Thyroid Stem Cells and Cancer

Abstract

Background:

Thyroid gland development and function are essential for life, and recent findings indicate the presence of stem/progenitor cells within the thyroid gland as a potential source of tissue regeneration and cancer formation.

Summary:

This review summarizes the current knowledge on early differentiation of thyroid cells from embryonic stem cells and highlights exciting concepts and recent novel findings on adult thyroid stem/progenitor cells in the normal thyroid gland and in thyroid cancer. Other potential sources and markers of stem/progenitor cells in the thyroid include bone marrow, microchimerism, and embryological remnant-derived multifocal solid cell nests. Finally, we discuss new therapeutic strategies that target thyroid cancer stem cells.

Conclusions:

Thyroid stem/progenitor cell populations are present in the normal and diseased thyroid gland. Advances in normal and cancer thyroid stem cell biology will be essential for future targeted therapies.

Introduction

S tem cells are pluripotent, clonogenic, and self-renewing, show plasticity to differentiate into cell types of a particular tissue they reside in, and often transdifferentiate (1). These qualities and the capacity of stem cells for tissue renewal and damage repair make them attractive targets (2). However, the combination of mutations affecting stem cell differentiation potential and unlimited growth potential may generate cancer stem cells (CSCs). Specific stem cell populations have been identified in numerous normal and diseased organs and tissues (3,4).

Thyroid cancer is the most common carcinoma of endocrine glands, comprising ∼1% of all malignancies, with a 3:1 female/male ratio, and North America has one of the highest incidences of this cancer among all malignancies (5,6). Four types of thyroid carcinoma, namely papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, anaplastic, undifferentiated thyroid carcinoma (UTC), and medullary thyroid carcinoma comprise more than 98% of all thyroid malignancies. Different and sometimes exclusive cytogenetic events and oncogenic mechanisms are effective in thyroid carcinoma of distinct histological types (7 –9).

Here, we review developmental processes in relation to thyroid stem/progenitor differentiation, comment on adult stem cells within the thyroid gland, and provide evidence for the presence of CSC in thyroid cancer.

Differentiation Markers in Thyroid Development

The two distinct endocrine cell populations of the thyroid gland develop from different origins: the thyroid hormone-producing follicular thyroid epithelial cells derive from the endoderm of the pharyngeal floor and the calcitonin-producing interfollicular C cells derive from neural crest cells within the ultimobranchial bodies (UBB) of the fourth pharyngeal arch (10,11). Thyroid organogenesis requires the specification of thyroid precursor cells from endoderm cells of the foregut, the subsequent migration and proliferation of thyroid precursors, the fusion of derivatives of the thyroid primordium and the UBB, and the final stage of functional differentiation (Fig. 1).

FIG. 1.

Summary of milestones of thyroid organogenesis in the mouse embryo until the beginning of functional differentiation (24,28,136,157). The equivalent human developmental stages according to Carnegie staging (CS) and/or weeks of development are shown in blue (158). Fgfr2, fibroblast growth factor receptor 2; Tg, thyroglobulin; TPO, thyroid peroxidase; TAZ (transcriptional coactivator with PDZ-binding motif ), a transcriptional regulator of the Pax8 and Titf-1 gene (159); TSH, thyroid stimulating hormone; TSHR, TSH receptor; T4, tetraiodothyronine or thyroxine; NIS, sodium/iodide (Na⁺/I⁻)-symporter.

In the mouse embryo, definitive endoderm originates as early as E6–E7.5 from the posterior epiblast, where gastrulation begins with the expression of fibroblast growth factor 8 (FGF8), brachyury, Wnt3, and eomesodermin (12 –16). Endoderm development in vertebrates is initiated by the transforming growth factor β (TGFβ)/nodal-related pathway (12,17) and gives rise to the epithelial lining of the gut and the respiratory system and all its derivatives, such as the thyroid, lung, liver, gallbladder, and pancreas. Canonical Wnt signaling was shown to promote the early expression of nodal ligands (12,18,19), and high levels of nodal signaling initiate definitive endoderm formation. Nodal activity in the epiblast is confined spatially and temporarily by expression of the nodal antagonists Lefty and Cer1 (20). In embryoid bodies (EBs), endoderm development can be induced by exposure to high levels of the TGFβ family member activin-A from a brachyury-positive cell population (21).

Among nodal targets, the homeodomain-like protein Mixl-1 and the transcriptional activators, GATA4 and GATA6, are key components in visceral endoderm derivatives. The high-mobility group (HMG)–box transcription factor (TF) Sox 17 and the hepatic nuclear factors Hnf3α (Foxa1) and Hnf3β (Foxa2) are temporarily expressed during thyroid organogenesis.

Thyroid organogenesis begins with the specification of distinct endodermal cells within the floor of the primitive pharynx on E8.5 (mouse) and formation of the thyroid anlage. Thyroid precursor cells are characterized by the unique coexpression of the four TFs, Titf1/Nkx2.1 (TTF1), Pax8, Foxe1 (TTF2), and Hhex, which are expressed simultaneously in thyroid precursor cells and in the differentiated follicular cells (22). The factors initiating thyroid specification of endoderm are unknown (Fig. 2). Although in vitro activation of the thyroid-stimulating hormone (TSH)/TSH receptor (TSHR) pathway regulates the expression of Titf1, Pax8, and Foxe1, their expression in vivo is independent of TSH/TSHR signaling (23,24) as shown in mouse models with impaired TSH signaling, such as Tshr^hyt/Tshr^hyt mice (25,26) or pit^dw/pit^dw mice (27). Studies with mouse mutants have shown that the four TFs have distinct roles during early organogenesis: Titf1 and Pax8 are essential for the survival of thyroid precursor cells, Foxe1 promotes migration of thyroid precursor cells, and Hhex may be important as a repressor of differentiation and for the maintenance of gene expression of Ttif1, Foxe1, and Pax8 in the thyroid primordium (Table 1).

FIG. 2.

Schematic drawings of early thyroid development. At E8.5, the thyroid anlage is formed as a thickening of the endoderm layer on the bottom of the primitive pharynx by specification of endoderm cells for the thyroid lineage. Factors initiating this specification process are unknown. Thyroid precursor cells coexpress the four transcription factors Titf-1/Nkx2.1, Pax8, Foxe1, and Hhex during development and functional differentiation and in adult thyroid follicular cells. At E10.5, proliferation of thyroid precursor cells has shaped the thyroid diverticulum. Migration of the thyroid precursors away from the endoderm lining temporarily forms the thyroglossal duct, which regresses at E11.5. At E11.5, the thyroid primordium has further descended and the UBB has formed from the fourth pharyngeal arch. At E13.0, the thyroid primordium has formed two lobes, reached the final location in front of the trachea and contacts the UBB. FGF10, fibroblast growth factor 10; FGFR2, FGF receptor 2; UBB, ultimobranchial body.

Table 1.

Developmental Markers and Thyroid Phenotypes in Mutant Mice

Mouse mutants	Thyroid phenotype	References
Pax8−/−	Regression of thyroid primordium at E11.5; thyroid precursor cells disappear at E12; no follicles, no Tg, no TPO; thyroids exclusively consist of C-cells (Ttif1 positive).	132
Ttif1/Nkx2.1−/−	Thyroid precursor cells disappear at E10.5–11.5; mice lack C cells and thyroid follicular cells.	133, 22
Hhex−/−	Thyroid anlage present at E9; absence of thyroid primordium at E10.	134
Foxe1−/− (= Titf2−/−)	Lack of migration of thyroid precursor cells; thyroid precursors remain attached to pharyngeal floor; precursors disappear at E11.5 in 50%.	135
FRS2α^2F/2F mutants	Aplasia or hypoplasia of thyroid lobes; thyroid lobes remain affiliated with pharyngeal epithelium.	136
Eya1−/−	Lack of fusion between UBB and thyroid lobes; thyroid hypoplasia; severe reduction in number of C cells and follicular cells.	137
Hoxa3−/−	Thyroid hypoplasia; persistent UBB with C cells.	138
Pax9−/−	Lack of UBB formation.	139
Hoxa5−/−	Disorganized follicle formation; decreased TPO; Nkx2.1, Titf2, Pax8 gene expression altered, but normal serum T4 level.	140

UBB, ultimobranchial bodies; TPO, thyroid peroxidase; Tg, thyroglobulin.

