Projects and Grants
Current Research
Thyroid stem cell biology and development
Murine embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of the blastocyst-stage embryos that can be indefinitely maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). When LIF is withdrawn, ES cells differentiate and give rise to three-dimensional embryo-like structures known as embryoid bodies (EBs) that are composed of derivatives of the three primary germ layers and have the potential to differentiate into cells of all lineages. These properties not only make ES cells a novel and unlimited source of cells for the treatment of a broad spectrum of diseases, but they also make them an ideal model of early embryonic development. This area of our research program is focused on understanding the mechanisms that regulate the earliest stages of commitment to the thyrocyte lineage.
Our laboratory was the first to demonstrate that mouse ES cells can differentiate into thyrocyte-like cells when cultured with TSH. To further investigate the relationship of TSH receptor (TSHR) to thyrocyte development, we analyzed thyrocyte progenitors in EBs differentiated from ES cells in which green fluorescent protein (GFP) cDNA had been targeted to the TSHR gene. Early EBs generated from the GFP-TSHR ES cells were found to contain a subpopulation of GFP-expressing cells. We have recently succeeded in isolating the GFP-TSHR-positive fraction, which can differentiate into thyroid neofollicle-like clusters following additional culture. Ongoing projects in this area include: (1) Identification of factors necessary to direct EBs to thyrocyte progenitors, (2) Characterization of the development and maturation of the thyrocyte lineage in the EB differentiation cultures, and (3) Functional analysis of the ES cell-derived thyrocytes in vivo.
Differentiation and development of thyrocyte lineage from human ES cells
We are actively pursuing research on human ES cells to confirm our mouse ES cell findings. This part of our research program is focused on defining optimal growth and differentiation conditions for the efficient induction of the thyroid lineage from human ES cells. Thyrocytes, like pancreas, liver and lung cells, are derived from the definitive endodermal embryonic germ layer. Our project has focused on directing ES cells to the definitive endoderm lineage, as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Our goal is to understand the molecular mechanisms regulating endoderm induction and its specification to the thyrocyte lineage during human ES cell differentiation. Our access to human thyroid stem/progenitor cell populations will allow the first detailed molecular analysis of the development of this lineage. Ongoing projects in this area include: (1) Characterization of conditions for the efficient growth of human ES cells; (2) Stimulation of the differentiation of human ES cells to endoderm derivatives; and (3) Specification of the differentiation of human ES cells to thyrocytes.
TSH signaling in embryonic thyroid development
The binding of TSH to its own receptor TSHR is a major trophic stimulus to the thyroid gland. The TSHR belongs to a superfamily of G protein-coupled glycoprotein receptors that have a common structure consisting of seven transmembrane domains. This binding promotes the exchange of GDP for GTP on the alpha subunit of the G protein. The result is activation of the protein kinase A signaling pathway mediated by adenylate cyclase and cAMP, which in turn activates multiple signaling pathways that regulate thyroid differentiation, proliferation and function. TSH resistance is one of the causes of congenital hypothyroidism - the most prevalent inborn endocrine disorder. One in every 3,500 newborns is affected by this condition, which is due primarily to developmental defects leading to an absent, ectopic or hypoplastic thyroid gland. Although the importance of both TSH and TSHR in the postnatal maintenance of the size and structure of the thyroid gland is well established, the specific mechanisms involved during early embryogenesis remain unclear.
Activation of the TSH/TSHR signaling pathway occurs in mice at E15. TSHR knockout mice die within one week of weaning, while heterozygotes are unaffected. TSHR knockout mice have developmental and growths delays and are severely hypothyroid with no detectable thyroid hormone and elevated TSH. By comparing the TSHR knockout mice with mutant mouse lines deprived of either TSH (pitdw/pitdw) or a functional TSHR (tshrhyt/tshrhyt), it is reported that the thyroid hormone synthetic pathway in mice can be divided into TSH-dependent and -independent steps. Ongoing projects in this area of our research program include: (1) The characterization of the thyroid phenotypes of TSHR knockout mouse embryos during post-implantation development, and (2) The determination whether cellular defects in TSHR knockout mice are rescued by adenoviral TSHR transfer.
Molecular basis for thyroid cancer development
In collaboration with Dr. Igor Matushansky at the Memorial Sloan-Kettering Cancer Center, we are establishing a collaborative project in which we will examine the relationship between progenitor cells differentiating into thyrocytes and the various types of thyroid cancers. We have access to a significant number of thyroid cancers of numerous types. Our goal is to have a better understanding of the abnormalities of differentiation that occur during thyroid tumorigenesis. Our ES cell in vitro differentiation model will allow us to identify the molecules responsible for programming thyroid stem cells, which could lead the way to more targeted treatment for thyroid disorders.
