Differentiating Thyroid Cells

T he root cause of a variety of thyroid diseases is abnormal thyroid cell proliferation and differentiation leading to both developmental abnormalities and benign and malignant tumor formation. The early cellular and molecular mechanisms leading to such abnormalities remain largely elusive. Although many insights into thyrocyte differentiation have been provided by studies using immortal thyroid cell lines, such lines are limited by their inability to model embryonic development and hence provide few clues about the developmental aspects of disease. In this regard, pluripotent embryonic stem cells, which can differentiate into all cell lineages, have the potential to provide a key thyroid developmental model. The ability to access cell populations representing the early stages of embryonic thyroid stem cell differentiation provides an exciting and unparalleled opportunity to model human thyroid development. In the long term, the ability to generate a patient's thyroid cells would allow the clinician to test the effect of therapeutic drugs as well as allowing the development of clinically relevant screening assays.

The first embryonic stem cells were isolated from mouse blastocysts in 1981 (1,2). Now, almost three decades later, the first ever clinical trial involving embryonic stem cell use on humans has been approved by the U.S. Food and Drug Administration to treat patients with spinal cord injury. However, thanks to the availability of an effective, economical, and well-tolerated hormone replacement therapy, the demand for cell replacement therapy to treat hypothyroidism is not as high as for many debilitating diseases such as Parkinson's disease, myocardial infarction, and type 1 diabetes mellitus. Nevertheless, the fact that in vitro differentiation of embryonic stem cells can generate a variety of mature cell types makes it a perfect model for studies on thyroid development and may provide the basis for further pharmaceutical and clinical applications.

Thyroid endoderm can be differentiated from embryonic stem cells by activin A. Activin A is a member of the transforming growth factor β superfamily, which has been shown to be critical in regulating endoderm formation in vitro and in vivo. With activin A alone, a small population of cells expressing Pax-8, thyrotropin receptor (TSHR) and the sodium-iodide symporter can be generated with no influence seen on the differentiation process by the addition of thyrotropin (TSH) or insulin-like growth factor (IGF)-1 (3). The data parallel several reports indicating that thyroid cell development may occur in the absence of TSH. However, no thyroglobulin transcription activity was found in these cultures. If the embryonic stem cell differentiation system is to be used as a reliable model of thyroid development, it is necessary to develop approaches to study the early stages of thyrocyte lineage commitment in a more predictable way. One approach has been our use of a mouse embryonic stem cell reporter line in which enhanced green fluorescent protein cDNA was targeted to the TSHR locus which allowed us to monitor TSHR expressing cells in differentiating cultures and to isolate cells that include representatives of early developmental stages. Studies with this reporter embryonic stem cell line suggest that the early development of a mouse thyrocyte is clearly regulated by TSH itself (4). By monitoring TSHR expression, we demonstrated that TSH alone also promoted the development of mature thyrocytes that can take up radioiodine as a result of the expression of functional sodium-iodide symporters but again thyroglobulin expression was absent or minor (4). Therefore, these key observations suggest that additional factors are necessary to proceed to generating fully functional thyrocytes. One of these factors is likely to be IGF-1 provided at the appropriate time, as demonstrated by our recent work showing that the addition of insulin and IGF-1 together to late embryonic stem cell cultures enabled the long-term propagation and differentiation of mature thyrocytes expressing thyroglobulin at the protein level (5). Together, these studies indicate that with appropriate combinations, concentrations, and timing of addition of growth factors and hormones, it is possible to direct differentiation toward the thyrocyte lineage in a tissue culture dish.

In this issue of Thyroid, Jiang et al. (6) use a mouse E14 embryonic stem cell line to confirm the feasibility of inducing differentiation of this cell line into thyrocytes. They also take one step forward and demonstrate ultrastructural features similar to adult thyroid cells using electron microscopy. They clearly demonstrate that under the influence of dual stimulators, TSH and insulin, some differentiated cells co-expressing TSHR, sodium-iodide symporter, thyroperoxidase, and thyroglobulin mRNA can be generated. This observation is similar to the previous reports of thyrocyte differentiation from another mouse embryonic stem cell line CCE (7), indicating this potential is possessed by many embryonic stem cell lines. Jiang et al. (6) also detected two thyroid transcription factors, TTF-1 and Pax-8, but the timing of the expression of the transcription factors and the thyroid-specific genes remains to be carefully delineated as does the number of cells successfully differentiated. According to elegant studies of thyroid embryogenesis, thyroid transcription factors TTF-1, TTF-2, and Pax-8 are expressed early in thyroid development, when the thyroid begins to bud from the floor of the primitive pharynx (8,9). Therefore, these “early” thyroid markers are likely indicative of thyroid progenitor cells. In contrast, the expression of thyroid proteins such as TSHR, sodium-iodide symporter, thyroperoxidase, and thyroglobulin are indicative of fully differentiated thyrocytes. This further highlights the importance of understanding the specific stages of thyrocyte development and the signaling pathways that regulate them as a prerequisite for predictably and reliably generating appropriate thyrocyte lineages. There is no doubt that access to markers that distinguish early thyroid progenitor populations from the later differentiated populations will be important for isolating specific thyrocyte lineages from embryonic stem cells.

Although the studies of Jiang et al. (6) demonstrate some success in the generation of thyrocytes from mouse embryonic stem cells, consistent with previous reports (3 –5,7), the investigators also need to confirm thyroid-specific protein expression and not just transcription. The fact that the mouse E14 embryonic stem cell-derived thyrocytes failed to produce thyroid hormones suggests that the cells were far from functional. In keeping with this, electron microscopy studies showed that the cells from TSH-stimulated cultures had no signs of secretory vesicles. Hence, it appears that their differentiated populations of cells were still highly heterogeneous, and current conditions must be further optimized to allow for in vitro differentiation of embryonic stem cells into functional thyrocytes.

The presence of thyroid stem cells within the mature thyroid gland has been well demonstrated (10,11) and their role in cell renewal and neoplastic change is currently under review. Understanding the biology of these cells by modeling their differentiation in vitro has the potential to greatly advance our knowledge of thyroid cell biology.