Factors derived from the surrounding mesenchyme are essential for thyroid development. The growth factors epidermal growth factor (EGF) and fibroblast growth factor (FGF) promote thyroid cell proliferation and repress differentiation in vitro (28). FGFs upregulate the expression of Titf1 in the early anterior foregut (29) and FGF10 was shown to downregulate Sox2 and p63 in the endoderm of the future esophagus (30). Mouse embryos deficient for the FGF receptor 2 IIIb (31) and FGF10 knockout mice (32) show thyroid agenesis, suggesting that thyroid precursor cells respond to FGF10 from the surrounding mesenchyme (32,33). Interestingly, E-cadherin is expressed during all stages of thyroid organogenesis and N-cadherin is absent at all times (34), indicating that the migration of thyroid precursor cells occur independent of epithelial to mesenchymal transition.

The UBB contains two different cell types: Titf1⁺ cells (majority of cells) and rare p63⁺ cells (35). P63⁺ cells can survive without Titf1 and give rise to vesicular structures lined by p63⁺ basal-type epithelial cells. Solid cell nests (SCNs) in the mature human thyroid gland are considered developmental remnants of the UBB and contain p63⁺, Bcl-2⁺, and telomerase⁺ main cells (36 –38).

Thyroid Stem/Progenitor Cells from Embryonic Stem Cells

Pluripotent embryonic stem (ES) cells can develop three-dimensional embryoblast-like EBs under suitable culture conditions. Cells within the EB can spontaneously differentiate into more advanced embryonic stages of all lineages (39,40). Apart from three major advantages of the EB model, namely (i) the characterization of precursor cell functions, (ii) the in vitro assessment of the effect of null mutations of specific genes or the testing of lethal gene knockouts on ES cell viability, and (iii) the suitability as a cell source for tissue engineering, this EB model has now also been used to promote early thyroid cell differentiation (41).

CCE ES cells and TSHR+/− ES cells (W9.5) with a green fluorescent protein (GFP)-neomycin cassette in mouse TSHR exon 1 coding sequence resulting in TSHR promoter-mediated GFP expression (GFP⁺) have been employed in ES studies on thyroid differentiation (41 –43). Generating thyroid-like cells from CCE and TSHR+/− ES cell-derived EBs is a multistep/multifactorial process. It involves initial induction of endoderm differentiation by limited exposure of EBs to serum or activin-A, a TGFβ family member and nodal ligand (21,42 –44). When EBs are allowed to grow out as an adherent cell layer, thyroid-like cells appear and express follicular thyroid markers PAX8, Na⁺/I⁻ symporter (NIS), functional TSHR, thyroid peroxidase (TPO), and thyroglobulin (Tg).

Activin-A induces human ES cells to undergo differentiation into definitive endoderm cells (21,45). Upon 5 days of exposure to activin-A, EBs derived from TSHR+/− ES cells (W9.5) were reported to show markedly decreased transcript levels for the Oct-4 and Rex-1 stem cell markers and significantly increased gene activity for the endoderm markers Gata-4, CXCR-4, Foxa-2, and α-fetoprotein (44). Similar treatment also resulted in ∼2% of GFP⁺ and functional TSHR⁺ ES cells within the EB expressing, identifying activin-A as an inducer of NIS⁺ , PAX8⁺ , and TSHR⁺ thyroid progenitor cells in the absence of TSH (44).

Recently, the importance of step-wise exposure of ES cells to a coordinated sequence of endocrine factors for thyroid-specific determination and thyrocyte maturation has advanced our understanding of in vitro thyroid differentiation from pluripotent ES cells. Although activin-A increased the expression of the endoderm-specific genes Foxa2 and Sox17, its transient effect on TSHR expression at day 5 was extended for an additional day in the presence of TSH, the endocrine factor known to affect TSHR gene regulation during EB differentiation (41,43). TSH-mediated thyroid lineage induction and determination also included a temporary upregulation of NIS in replated cells from EBs under serum-free conditions. However, both activin-A and TSH failed to sustain a stable differentiated thyrocyte phenotype (43). Propagation of EB-derived mature thyrocytes with the expression and partial colocalization of the follicular thyroid markers TSHR (number of GFP⁺ cells) and NIS and the appearance of Tg required the additional presence of insulin and/or insulin-like growth factor-I at later stages during EB culture (43).

Thyroid Stem/Progenitor Cells in the Adult Thyroid Gland

Resident thyroid stem/progenitor cell populations

Although the ES/EB system is a suitable in vitro model to recapitulate steps in follicular thyrocyte differentiation and despite sophisticated new culture conditions, like the activin-A–TSH–insulin/insulin-like growth factor-I treatment regime, the thyroid-like cell progenitors generated are usually transient, variable, and too low in cell number (1–2%) for additional functional studies. The adult thyroid gland has been implicated as another source of stem/progenitor cells. Being a low-proliferation tissue (46,47), the high frequency of benign and malignant nodular disease in the thyroid gland is not sufficiently and convincingly explained by genetic predisposition, iodine deficiency, mutagenesis, perturbed endocrine actions, and/or ligand–receptor deregulation events alone (48,49). Earlier results from transplantation experiments in nude mice also suggest the presence within nodular goiter tissues of autonomously growing thyroid-derived cells (50). Recently, first evidence for the presence of Oct4⁺ adult stem cells and precursor cells of endoderm origin with an Oct4⁺, GATA-4⁺, HNF4a⁺, PAX8⁺, and Tg⁻ phenotype was obtained in tissues and cultured cells derived from adult human goiter (51). Based on the expression of a functionally active ABCG2, fluorescent-activated cell sorting (FACS) revealed a small side population (SP; 0.1% of total cells) in cultured thyrocytes from human goiter tissues with a high nuclear/cytoplasmic ratio and an ABCG2⁺, Oct4⁺ phenotype. This SP cell fraction failed to express the endoderm markers GATA-4, PAX8, and HNF4a, or the typical thyroid differentiation markers TSHR, TPO, NIS, and Tg which were all detected in the non-SP fraction (52). The SP cell population remained viable in monolayer cultures for 14 days, but only in nonadherent suspension cultures and in the presence of EGF and FGF did they create spheres. These thyrospheres were composed of up to 5% of a proliferative ABCG2⁺, Oct4⁺ (SP) or GATA-4⁺, HNF4a⁺ (non-SP) thyroid stem/progenitor cell population (52). TSH completely inhibited sphere formation. The addition of serum-induced TSHR gene activity and subsequent dual action of serum plus TSH over a 3-week period further enhanced TSHR expression and caused the sphere-derived cells to differentiate into adult PAX8⁺, TSHR⁺, NIS⁺, TPO⁺, and Tg⁺ thyrocytes with loss of stem and endoderm markers. Importantly, these cells showed the hallmarks of differentiated thyrocytes by being capable of generating thyroid follicle-like structures and showing TSH-dependent iodine uptake (52).

Clusters of clonally derived human Oct4⁺, Nanog⁺ cells with large nuclei and small cytoplasm similar to thyroid stem cells described by Lan et al. (52) were recently also reported to generate spheres in the presence of basic FGF and EGF but in the absence of serum and TSH (53). When these thyrospheres were seeded into a three-dimensional collagen matrix adopted for differentiated thyrocytes (54,55), ∼50% of spheres created T₄-secreting follicle-like structures of Tg⁺ cells lining the lumen and peripherally located TPO⁺ cells, but TSHR was absent from these follicles (53). The formation of follicles in the presence of serum was TSH independent but required the absence of EGF and FGF. Spheres cocultured with the neuroblastoma cell line LAN5 (56) displayed larger morphology, showed expression of the neuronal marker β-tubulin III, and also initiated adipocyte differentiation, indicating a high level of plasticity of the cells within the thyrospheres (53). When injected into SCID mice, thyrocytes failed to generate tumors (57).

In the adult mouse thyroid, stem cells expressing the ES cell genes Oct4 and nucleostemin also reside in the SP which makes up 0.3–1.4% of the total population of thyroid cells (58 –60). Similar to the human thyroid SP, mouse ABCG2⁺ cells represent a heterogeneous population with ∼50% expressing Sca1⁺ (61,62) thyroid stem/progenitor cell marker of an otherwise ABCG2⁺, Oct4⁺, nucleostemin⁺, c-kit⁻/CD117⁻, and CD45⁻ phenotype. Similar to the human thyroid SP, mouse thyroid SP cells were devoid of transcripts encoding for PAX8, TPO, and Tg (58). However, when the SP was further subdivided into SP1 and SP2 fractions resembling FACS-gated areas of higher and lower Hochst33342 efflux, respectively, the SP2 of ABCG2⁺, Oct4⁺ cells was weakly positive for TSHR and Titf1, suggesting a thyroid progenitor lineage to be present in SP2 (58). Small numbers of ABCG2⁺ cells (∼1%) were localized to the interfollicular space and coexpressed Sca1 or the mesenchymal marker vimentin. Interestingly, a few calcitonin-positive C cells were also ABCG2⁺, suggesting the presence of a C-cell progenitor lineage among the ABCG2⁺ thyroid cells in the mouse (58).

