by various insults such as chemicals, radiation, and surgery.

Pathway I: clear immature cells with characteristics of C cells or follicular cells appear at ∼7–14 days post PTx in mice . Clear cells are shown by pale yellow color. Pathway II: Sca1(+) cells appear in mesenchymal areas at ∼7–14 days post PTx, followed by the appearance of Sca1(+) cells in follicles at ∼14–35 days post PTx. Sca1(+) cell is shown by dark-red color.