Homepage of This Laboratory

Professor

Yasufumi SATO

Professor

Yasuhisa MATSUI

Assistant Professor

Daiji OKAMURA

Assistant Professor

Ikuma MAEDA

Technical Staff

Ikue AIHARA

Technical staff

Yasuhiro SUGAWARA

Technical Assistant

Yuko TOKITAKE

Technical Assistant

Fujimi KOIZUMI

Technical Assistant

Makiko IKEDA

Graduate Student

Kentarou MOCHIZUKI

Graduate Student

Ryo-hei HAMADA

1. Cell Bank

The purpose of the Cell Resource Center for Biomedical Research is the collection, establishment, quality control, distribution of useful cell lines, and construction of database for researchers. The cell lines include transplantable animal cell lines, such as Yoshida sarcoma and rat ascites hepatoma (AH series) cell lines as well as human, murine cell lines and hybridoma cells. The Cell Line Catalog is available on the web site (http://www2.idac.tohoku.ac.jp/dep/ccr/).

2. Research Interest

Our goal is to elucidate the molecular mechanisms of mouse primordial germ cell (PGC) formation and their subsequent differentiation. These studies are meaningful not only for basic research interests for germ cell development, but also for furthering our understanding the possible applications of germ cells and pluripotent stem cells in practical science.

Heterogeneity of mouse primordial germ cells (PGCs) during their differentiation, and identification of PGC subpopulations that have greater ability to develop into pluripotential stem cells: Dev. Growth Differ. 51, 567-584 (2009); Dev. Growth Differ., in press.

Primordial germ cells (PGCs) in mouse embryos likely include heterogeneous cells having distinct cellular properties. We found that heterogeneity of PGCs can be defined by the expression of integrin α6 and c-Kit. The changes in integrin α6 and c-Kit expression in PGCs were obvious as embryonic development progressed, and the PGCs became a mixture of populations consisting of cells with distinct levels of cell surface protein expression. The changes and heterogeneity of cell surface protein expression mainly reflected asynchronous differentiation of PGCs. Apoptosis of PGCs was biased in populations of c-Kit or integrin α6 negative PGCs at particular developmental stages, suggesting possible linkage between PGC apoptosis and the levels of expression of these cell surface proteins. This study enables us to analyze and isolate populations of living PGCs showing a distinct status of differentiation, or different properties of proliferation or of cell death in individual embryos, and provides a new strategy to examine the mechanisms of PGC development.

We previously found that some mouse PGCs develop into pluripotential cells (EG cells) when cultured on a feeder layer expressing the membrane bound form of Steel factor with culture medium containing LIF and bFGF. To understand the mechanisms of the conversion of PGCs into EG cells, we attempted to identify PGC subpopulations that have the ability to develop into EG cells. Using flow cytometry, we fractionated PGCs by the expression of the cell surface antigen integrin α6, as well as by the detection of side-population (SP) cells in which stem cells are enriched in various tissues. PGCs with negative or low integrin α6 expression and with SP cell phenotype showed higher potential to convert to EG cells. Negative or low integrin α6 expression in PGCs was also correlated with lower expression of Ddx4, which is specifically expressed in PGCs after E10.5. The results indicate that the primitive PGC population showing the SP cell phenotype among undifferentiated PGCs have higher ability to be converted into EG cells. Thus, conversion of PGCs into pluripotential stem cells may be regulated by being influenced by the natural status of individual PGCs as well as the reprogramming process after starting culture.

Figure 1

Heterogeneity of mouse primordial germ cells (PGCs)
The expression of cell surface proteins, integrin a6 and c-Kit is dynamically changed during PGC differentiation, and PGCs can be classified by their expression (upper figure).

Primordial germ cells (PGCs) with low level expression of c-Kit frequently undergo apoptosis (lower picture, Green; PGCs, Blue; active caspase 3, Red; c-Kit).

 

Requirement of Oct3/4 for germ cell specification: Dev. Biol. 317, 576-584 (2008).

Mammalian fertilized eggs first develop into a population of pluripotent cells such as those of an inner cell mass or epiblast, from which all somatic and germ cells are then derived. The restriction of germ cell lineage from epiblast seems to occur after the mid-gastrula stage of development in mouse embryos, and primordial germ cells (PGCs ) are first identified histologically within the posterior extraembryonic mesoderm at 7.25 dpc as a cluster of cells expressing tissue-nonspecific alkaline phosphatase (TNAP). Previous experiments indicated that PGC precursors exist in the proximal region of epiblats and those precursors were induced by BMP signals from the extraembryonic ectoderm before 6.0 dpc. PGC precursors then form a cluster of cells within a region of extraembryonic mesoderm at 6.75 dpc, and we have shown that E-cadherin-mediated cell-cell interaction among these precursors was required for germ cell specification.

In addition to the extracellular signals, the PGC precursors should need activation of intracellular molecular cascades to be specified to PGCs, and we supposed that Oct3/4 could function as an activator for PGC specification because of its germ cell-specific expression. Oct3/4 is specifically expressed in pluripotential cells as well, and play a crucial role on maintenance of pluripotency．To examine a possible function of Oct3/4 in germ cell formation, we used a ES cell line in which Oct3/4 gene was manipulated. In this ES cell, both alleles of the endogenous Oct3/4 gene were disrupted by gene targeting, and its pluripotency was maintained by a Oct3/4 transgene under the control of the Tet-Off promoter. This ES cell line has another expression vector of Oct3/4 fused to GFP and the ligand binding domain of glucocorticoid receptor, which is activated by dexamethasone. In the absence of the drugs, the ES cells can maintain pluripotency by the Oct3/4 protein from the Tet-Off transgene. In the presence of tetracycline or its derivative, Doxycycline, the cells lose Oct3/4 protein, while Oct3/4 activity is restored by additional dexamethasone.

By using this ES cell, we generated chimeric embyos, and cultured the PGC precursors obtained from the chimeric embryos with doxycycline or with both doxycycline and dexamethasone. The ES-derived cells can be distinguished by GFP signal. With doxycycline, Oct3/4 protein was present only in cytoplasm of the ES-derived cells as indicated by GFP signal, and all of the specified PGCs expressing Stella were GFP negative host derived cells. By contrast, with both doxycycline and dexamethasone, Oct3/4 protein was restored in nucleus, and a lot of the ES-derived cells became stella expressing PGCs. The results indicate that PGC specification from the precursor cells depends on the activity of Oct3/4.

Figure 2
Oct3/4-dependent PGC specification
In the presence of Dox (+Dox), most of the PGC7/Stella-positive cells (red) were derived from the GFP-negative recipient cells (arrowheads). In the presence of Dex as well as Dox (+Dox +Dex), the GFP-expressing 4EOGR1 ES-derived cells (green) efficiently contributed to PGC7/Stella positive cells (arrowheads).

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