Other Sources of Stem/Progenitor Cells in the Thyroid

Bone marrow

In many high- and low-proliferating organs, stem/progenitor cells are replenished by the bone marrow (63,64). Patients who had received hematopoietic stem cell transplantation have an increased risk for thyroid carcinoma (65,66). Young age at transplantation was the strongest risk factor, followed by irradiation, female sex, and graft-versus-host disease. The lack of CD45 and/or CD117 hematopoietic marker expression in SP cells derived from the human (53) and mouse adult thyroid (58), respectively, may indicate either of two things: (i) stem cells in the thyroid are not replenished by the bone marrow, or (ii) as a result of low cell turnover rates in the thyroid (46,47), human CD45⁺ and mouse CD45⁺, CD117⁺ stem cells derived from bone marrow lose the expression of both markers while residing in the thyroid for longer times.

Microchimerism

Microchimerism describes the bidirectional trafficking and persistence of a small number of allogeneic cells in a genetically different organism (67). This fetal–maternal exchange of cells is a common phenomenon in likely all placental species (68 –72). The detection of allogeneic fetal cells in maternal tissues, called fetal microchimerism, is frequently observed as early as 4–6 weeks of gestation (71,73,74) and at term virtually all pregnant women contain low numbers of circulating fetal cells (75 –77). Long-term survival of fetal cells, for example, CD34⁺ hematopoietic stem/progenitor cells, CD45⁺ cells, and desmin⁺ mesenchymal stem cells, has been reported in maternal tissues (77 –80). Male fetal cells were detected in follicular adenoma and the normal thyroid gland of mothers (81 –84) and these cells frequently populate the thyroid tissues of autoimmune Graves' disease and Hashimoto thyroiditis (85). A subset population of human CD34⁺, CD45⁻ stem/progenitor cells (3–19%) with high plasticity was recently detected in seven spheroid lines generated from the normal thyroid and perinodular normal thyroid tissues (53). Of these cell lines, three were tested Tg⁺ and two were Oct4⁺ and Nanog⁺ (53), but the potential fetal microchimeric origin of these stem/progenitor thyroid cells was not reported.

Embryological remnant-derived SCNs

Nonfollicular, multicellular, single, or multifocal SCNs are frequently (61–89%) detected in the thyroid and were postulated to be embryological remnants of the UBB and the thyroid diverticulum (36,86 –88). SCN, thyroglossal duct cysts, and lingual thyroid remnants are believed to harbor pluripotent stem cells (36,86,87). SCNs are composed of two distinct cell types, main cells and C cells, and display a wide variety of histopathological features making it sometimes difficult to distinguish SCNs from primary, secondary, or metastasizing squamous metaplasia or carcinoma, thyroglossal cust, PTC, medullary microcarcinoma, and C-cell hyperplasia (36,86). Separate from SCNs but often (81%) admixed with SCNs are mixed follicles which consist of main-like cells and regular follicular cells lining a follicular lumen (36,89). Two recent reports highlighted the importance of the basal/stem cell marker p63 as a suitable marker for main cells of SCN, main-like cells within mixed follicles, and a rare cell population in thyroglossal duct cyst and thyroid isthmus, the latter being a derivative of the thyroid diverticulum/thyroglossal duct (37,88). The nuclear TF p63 shares structural and functional similarities with the tumor suppressor p53. Alternative promoter usage and splicing events generate multiple p63 isotypes with N- and C-terminal alterations, respectively, some of which are known to counteract the actions of p53 (90,91). P63 is a marker of basal and progenitor epithelial cell layers in numerous tissues (91,92). The major defects in limb and craniofacial development and deficient epithelial regeneration due to depletion of a basal progenitor cell population in mice deficient in p63 also point toward a role for p63 in stem cell maintenance and the initiation of squamous cell differentiation (93,94). P63 and the cytokeratin (CK) 34βE12 were identified as specific markers for main cells of SCNs and within mixed follicles, but these cells lacked the thyroid markers Titf-1, Tg, calcitonin, or calcitonin-related peptide (CRP) (38,86). By contrast, C cells of SCNs displayed a P63⁻, Galectin-3⁺, Titf-1⁺, calcitonin⁺, CRP⁺, and chromogranin⁺ phenotype which is thought to reflect progressive parafollicular C-cell differentiation (37). Galectin-3 was identified as a suitable marker to distinguish SCN from normal parafollicular C cells (95).

Thyroid Cancer Stem/Progenitor Cells

Emerging concepts in thyroid carcinogenesis

Thyroid cancer is a diverse entity with respect to causative factors, thyroid cell types involved, histopathological phenotypes at different frequencies, and clinical outcome. Based on epidemiological, genetic, and animal data, a multistep, progressive thyroid carcinogenesis model suggested an orchestrated step-wise dedifferentiation from benign thyroid disease to differentiated thyroid carcinoma to anaplastic thyroid cancer as a result of accumulating somatic mutations in mature thyroid cells (8). This multistep thyroid carcinogenesis hypothesis has been challenged for a number of reasons: (i) a low turnover rate of the thyroid gland of 1 per 5–10 years or six to eight renewals per lifetime limits the accumulation of multiple mutations (46,47); (ii) unique genetic alterations specifically found only in a certain type of thyroid cancer question the multistep carcinogenesis model (96); (iii) transgenic rearranged during transfection/papillary thyroid carcinoma (RET/PTC) mice develop PTC but mature thyrocytes fail to transform when overexpressing the same fusion protein (97) (98); (iv) the monoclonal origin of thyroid cancer cells combined with strikingly different histomorphology may reflect distinct developmental morphogenesis programs to be active in specific thyroid cancer cells (99); (v) specific differentiation marker profiles, like oncofetal fibronectin, in PTC and UTC suggest common fetal ancestry (100 –103); (vi) the frequent presence of SCN within the thyroid points toward an involvement of developmental factors in thyroid cancer (86,88). The fetal cell thyroid carcinogenesis hypothesis postulates a hierarchy of fetal cell remnants within the adult thyroid which may act as a source for specific thyroid CSC generating distinct thyroid cancer phenotypes (104,105) (Fig. 3). Such CSC and specific CSC markers have been identified for different cancers (4,106). Carcinogenic thyroid stem cells and more differentiated thyroblasts are postulated to generate UTC and PTC, respectively, whereas follicular thyroid carcinoma is proposed to be generated from both stem cell populations and further advanced prothyrocytes (104,107). The degree of malignancy in thyroid cancer is likely higher with CSC derived from more immature stem cell populations (107). A modified hierarchical scheme of thyroid stem cell development incorporates SCN-derived embryonic remnant cells (p63⁺, CEA⁺, and CK34βE12⁺) and, supported by histopathological findings, postulates a bipotential Titf1⁺ and TSHR⁺ precursor as the cellular source for specific progenitors of the follicular (Titf1⁺ and PAX8⁺) and C-cell lineage (P63⁻ but possibly Galectin-3⁺ and Titf-1⁺) (86,88,102).

FIG. 3.

Schematic representation showing a developmental hierarchy of fetal thyroid stem/progenitor cells as a potential cause of specific follicular thyroid cancer types [modified according to Takano (104,107)]. Examples of mutations associated with and factors expressed in certain thyroid cancer types are depicted. Trefoil factor 3 (TFF3) may be used to distinguish foci of thyroid TTF3⁺ adenoma from TTF3^weak/− FTC (160). FTC, follicular thyroid carcinoma; PTC, papillary thyroid carcinoma; UTC, undifferentiated anaplastic thyroid carcinoma.

