Differentiation of stem cells into thyroid tissue
Embodiments herein relate in vitro methods of stem cell differentiation into thyroid hormone producing cells and tissues, and methods of use of these cells.
1. An ex vivo or in vitro method for producing a mature thyroid follicular epithelial cell comprising:
a) contacting an endodermal cell with a thyroid lineage culture medium to differentiate the endodermal cell into the thyroid lineage without exogenously delivered nucleic acid sequences resulting in the forced over-expression of the Nkx2-1 protein and the Pax8 protein in the endodermal cell, thereby producing a Nkx2.1+ and Pax8+ thyroid progenitor cell, wherein the thyroid lineage culture medium comprises:
i) bone morphogenetic protein 4 (BMP4), and
ii) fibroblast growth factor 2 (basic) (FGF2), and
wherein the thyroid lineage culture medium does not contain:
iii) Wingless-Type MMTV Integration Site Family protein, member 3A, (Wnt3A),
iv) fibroblast Growth Factor 10 ((FGF10),
v) keratinocyte Growth Factor (KGF), and
vi) epidermal growth factor (EGF),
b) isolating and culturing the Nkx2-1+/Pax8+ thyroid progenitor cell of step (a) in a differentiation medium comprising fibroblast growth factor 2 (basic) (FGF2) for a time sufficient to permit differentiation of the Nkx2-1+/Pax8+ thyroid progenitor cell along the thyroid lineage; and
c) culturing the further differentiated cell produced in step (b) in a maturation medium comprising dexamethasone and thyroid stimulating hormone (TSH), under conditions and for a time sufficient to produce a Nkx2-1+/Pax8+ co-expressing cell that also expresses early and mature thyroid markers: thyroglobulin (Tg+), thyroid stimulating hormone receptor (Tsh+), sodium iodine symporter (Nis+), and thyroid peroxidase (Tpo+),
thereby producing a mature thyroid follicular epithelial cell.
2. The method according to claim 1 , wherein the endodermal cell is an anterior/foregut endodermal cell.
3. The method according to claim 1 , wherein the differentiation medium further comprises one or more factors selected from the group consisting of a fibroblast growth factor, BMP4, and TSH.
4. The method according to claim 3 , wherein the fibroblast growth factor is FGF2 or FGF10.
5. The method according to claim 1 , wherein the resultant Nkx2-1+/Pax8+/Tg+/Tsh+/Nis+/Tpo+ cells do not express differentiated lung markers: surfactant, pulmonary-associated protein C (Sftpc) and secretoglobin family 1A member 1 (Scgb1a1).
6. The method according to claim 1 , wherein the resultant Nkx2-1+/Pax8+/Tg+/Tsh+/Nis+/Tpo+ cells also express Forkhead Box E1 protein (Foxe1) and thyroidally-expressed homeobox protein (Hhex).
7. The method according to claim 1 , wherein the resultant Nkx2-1+/Pax8+/Tg+/Tsh+/Nis+/Tpo+ cells also express epithelial markers E-Cadhedrin (E-Cad) and epithelial cell adhesion molecule (EpCam).
8. The method according to claim 1 , the method further comprising selecting for Nkx2-1+/Pax8+ expressing cells by removing Nkx2-1+/Pax8−, Nkx2-1−/Pax8−, and Nkx2-1−/Pax8+ expressing cells.
9. The method according to claim 1 , the method further comprising determining diploid expression of Nkx2-1 in the resultant cells.
10. The method according to claim 1 , the method further comprising selecting for Nkx2-1+/Pax8+/Tg+/Tsh+/Nis+/Tpo+/expressing cells obtained.
11. The method according to claim 10 , the method further comprising selecting for (I) Foxe1+/Hhex+expressing cells; (II) E-Cad+/EpCam+ expressing cells; (III) Sftpc−/Scgb1a1− expressing cells obtained; or a combination of (I), (II) and (III).
12. The method according to claim 1 , wherein in the Nkx2-1+/Pax8+ expressing cells are cultured in a 3D matrix support material.
13. The method according to claim 12 , wherein the 3D matrix support material is matrigel.
14. The method of claim 1 , wherein the thyroid follicular epithelial cell is functional.
15. The method of claim 14 , wherein the functional thyroid follicular epithelial cell can metabolize iodine, secretes thyroid hormones, expresses enzymes necessary for thyroid hormone synthesis and iodide metabolism and responds to the presence of TSH by secreting thyroid hormones.
16. The method of claim 1 , wherein the time sufficient to permit differentiation of the Nkx2-1+/Pax8+ thyroid progenitor cell along the thyroid lineage is 8 days.
17. The method of claim 1 , wherein step (c) is initiated when the cells are at days 22-26 of the method.