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Borough of Bronx, NY, United States

Ma R.,Thyroid Research Unit | Latif R.,Thyroid Research Unit | Davies T.F.,Thyroid Research Unit
Thyroid | Year: 2015

Objective: The molecular events that lead to human thyroid cell speciation remain incompletely characterized. It has been shown that overexpression of the regulatory transcription factors Pax8 and Nkx2-1 (ttf-1) directs murine embryonic stem (mES) cells to differentiate into thyroid follicular cells by initiating a transcriptional regulatory network. Such cells subsequently organized into three-dimensional follicular structures in the presence of extracellular matrix. In the current study, human embryonic stem (hES) cells were studied with the aim of recapitulating this scenario and producing functional human thyroid cell lines. Methods: Reporter gene tagged pEZ-lentiviral vectors were used to express human PAX8-eGFP and NKX2-1-mCherry in the H9 hES cell line followed by differentiation into thyroid cells directed by Activin A and thyrotropin (TSH). Results: Both transcription factors were expressed efficiently in hES cells expressing either PAX8, NKX2-1, or in combination in the hES cells, which had low endogenous expression of these transcription factors. Further differentiation of the double transfected cells showed the expression of thyroid-specific genes, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium/iodide symporter (NIS), and the TSH receptor (TSHR) as assessed by reverse transcription polymerase chain reaction and immunostaining. Most notably, the Activin/TSH-induced differentiation approach resulted in thyroid follicle formation and abundant TG protein expression within the follicular lumens. On stimulation with TSH, these hES-derived follicles were also capable of dose-dependent cAMP generation and radioiodine uptake, indicating functional thyroid epithelial cells. Conclusion: The induced expression of PAX8 and NKX2-1 in hES cells was followed by differentiation into thyroid epithelial cells and their commitment to form functional three-dimensional neo-follicular structures. The data provide proof of principal that hES cells can be committed to thyroid cell speciation under appropriate conditions. © 2015 Mary Ann Liebert, Inc. Source

Yin X.,Thyroid Research Unit | Sachidanandam R.,The New School | Morshed S.,Thyroid Research Unit | Latif R.,Thyroid Research Unit | And 2 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2014

Context: The immune response in autoimmune thyroid disease has been shown to occur primarily within the thyroid gland in which the most abundant antigens can be found. A variety of capture molecules are known to beexpressedby thyroid epithelial cells and servetoattract and help retain an intrathyroidal immune infiltrate.Objective: To explore the entire repertoire of expressed genes in human thyroid tissue, we have deep sequenced the transcriptome (referred to as mRNA-Seq).Design and Patients: We applied mRNA-Seq to thyroid tissue from nine patients with Graves' disease subjected to total thyroidectomy and compared the data with12 samples of normal thyroid tissue obtained from patients having a thyroid nodule removed. The expression for each gene was calculated from the sequencing databy taking the median of the coverage across the lengthof the gene.The expressionlevels were quantile normalized and a gene signature was derived from these.Results: On comparison of expression levels in tissues derived from Graves' patients and controls, there was clear evidence for overexpression of the antigen presentation pathway consisting of HLA and associated genes. We also found a robust disease signature and discovered active innate and adaptive immune signaling networks.Conclusions: These data reveal anactive immune defense system in Graves' disease, which involves novel molecular mechanisms in its pathogenesis and development. Copyright © 2014 by the Endocrine Society. Source

Latif R.,Mount Sinai School of Medicine | Latif R.,Thyroid Research Unit | Teixeira A.,Mount Sinai School of Medicine | Michalek K.,Poznan University of Medical Sciences | And 10 more authors.
PLoS ONE | Year: 2012

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity. Source

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