No CSCs have so far been isolated from thyroid cancer tissues, but we were able to detect Oct4⁺ cells within human UTC tissues (Fig. 4A). The isolation of CSC-like cells enriched in ABCG2⁺ SP or CD133⁺ cell populations has recently been reported using cultured human thyroid cancer cell lines. Comprehensive microarray chip analysis revealed genes related to the Wnt and Notch signaling pathways and known to play important roles in normal stem cell development to be upregulated in the SP fraction of ARO cells (4,108). However, both SP and non-SP fractions generated nude mouse tumors, indicating that the non-SP fraction still contained CSCs (108). Recently, CD133 (prominin-1) has been implicated as a specific marker of CSCs in the human anaplastic thyroid cancer cell lines ARO, KAT-4, and FRO (109,110). In comparison to CD133⁻ ARO, the CD133⁺ ARO population showed increased expression of Oct-4 and Titf1 and significantly higher resistance to chemotherapy-induced apoptosis against chemotherapeutics used to treat UTC, such as cisplatin, doxorubicin, and etoposide (109). Increased Oct-4 expression in the CD133⁺ ARO was confirmed in a recent study which also showed a strong increase in TSHR mRNA and further enhancement in Oct-4 and TSHR gene activities and presence of CD133 upon TSH-induced proliferation of CD133⁺ ARO (110). CD133^+/high ARO subpopulation revealed higher Oct-4 and TSHR expression levels than CD133^+/low or CD133⁻ ARO. When injected subcutaneously into NOD/SCID mice, CD133^+/high ARO repeatedly formed aggressive tumors at 10-fold lower cell numbers than CD133^+/low ARO, with CD133⁻ ARO being unable to generate any tumor xenografts (110). Strong immunoreactivity for CD133 specific for human UTC tissues suggests an in vivo role for this five-transmembrane domain glycoprotein (110).

FIG. 4.

(A) OCT-4 immunostaining in a human UTC was detected in the peripheral regions of UTC. (B) Representative FACS analysis of an Aldefluor assay for the detection of an ALDH1⁺ cell subpopulation with potential stem-like characteristics in the anaplastic human thyroid cell line UTC-8505C. UTC, undifferentiated thyroid carcinoma; ALDH, aldehyde dehydrogenase. Color images available online at www.liebertonline.com/thy.

Although the reported results on UTC-derived CSC are interesting, it should be noted that in all three studies the main experimental work was done with the ARO81-1 cell line (108 –110). A recent DNA profiling analysis of 40 human thyroid cancer cell lines has raised concerns about the ARO cell line as a valid thyroid cell model. ARO81-1 and KAT-4, also used in one of the studies (109), were reported to be likely of nonthyroid origin and a result from a cross-contamination with the human colon cancer cell line HT-29 (111). For ARO81-1, this conclusion was drawn based on the results from two independent labs (111).

We used a different FACS-based functional approach to identify CSCs within the human UTC-8505C cell line, which is considered unique among the 40 thyroid cell lines tested (111). The ALDEFLUOR assay is based on the fact that human normal and cancer mammary cells with increased aldehyde dehydrogenase (ALDH1) activity were found to have stem/progenitor properties (112). Stem cell populations in multiple myeloma and acute leukemia also show increased ALDH1 activity (113,114). The ALDH1 substrate BAAA is specifically metabolized by cells harboring ALDH1 to create a fluorescent dye which remains trapped within the cells, thus facilitating positive selection in FACS. The specificity of the enzymatic reaction is determined using the specific ALDH1 inhibitor diethyl-aminobenzaldehyde. Employing this method, we could demonstrate a 17–38% ALDH1⁺ cell fraction in UTC-8505C which was almost completely inhibited in the presence of diethyl-aminobenzaldehyde, suggesting the presence of a resident stem/progenitor population in human UTC-8505C (Fig. 4B).

Other cell types involved in thyroid cancer

Recent evidence shows an inverse association between pregnancy, microchimerism, and cancer (72,115). Parous women with fetal microchimerism are significantly less likely to develop cancer than parous women not harboring fetal cells (116); survival times and therapeutic response rates are better in microchimerism-positive patients (117). Fetal cells acquired during pregnancy and potentially of stem/progenitor phenotype have been identified in thyroid cancer tissues (82,118). Although their function is not yet known, evidence from skin, breast, and thyroid cancer favors the involvement of these fetal cells in repair mechanisms rather than a role as CSC in cancer propagation (118 –120). Nevertheless, as recently shown for Kaposi sarcoma and evidenced by the blood vessel-forming capacity of fetal progenitor cells, a role for microchimeric cells in tumor formation and/or tumor propagation should also be considered (121).

The persistence within the adult thyroid of embryological remnants forming SCNs and mixed follicles and the pluripotent undifferentiated p63⁺ stem/progenitor cells they harbor have been implicated in the pathogenesis of Hashimoto autoimmune thyroiditis and thyroid cancer (88,122,123).

New Therapeutic Strategies for Thyroid Cancer

There is no treatment that effectively targets the CSC compartment in thyroid cancer. Current antithyroid therapies, such as surgery, chemotherapy, and radiation, destroy rapidly growing differentiated thyroid tumor cells, thus reducing tumor mass, but leave behind cancer initiator cells. An ideal treatment regime should kill differentiated cancer cells, and at the same time, specifically, selectively, and quickly destroy thyroid CSCs to avoid toxic side effects to other cell types, and counteract the mutagenic evasive potential of these CSCs. In SP the presence of active transmembrane ABC transporter family members, multidrug resistance transporter 1 and breast cancer resistance protein 1/ABCG2/MRP, not only facilitates the efflux of DNA-binding dyes like Hoechst 33342 but can also promote chemoresistance through efflux of anticancer drugs, such as mitoxanthrone, gemcitabine, doxorubicin, and 5-fluorouracil, from cells with (cancer) stem cell activity (124,125). Such SPs have been identified in numerous cancers, including normal and neoplastic thyroid cells (52,108). Targeted inactivation of the ABCG2 may increase resistance of CSCs through upregulation of the transporter as shown for neuroblastoma and CD133⁺ brain tumor stem cells (125,126). The Wnt/β-catenin pathway has important roles in stem cell renewal and chemoresistance of CSCs. Deregulation of this pathway is associated with thyroid cancer (127). Shown to target β-catenin and cause reduced invasiveness and proliferation of thyroid cancer cells (128), the selective tyrosine kinase inhibitor imatinib mesylate (Gleevec, Novartis, NY; STI571) was ineffective as monotherapy in UTC patients (129). Future advances in targeting CSCs critically depend on new stem cell isolation protocols to identify suitable markers for thyroid CSCs, and such cells isolated from existing thyroid cancer mouse models may provide important clues as to the potential CSC subtypes (Table 2). This will aid in the discovery of novel therapeutic agents and strategies that effectively target thyroid cancer stem/progenitor populations. At the same time, new antagonists directed at epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), v-raf murine sarcoma viral oncogene homologue B1 (BRAF), rearranged during transfection (RET), cyclooxygenase 2 (COX-2), or vascular endothelial (VE)-cadherin and novel, more targeted delivery systems possibly involving coated multipurpose nanoparticles will be useful to block the growth of differentiated tumor cells (130,131).

Table 2.

Mouse Models of Thyroid Cancer as a Potential Source of Thyroid Stem/Progenitor Cancer Cells

Mouse model	Tumor type	References
Tg-c-Ha-ras	Papillary	141
Tg-TRK-T1		142
Tg-Ret/PTC1		97
Tg-Ret/PTC1 × p53^−/−		143
Tg-Ret/PTC3		144
Tg-Ret/PTC3 × p53^−/−		145
Tg-BRAF^V600E		146
TRβ^PV/PV	Follicular	147
TRβ^PV/PV × Pten^+/−		148
Tg-Rap1b^G12V		149
Pten^−/− × K-ras^G12D		150
Pten^−/−		151
Tg-SV40 LT	Undifferentiated/anaplastic	152
CGRP-Ret	Medullary	153
CGRP-v-Ha-ras		154
Rb+/− and Rb+/− × p53^+/−		155, 156

PTC, papillary thyroid carcinoma.

Conclusions

The recent exciting discoveries in thyroid stem cell and thyroid CSCs research mark the dawn of a new era with potentially widespread impact on our understanding of thyroid pathophysiology, carcinogenesis, and treatment. Understanding the complexity of stem cells and their malignant CSC counterpart will undoubtedly be an essential step in the development of more targeted, selective, and individualized treatment regimes for thyroid cancer.

Footnotes

Acknowledgment

The authors express their gratitude to the following grant agencies: Natural Science and Engineering Research Council of Canada and Manitoba Health Research Council (to T.K. and S.H.K.) and Deutsche Forschungsgemeinschaft (to C.H.V.).

Disclosure Statement

The authors declare that no competing financial interests exist.

References

Filip

, English

, Mokry

. 2004. Issues in stem cell plasticity. J Cell Mol Med, 8:572–577.

