<P>To determine whether stem/progenitor cells are present in adult mouse thyroid, we focused on Side Population (SP) cells. SP cells are characterized by their ability to efflux the vital dye Hoechst 33342 when analyzed by flow cytometry, due to expression of the ATP binding cassette (ABC)-dependent transporter ABCG2. SP cells have been identified in various hematopoietic and non-hematopoietic adult tissues, the latter including the liver, skeletal muscle, lung, kidney, and mammary gland. SP cells are thought to be highly enriched for stem/progenitor cell activity.</P> <P>We previously identified SP cells in the adult mouse thyroid ranging from 0.3-1.4% of the total population of cells, which highly express ABCG2 and the stem cell marker genes such as those encoding nucleostemin and Oct4, but not thyroid differentiation markers such as thyroid peroxidase (TPO) and thyroglobulin, as examined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Flow cytometry demonstrated that approximately a half of the thyroid SP cells are positive for Stem cell antigen 1 (Sca1), a marker for stem cells. Because of the size of thyroid and the small number of SP cells obtainable, we performed partial thyroidectomy on mice in order to enhance the population of thyroid SP cells. This was based on the hypothesis that partial thyroidectomy mimics acute thyroid injury and thyroid stem/progenitor cells may be required to actively participate in the regeneration of injured thyroid. Partial thyroidectomized thyroids were removed two days after the surgery, and the thyroid cells subjected to flow cytometric analysis. The percentage of SP cells increased after partial thyroidectomy, suggesting that stem/progenitor cells in the SP cells may be activated by partial thyroidectomy, resulting in the increased population. We also found that the homeodomain transcription factor NKX2-1, which is the critical transcription factor for the genesis, homeostasis, and function of the thyroid, affects the number of SP cells;NKX2-1 heterozygous mouse thyroids exhibited approximately a half the number of SP cells as compared to wild-type mouse thyroids. The reason for this is unknown, however, it might be relevant to the pathogensis of thyroid cancer because the recent study showed that NKX2-1 plays a role in thyroid carcinogenesis.</P> <P>In order to determine the identity of genes that are specifically expressed in thyroid SP cells, and/or those affected by partial thyroidectomy, SP cells and non-SP cells obtained from mock operated and partial thyroidectomized thyroids were subjected to microarray analysis. Several genes were highly up-regulated in SP cells as compared to non-SP cells. Among those are Duffy blood group, chemokine receptor (DARC), also known as CD234, von Willebrand factor (Vwf), transmembrane 4 superfamily member 1 (Tm4sf1), leucine rich repeat containing 15 (Lrrc15), stanniocalcin 1 (Stc1), and SRY (sex determining region Y)-box 7 (Sox7). qRT-PCR analysis confirmed that they are indeed highly expressed only in SP cells. Whether these genes can be used as a marker for thyroid SP cells needs further experimentation.</P><P>In order to determine genes increased in their expression after partial thyroidectomy and the pattern of changes in the expression, we have also taken another approach, in that thyroids from beta-galactosidase reporter mice are subjected to histological and immunohistological analyses at various time points after partial thyroidectomy. This reporter mouse carries beta-galactosidase gene flanked by LoxP sites and the thyroid peroxidase (TPO) promoter-driven Cre recombinase, in which beta-galactosidase is expressed upon activation of TPO, in turn driving Cre expression. Since TPO is expressed only in matured follicular cells, this mouse can be used to trace a lineage for mature follicular cells. Bromo-deoxyuridine (BrdU) pulse labeling is also employed to examine long label-retaining cells, which are considered to be representing stem/progenitor cells. We have focused on cells expressing Sca1, beta-galactosidase, thyroid differentiation markers such as TPO and thyroglobulin, and those retaining BrdU expression to identify newly generated cells after partial thyroidectomy and their possible origins. The experiments are currently in progress.</P>
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