Mimeault

, Hauke

, Mehta

, Batra

. 2007. Recent advances in cancer stem/progenitor cell research: therapeutic implications for overcoming resistance to the most aggressive cancers. J Cell Mol Med, 11:981–1011.

Hombach-Klonisch

, Panigrahi

, Rashedi

, Seifert

, Alberti

, Pocar

, Kurpisz

, Schulze-Osthoff

, Mackiewicz

, Los

. 2008. Adult stem cells and their trans-differentiation potential—perspectives and therapeutic applications. J Mol Med, 86:1301–1314.

Klonisch

, Wiechec

, Hombach-Klonisch

, Ande

, Wesselborg

, Schulze-Osthoff

, Los

. 2008. Cancer stem cell markers in common cancers—therapeutic implications. Trends Mol Med, 14:450–460.

Jemal

, Siegel

, Ward

, Murray

, Xu

, Thun

. 2007. Cancer statistics, 2007. CA Cancer J Clin, 57:43–66.

Davies

, Welch

. 2006. Increasing incidence of thyroid cancer in the United States, 1973–2002. JAMA, 295:2164–2167.

de Groot

, Links

, Plukker

, Lips

, Hofstra

. 2006. RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors. Endocr Rev, 27:535–560.

Kondo

, Ezzat

, Asa

. 2006. Pathogenetic mechanisms in thyroid follicular-cell neoplasia. Nat Rev Cancer, 6:292–306.

Smallridge

, Marlow

, Copland

. 2009. Anaplastic thyroid cancer: molecular pathogenesis and emerging therapies. Endocr Relat Cancer, 16:17–44.

10.

Douarin N

, Fontaine

, Le Lievre

. 1974. New studies on the neural crest origin of the avian ultimobranchial glandular cells—interspecific combinations and cytochemical characterization of C cells based on the uptake of biogenic amine precursors. Histochemistry, 38:297–305.

11.

Fontaine

. 1979. Multistep migration of calcitonin cell precursors during ontogeny of the mouse pharynx. Gen Comp Endocrinol, 37:81–92.

12.

Zorn

, Wells

. 2007. Molecular basis of vertebrate endoderm development. Int Rev Cytol, 259:49–111.

13.

Kimelman

, Griffin

. 2000. Vertebrate mesendoderm induction and patterning. Curr Opin Genet Dev, 10:350–356.

14.

Rodaway

, Patient

. 2001. Mesendoderm. An ancient germ layer? Cell, 105:169–172.

15.

Lawson

, Pedersen

. 1987. Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse. Development, 101:627–652.

16.

Lawson

, Meneses

, Pedersen

. 1991. Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development, 113:891–911.

17.

Ninomiya

, Takahashi

, Tanegashima

, Yokota

, Asashima

. 1999. Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus. Dev Growth Differ, 41:391–400.

18.

Norris

, Robertson

. 1999. Asymmetric and node-specific nodal expression patterns are controlled by two distinct cis-acting regulatory elements. Genes Dev, 13:1575–1588.

19.

Morkel

, Huelsken

, Wakamiya

, Ding

, van de Wetering

, Clevers

, Taketo

, Behringer

, Shen

, Birchmeier

. 2003. Beta-catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation. Development, 130:6283–6294.

20.

Yamamoto

, Saijoh

, Perea-Gomez

, Shawlot

, Behringer

, Ang

, Hamada

, Meno

. 2004. Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo. Nature, 428:387–392.

21.

Kubo

, Shinozaki

, Shannon

, Kouskoff

, Kennedy

, Woo

, Fehling

, Keller

. 2004. Development of definitive endoderm from embryonic stem cells in culture. Development, 131:1651–1662.

22.

Lazzaro

, Price

, de Felice

, Di Lauro

. 1991. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development, 113:1093–1104.

23.

De Felice

, Postiglione

, Di Lauro

. 2004. Minireview: thyrotropin receptor signaling in development and differentiation of the thyroid gland: insights from mouse models and human diseases. Endocrinology, 145:4062–4067.

24.

Postiglione

, Parlato

, Rodriguez-Mallon

, Rosica

, Mithbaokar

, Maresca

, Marians

, Davies

, Zannini

, De Felice

, Di Lauro

. 2002. Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland. Proc Natl Acad Sci USA, 99:15462–15467.

25.

Beamer

, Eicher

, Maltais

, Southard

. 1981. Inherited primary hypothyroidism in mice. Science, 212:61–63.

26.

, Du

, Kopp

, Rentoumis

, Albanese

, Kohn

, Madison

, Jameson

. 1995. The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding. Endocrinology, 136:3146–3153.

27.

, Crenshaw

3rd , Rawson

, Simmons

, Swanson

, Rosenfeld

. 1990. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature, 347:528–533.

28.

De Felice

, Di Lauro

. 2004. Thyroid development and its disorders: genetics and molecular mechanisms. Endocr Rev, 25:722–746.

29.

Serls

, Doherty

, Parvatiyar

, Wells

, Deutsch

. 2005. Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Development, 132:35–47.

30.

Que

, Okubo

, Goldenring

, Nam

, Kurotani

, Morrisey

, Taranova

, Pevny

, Hogan

. 2007. Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm. Development, 134:2521–2531.

31.

Revest

, Spencer-Dene

, Kerr

, De Moerlooze

, Rosewell

, Dickson

. 2001. Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev Biol, 231:47–62.

32.

Ohuchi

, Hori

, Yamasaki

, Harada

, Sekine

, Kato

, Itoh

. 2000. FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem Biophys Res Commun, 277:643–649.

33.

Celli

, LaRochelle

, Mackem

, Sharp

, Merlino

. 1998. Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning. EMBO J, 17:1642–1655.

34.

Fagman

, Grande

, Edsbagge

, Semb

, Nilsson

. 2003. Expression of classical cadherins in thyroid development: maintenance of an epithelial phenotype throughout organogenesis. Endocrinology, 144:3618–3624.

35.

Kusakabe

, Hoshi

, Kimura

. 2006. Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1 expression is required for development and fusion of the ultimobranchial body to the thyroid. Dev Dyn, 235:1300–1309.

36.

Harach

. 1988. Solid cell nests of the thyroid. J Pathol, 155:191–200.

37.

Reis-Filho

, Preto

, Soares

, Ricardo

, Cameselle-Teijeiro

, Sobrinho-Simoes

. 2003. p63 expression in solid cell nests of the thyroid: further evidence for a stem cell origin. Mod Pathol, 16:43–48.

38.

Preto

, Cameselle-Teijeiro

, Moldes-Boullosa

, Soares

, Cameselle-Teijeiro

, Silva

, Reis-Filho

, Reyes-Santias

, Alfonsin-Barreiro

, Forteza

, Sobrinho-Simoes

. 2004. Telomerase expression and proliferative activity suggest a stem cell role for thyroid solid cell nests. Mod Pathol, 17:819–826.

39.

Desbaillets

, Ziegler

, Groscurth

, Gassmann

. 2000. Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol, 85:645–651.

40.

Trounson

. 2006. The production and directed differentiation of human embryonic stem cells. Endocr Rev, 27:208–219.

41.

Lin

, Kubo

, Keller

, Davies

. 2003. Committing embryonic stem cells to differentiate into thyrocyte-like cells in vitro. Endocrinology, 144:2644–2649.

42.

Lin

, Davies

. 2006. Derivation and characterization of thyrocyte-like cells from embryonic stem cells in vitro. Methods Mol Biol, 330:249–261.

43.

Arufe

, Lu

, Lin

. 2009. Differentiation of murine embryonic stem cells to thyrocytes requires insulin and insulin-like growth factor-1. Biochem Biophys Res Commun, 381:264–270.

44.

, Latif

, Davies

. 2009. Thyrotropin-independent induction of thyroid endoderm from embryonic stem cells by activin A. Endocrinology, 150:1970–1975.

45.

D'Amour

, Agulnick

, Eliazer

, Kelly

, Kroon

, Baetge

. 2005. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol, 23:1534–1541.

46.

Coclet

, Foureau

, Ketelbant

, Galand

, Dumont

. 1989. Cell population kinetics in dog and human adult thyroid. Clin Endocrinol (Oxf ), 31:655–665.

47.

Dumont

, Lamy

, Roger

, Maenhaut

. 1992. Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors. Physiol Rev, 72:667–697.

48.

Derwahl

, Studer

. 2002. Hyperplasia versus adenoma in endocrine tissues: are they different? Trends Endocrinol Metab, 13:23–28.

49.

Krohn

, Fuhrer

, Bayer

, Eszlinger

, Brauer

, Neumann

, Paschke

. 2005. Molecular pathogenesis of euthyroid and toxic multinodular goiter. Endocr Rev, 26:504–524.

50.

Peter

, Gerber

, Studer

, Smeds

. 1985. Pathogenesis of heterogeneity in human multinodular goiter. A study on growth and function of thyroid tissue transplanted onto nude mice. J Clin Invest, 76:1992–2002.

51.

Thomas

, Nowka

, Lan

, Derwahl

. 2006. Expression of endoderm stem cell markers: evidence for the presence of adult stem cells in human thyroid glands. Thyroid, 16:537–544.

52.

Lan

, Cui

, Nowka

, Derwahl

. 2007. Stem cells derived from goiters in adults form spheres in response to intense growth stimulation and require thyrotropin for differentiation into thyrocytes. J Clin Endocrinol Metab, 92:3681–3688.

53.

Fierabracci

, Puglisi

, Giuliani

, Mattarocci

, Gallinella-Muzi

. 2008. Identification of an adult stem/progenitor cell-like population in the human thyroid. J Endocrinol, 198:471–487.

54.

Toda

, Watanabe

, Yokoi

, Matsumura

, Suzuki

, Ootani

, Aoki

, Koike

, Sugihara

. 2002. A new organotypic culture of thyroid tissue maintains three-dimensional follicles with C cells for a long term. Biochem Biophys Res Commun, 294:906–911.

55.

Toda

, Koike

, Sugihara

. 2001. Thyrocyte integration, and thyroid folliculogenesis and tissue regeneration: perspective for thyroid tissue engineering. Pathol Int, 51:403–417.

56.

De Tullio

, Averna

, Salamino

, Pontremoli

, Melloni

. 2000. Differential degradation of calpastatin by mu- and m-calpain in Ca(2+)-enriched human neuroblastoma LAN-5 cells. FEBS Lett, 475:17–21.

57.

Greiner

, Hesselton

, Shultz

. 1998. SCID mouse models of human stem cell engraftment. Stem Cells, 16:166–177.

58.

Hoshi

, Kusakabe

, Taylor

, Kimura

. 2007. Side population cells in the mouse thyroid exhibit stem/progenitor cell-like characteristics. Endocrinology, 148:4251–4258.

59.

Tsai

, McKay

. 2002. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev, 16:2991–3003.

60.

Niwa

, Miyazaki

, Smith

. 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet, 24:372–376.

61.

Xin

, Lawson

, Witte

. 2005. The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis. Proc Natl Acad Sci USA, 102:6942–6947.

62.

van de Rijn

, Heimfeld

, Spangrude

, Weissman

. 1989. Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci USA, 86:4634–4638.

63.

Wulf

, Luo

, Jackson

, Brenner

, Goodell

. 2003. Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica, 88:368–378.

64.

Sherwood

, Christensen

, Weissman

, Wagers

. 2004. Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells. Stem Cells, 22:1292–1304.

65.

Cohen

, Rovelli

, Merlo

, van Lint

, Lanino

, Bresters

, Ceppi

, Bocchini

, Tichelli

, Socie

. 2007. Risk for secondary thyroid carcinoma after hematopoietic stem-cell transplantation: an EBMT Late Effects Working Party Study. J Clin Oncol, 25:2449–2454.

66.

Sanders

, Hoffmeister

, Woolfrey

, Carpenter

, Storer

, Storb

, Appelbaum

. 2009. Thyroid function following hematopoietic cell transplantation in children: 30 years' experience. Blood, 113:306–308.

67.

Liegeois

, Escourrou

, Ouvre

, Charreire

. 1977. Microchimerism: a stable state of low-ratio proliferation of allogeneic bone marrow. Transplant Proc, 9:273–276.

68.

Wang

, Iwatani

, Ito

, Horimoto

, Yamato

, Matsui

, Imai

, Hori

. 2004. Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury. Biochem Biophys Res Commun, 325:961–967.

69.

Turin

, Tribbioli

, Invernizzi

, Grati

, Crema

, Laible

, Riva

. 2007. Fetal microchimerism in normal and embryo transfer bovine pregnancies. Vet Res Commun, 31,Suppl 1:205–207.

70.

Jimenez

, Leapley

, Lee

, Ultsch

, Tarantal

. 2005. Fetal CD34+ cells in the maternal circulation and long-term microchimerism in rhesus monkeys (Macaca mulatta) Transplantation, 79:142–146.

71.

, Patel

, Sampietro

, Gillmer

, Fleming

, Wainscoat

. 1990. Detection of single-copy fetal DNA sequence from maternal blood. Lancet, 335:1463–1464.

72.

Klonisch

, Drouin

. 2009. Fetal-maternal exchange of multipotent stem/progenitor cells: microchimerism in diagnosis and diseases. Trends Mol Med, 15:510–518.

73.

Thomas

, Williamson

, Craft

, Yazdani

, Rodeck

. 1994. Y chromosome sequence DNA amplified from peripheral blood of women in early pregnancy. Lancet, 343:413–414.

74.

Ariga

, Ohto

, Busch

, Imamura

, Watson

, Reed

, Lee

. 2001. Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion, 41:1524–1530.

75.

Bianchi

, Farina

, Weber

, Delli-Bovi

, Deriso

, Williams

, Klinger

. 2001. Significant fetal-maternal hemorrhage after termination of pregnancy: implications for development of fetal cell microchimerism. Am J Obstet Gynecol, 184:703–706.

76.

Bianchi

, Williams

, Sullivan

, Hanson

, Klinger

, Shuber

. 1997. PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet, 61:822–829.

77.

Bianchi

, Zickwolf

, Weil

, Sylvester

, DeMaria

. 1996. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA, 93:705–708.

78.

Campagnoli

, Roberts

, Kumar

, Bennett

, Bellantuono

, Fisk

. 2001. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood, 98:2396–2402.

79.

Mikhail

, M'Hamdi

, Welsh

, Levicar

, Marley

, Nicholls

, Habib

, Louis

, Fisk

, Gordon

. 2008. High frequency of fetal cells within a primitive stem cell population in maternal blood. Hum Reprod, 23:928–933.

80.

Nguyen Huu

, Dubernard

, Aractingi

, Khosrotehrani

. 2006. Feto-maternal cell trafficking: a transfer of pregnancy associated progenitor cells. Stem Cell Rev, 2:111–116.

81.

Renne

, Ramos Lopez

, Steimle-Grauer

, Ziolkowski

, Pani

, Luther

, Holzer

, Encke

, Wahl

, Bechstein

, Usadel

, Hansmann

, Badenhoop

. 2004. Thyroid fetal male microchimerisms in mothers with thyroid disorders: presence of Y-chromosomal immunofluorescence in thyroid-infiltrating lymphocytes is more prevalent in Hashimoto's thyroiditis and Graves' disease than in follicular adenomas. J Clin Endocrinol Metab, 89:5810–5814.

82.

Srivatsa

, Srivatsa

, Johnson

, Samura

, Lee

, Bianchi

. 2001. Microchimerism of presumed fetal origin in thyroid specimens from women: a case–control study. Lancet, 358:2034–2038.

83.

Koopmans

, Kremer Hovinga

, Baelde

, Harvey

, de Heer

, Bruijn

, Bajema

. 2008. Chimerism occurs in thyroid, lung, skin and lymph nodes of women with sons. J Reprod Immunol, 78:68–75.

84.

Imaizumi

, Pritsker

, Unger

, Davies

. 2002. Intrathyroidal fetal microchimerism in pregnancy and postpartum. Endocrinology, 143:247–253.

85.

Klintschar

, Schwaiger

, Mannweiler

, Regauer

, Kleiber

. 2001. Evidence of fetal microchimerism in Hashimoto's thyroiditis. J Clin Endocrinol Metab, 86:2494–2498.

86.

Cameselle-Teijeiro

, Varela-Duran

, Sambade

, Villanueva

, Varela-Nunez

, Sobrinho-Simoes

. 1994. Solid cell nests of the thyroid: light microscopy and immunohistochemical profile. Hum Pathol, 25:684–693.

87.

Beckner

, Shultz

, Richardson

. 1990. Solid and cystic ultimobranchial body remnants in the thyroid. Arch Pathol Lab Med, 114:1049–1052.

88.

Burstein

, Nagi

, Wang

, Unger

. 2004. Immunohistochemical detection of p53 homolog p63 in solid cell nests, papillary thyroid carcinoma, and Hashimoto's thyroiditis: a stem cell hypothesis of papillary carcinoma oncogenesis. Hum Pathol, 35:465–473.

89.

Harach

. 1987. Mixed follicles of the human thyroid gland. Acta Anat (Basel), 129:27–30.

90.

Yang

, Kaghad

, Wang

, Gillett

, Fleming

, Dotsch

, Andrews

, Caput

, McKeon

. 1998. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell, 2:305–316.

91.

Yang

, McKeon

. 2000. P63 and P73: P53 mimics, menaces and more. Nat Rev Mol Cell Biol, 1:199–207.

92.

Pellegrini

, Dellambra

, Golisano

, Martinelli

, Fantozzi

, Bondanza

, Ponzin

, McKeon

, De Luca

. 2001. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA, 98:3156–3161.

93.

Mills

, Zheng

, Wang

, Vogel

, Roop

, Bradley

. 1999. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature, 398:708–713.

94.

Yang

, Schweitzer

, Sun

, Kaghad

, Walker

, Bronson

, Tabin

, Sharpe

, Caput

, Crum

, McKeon

. 1999. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature, 398:714–718.

95.

Faggiano

, Talbot

, Baudin

, Bidart

, Schlumberger

, Caillou

. 2003. Differential expression of galectin 3 in solid cell nests and C cells of human thyroid. J Clin Pathol, 56:142–143.

96.

Nikiforova

, Kimura

, Gandhi

, Biddinger

, Knauf

, Basolo

, Zhu

, Giannini

, Salvatore

, Fusco

, Santoro

, Fagin

, Nikiforov

. 2003. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab, 88:5399–5404.

97.

Jhiang

, Sagartz

, Tong

, Parker-Thornburg

, Capen

, Cho

, Xing

, Ledent

. 1996. Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas. Endocrinology, 137:375–378.

98.

Portella

, Vitagliano

, Borselli

, Melillo

, Salvatore

, Rothstein

, Vecchio

, Fusco

, Santoro

. 1999. Human N-ras, TRK-T1, and RET/PTC3 oncogenes, driven by a thyroglobulin promoter, differently affect the expression of differentiation markers and the proliferation of thyroid epithelial cells. Oncol Res, 11:421–427.

99.

Moniz

, Catarino

, Marques

, Cavaco

, Sobrinho

, Leite

. 2002. Clonal origin of non-medullary thyroid tumours assessed by non-random X-chromosome inactivation. Eur J Endocrinol, 146:27–33.

100.

Pagani

, Zagato

, Vergani

, Casari

, Sidoli

, Baralle

. 1991. Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat. J Cell Biol, 113:1223–1229.

101.

Takano

, Matsuzuka

, Miyauchi

, Yokozawa

, Liu

, Morita

, Kuma

, Amino

. 1998. Restricted expression of oncofetal fibronectin mRNA in thyroid papillary and anaplastic carcinoma: an in situ hybridization study. Br J Cancer, 78:221–224.

102.

Zhang

, Zuo

, Ozaki

, Nakagomi

, Kakudo

. 2006. Cancer stem cell hypothesis in thyroid cancer. Pathol Int, 56:485–489.

103.

Huang

, Prasad

, Lemon

, Hampel

, Wright

, Kornacker

, LiVolsi

, Frankel

, Kloos

, Eng

, Pellegata

, de la Chapelle

. 2001. Gene expression in papillary thyroid carcinoma reveals highly consistent profiles. Proc Natl Acad Sci USA, 98:15044–15049.

104.

Takano

. 2004. Fetal cell carcinogenesis of the thyroid: a hypothesis for better understanding of gene expression profile and genomic alternation in thyroid carcinoma. Endocr J, 51:509–515.

105.

Takano

, Amino

. 2005. Fetal cell carcinogenesis: a new hypothesis for better understanding of thyroid carcinoma. Thyroid, 15:432–438.

106.

Hombach-Klonisch

, Paranjothy

, Wiechec

, Pocar

, Mustafa

, Seifert

, Zahl

, Gerlach

, Biermann

, Steger

, Hoang-Vu

, Schulze-Osthoff

, Los

. 2008. Cancer stem cells as targets for cancer therapy: selected cancers as examples. Arch Immunol Ther Exp (Warsz), 56:165–180.

107.

Takano

. 2007. Fetal cell carcinogenesis of the thyroid: theory and practice. Semin Cancer Biol, 17:233–240.

108.

Mitsutake

, Iwao

, Nagai

, Namba

, Ohtsuru

, Saenko

, Yamashita

. 2007. Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology, 148:1797–1803.

109.

Zito

, Richiusa

, Bommarito

, Carissimi

, Russo

, Coppola

, Zerilli

, Rodolico

, Criscimanna

, Amato

, Pizzolanti

, Galluzzo

, Giordano

. 2008. In vitro identification, characterization of CD133(pos) cancer stem-like cells in anaplastic thyroid carcinoma cell lines. PLoS One, 3:e3544.

110.

Friedman

, Lu

, Schultz

, Thomas

, Lin

. 2009. CD133+ anaplastic thyroid cancer cells initiate tumors in immunodeficient mice, are regulated by thyrotropin. PLoS One, 4:e5395.

111.

Schweppe

, Klopper

, Korch

, Pugazhenthi

, Benezra

, Knauf

, Fagin

, Marlow

, Copland

, Smallridge

, Haugen

. 2008. Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab, 93:4331–4341.

112.

Ginestier

, Hur

, Charafe-Jauffret

, Monville

, Dutcher

, Brown

, Jacquemier

, Viens

, Kleer

, Liu

, Schott

, Hayes

, Birnbaum

, Wicha

, Dontu

. 2007. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell, 1:555–567.

113.

Matsui

, Huff

, Wang

, Malehorn

, Barber

, Tanhehco

, Smith

, Civin

, Jones

. 2004. Characterization of clonogenic multiple myeloma cells. Blood, 103:2332–2336.

114.

Pearce

, Taussig

, Simpson

, Allen

, Rohatiner

, Lister

, Bonnet

. 2005. Characterization of cells with a high aldehyde dehydrogenase activity from cord blood and acute myeloid leukemia samples. Stem Cells, 23:752–760.

115.

Sawicki

. 2008. Fetal microchimerism and cancer. Cancer Res, 68:9567–9569.

116.

, Ren

, Cao

, Li

, Hao

. 2008. Beneficial effects of fetal-maternal microchimerism on the activated haplo-identical peripheral blood stem cell treatment for cancer. Cytotherapy, 10:331–339.

117.

Gilmore

, Haq

, Shadduck

, Jasthy

, Lister

. 2008. Fetal-maternal microchimerism in normal parous females and parous female cancer patients. Exp Hematol, 36:1073–1077.

118.

Cirello

, Recalcati

, Muzza

, Rossi

, Perrino

, Vicentini

, Beck-Peccoz

, Finelli

, Fugazzola

. 2008. Fetal cell microchimerism in papillary thyroid cancer: a possible role in tumor damage and tissue repair. Cancer Res, 68:8482–8488.

119.

Dubernard

, Oster

, Chareyre

, Antoine

, Rouzier

, Uzan

, Aractingi

, Khosrotehrani

. 2009. Increased fetal cell microchimerism in high grade breast carcinomas occurring during pregnancy. Int J Cancer, 124:1054–1059.

120.

Nguyen Huu

, Khosrotehrani

, Oster

, Moguelet

, Espie

, Aractingi

. 2008. Early phase of maternal skin carcinogenesis recruits long-term engrafted fetal cells. Int J Cancer, 123:2512–2517.

121.

Aractingi

, Kanitakis

, Euvrard

, Le Danff

, Peguillet

, Khosrotehrani

, Lantz

, Carosella

. 2005. Skin carcinoma arising from donor cells in a kidney transplant recipient. Cancer Res, 65:1755–1760.

122.

Cameselle-Teijeiro

, Febles-Perez

, Sobrinho-Simoes

. 1995. Papillary and mucoepidermoid carcinoma of the thyroid with anaplastic transformation: a case report with histologic and immunohistochemical findings that support a provocative histogenetic hypothesis. Pathol Res Pract, 191:1214–1221.

123.

Malaguarnera

, Mandarino

, Mazzon

, Vella

, Gangemi

, Vancheri

, Vigneri

, Aloisi

, Vigneri

, Frasca

. 2005. The p53-homologue p63 may promote thyroid cancer progression. Endocr Relat Cancer, 12:953–971.

124.

Zhou

, Schuetz

, Bunting

, Colapietro

, Sampath

, Morris

, Lagutina

, Grosveld

, Osawa

, Nakauchi

, Sorrentino

. 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med, 7:1028–1034.

125.

Hirschmann-Jax

, Foster

, Wulf

, Nuchtern

, Jax

, Gobel

, Goodell

, Brenner

. 2004. A distinct “side population” of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA, 101:14228–14233.

126.

Liu

, Yuan

, Zeng

, Tunici

, Ng

, Abdulkadir

, Lu

, Irvin

, Black

, Yu

. 2006. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer, 5:67–79.

127.

Woodward

, Chen

, Behbod

, Alfaro

, Buchholz

, Rosen

. 2007. WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA, 104:618–623.

128.

Rao

, Kremenevskaja

, von Wasielewski

, Jakubcakova

, Kant

, Resch

, Brabant

. 2006. Wnt/beta-catenin signaling mediates antineoplastic effects of imatinib mesylate (gleevec) in anaplastic thyroid cancer. J Clin Endocrinol Metab, 91:159–168.

129.

Dziba

, Ain

. 2004. Imatinib mesylate (gleevec; STI571) monotherapy is ineffective in suppressing human anaplastic thyroid carcinoma cell growth in vitro. J Clin Endocrinol Metab, 89:2127–2135.

130.

de Martimprey

, Bertrand

, Fusco

, Santoro

, Couvreur

, Vauthier

, Malvy

. 2008. siRNA nanoformulation against the ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma. Nucleic Acids Res, 36:e2.

131.

Deshpande

, Gettinger

, Sosa

. 2008. Novel chemotherapy options for advanced thyroid tumors: small molecules offer great hope. Curr Opin Oncol, 20:19–24.

132.

Mansouri

, Chowdhury

, Gruss

. 1998. Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet, 19:87–90.

133.

Kimura

, Hara

, Pineau

, Fernandez-Salguero

, Fox

, Ward

, Gonzalez

. 1996. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev, 10:60–69.

134.

Martinez Barbera

, Clements

, Thomas

, Rodriguez

, Meloy

, Kioussis

, Beddington

. 2000. The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. Development, 127:2433–2445.

135.

De Felice

, Ovitt

, Biffali

, Rodriguez-Mallon

, Arra

, Anastassiadis

, Macchia

, Mattei

, Mariano

, Scholer

, Macchia

, Di Lauro

. 1998. A mouse model for hereditary thyroid dysgenesis and cleft palate. Nat Genet, 19:395–398.

136.

Kameda

, Ito

, Nishimaki

, Gotoh

. 2009. FRS2alpha is required for the separation, migration, and survival of pharyngeal-endoderm derived organs including thyroid, ultimobranchial body, parathyroid, and thymus. Dev Dyn, 238:503–513.

137.

, Zheng

, Laclef

, Maire

, Maas

, Peters

, Xu

. 2002. Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. Development, 129:3033–3044.

138.

Manley

, Capecchi

. 1995. The role of Hoxa-3 in mouse thymus and thyroid development. Development, 121:1989–2003.

139.

Peters

, Neubuser

, Kratochwil

, Balling

. 1998. Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities. Genes Dev, 12:2735–2747.

140.

Meunier

, Aubin

, Jeannotte

. 2003. Perturbed thyroid morphology and transient hypothyroidism symptoms in Hoxa5 mutant mice. Dev Dyn, 227:367–378.

141.

Rochefort

, Caillou

, Michiels

, Ledent

, Talbot

, Schlumberger

, Lavelle

, Monier

, Feunteun

. 1996. Thyroid pathologies in transgenic mice expressing a human activated Ras gene driven by a thyroglobulin promoter. Oncogene, 12:111–118.

142.

Russell

, Powell

, Cunnane

, Greco

, Portella

, Santoro

, Fusco

, Rothstein

. 2000. The TRK-T1 fusion protein induces neoplastic transformation of thyroid epithelium. Oncogene, 19:5729–5735.

143.

La Perle

, Jhiang

, Capen

. 2000. Loss of p53 promotes anaplasia and local invasion in ret/PTC1-induced thyroid carcinomas. Am J Pathol, 157:671–677.

144.

Powell

Jr. , Russell

, Nibu

, Li

, Rhee

, Liao

, Goldstein

, Keane

, Santoro

, Fusco

, Rothstein

. 1998. The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids. Cancer Res, 58:5523–5528.

145.

Powell

Jr. , Russell

, Li

, Kuo

, Fidanza

, Huebner

, Rothstein

. 2001. Altered gene expression in immunogenic poorly differentiated thyroid carcinomas from RET/PTC3p53-/- mice. Oncogene, 20:3235–3246.

146.

Knauf

, Ma

, Smith

, Zhang

, Mitsutake

, Liao

, Refetoff

, Nikiforov

, Fagin

. 2005. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res, 65:4238–4245.

147.

Suzuki

, Willingham

, Cheng

. 2002. Mice with a mutation in the thyroid hormone receptor beta gene spontaneously develop thyroid carcinoma: a mouse model of thyroid carcinogenesis. Thyroid, 12:963–969.

148.

Guigon

, Zhao

, Willingham

, Cheng

. 2009. PTEN deficiency accelerates tumour progression in a mouse model of thyroid cancer. Oncogene, 28:509–517.

149.

Ribeiro-Neto

, Leon

, Urbani-Brocard

, Lou

, Nyska

, Altschuler

. 2004. cAMP-dependent oncogenic action of Rap1b in the thyroid gland. J Biol Chem, 279:46868–46875.

150.

Miller

, Yeager

, Baker

, Liao

, Refetoff

, Di Cristofano

. 2009. Oncogenic Kras requires simultaneous PI3K signaling to induce ERK activation and transform thyroid epithelial cells in vivo. Cancer Res, 69:3689–3694.

151.

Yeager

, Klein-Szanto

, Kimura

, Di Cristofano

. 2007. Pten loss in the mouse thyroid causes goiter and follicular adenomas: insights into thyroid function and Cowden disease pathogenesis. Cancer Res, 67:959–966.

152.

Ledent

, Coppee

, Dumont

, Vassart

, Parmentier

. 1996. Transgenic models for proliferative and hyperfunctional thyroid diseases. Exp Clin Endocrinol Diabetes, 104,Suppl 3:43–46.

153.

Michiels

, Chappuis

, Caillou

, Pasini

, Talbot

, Monier

, Lenoir

, Feunteun

, Billaud

. 1997. Development of medullary thyroid carcinoma in transgenic mice expressing the RET protooncogene altered by a multiple endocrine neoplasia type 2A mutation. Proc Natl Acad Sci USA, 94:3330–3335.

154.

Johnston

, Hatzis

, Sunday

. 1998. Expression of v-Ha-ras driven by the calcitonin/calcitonin gene-related peptide promoter: a novel transgenic murine model for medullary thyroid carcinoma. Oncogene, 16:167–177.

155.

Harvey

, Vogel

, Lee

, Bradley

, Donehower

. 1995. Mice deficient in both p53 and Rb develop tumors primarily of endocrine origin. Cancer Res, 55:1146–1151.

156.

Coxon

, Ward

, Geradts

, Otterson

, Zajac-Kaye

, Kaye

. 1998. RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer. Oncogene, 17:1625–1628.

157.

Parlato

, Rosica

, Rodriguez-Mallon

, Affuso

, Postiglione

, Arra

, Mansouri

, Kimura

, Di Lauro

, De Felice

. 2004. An integrated regulatory network controlling survival and migration in thyroid organogenesis. Dev Biol, 276:464–475.

158.

Trueba

, Auge

, Mattei

, Etchevers

, Martinovic

, Czernichow

, Vekemans

, Polak

, Attie-Bitach

. 2005. PAX8, TITF1, and FOXE1 gene expression patterns during human development: new insights into human thyroid development and thyroid dysgenesis-associated malformations. J Clin Endocrinol Metab, 90:455–462.

159.

Di Palma

, D'Andrea

, Liguori

, Liguoro

, de Cristofaro

, Del Prete

, Pappalardo

, Mascia

, Zannini

. 2009. TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation. Exp Cell Res, 315:162–175.

160.

Takano

, Miyauchi

, Yoshida

, Kuma

, Amino

. 2004. High-throughput differential screening of mRNAs by serial analysis of gene expression: decreased expression of trefoil factor 3 mRNA in thyroid follicular carcinomas. Br J Cancer, 90:1600–1605.