Tadao Atsumi

A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells

A cell line, ATDC5, isolated from a differentiating culture of AT805 teratocarcinoma expressed a fibroblastic cell phenotype in a growing phase. With the addition of 10 micrograms/ml insulin to the medium, cells continued to grow even in a postconfluent phase, formed cartilage nodule-like cell aggregates, were stained with Alcian blue and produced cartilage-specific proteoglycan and type II collagen, typical marker molecules for chondrogenesis. Since ATDC5 cells also differentiated into unidentifiable pigmented cells, they are apparently composed of undetermined cells. ATDC5, therefore, provides a good model system with which to understand chondrogenic differentiation.

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites☆

Abstract The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca2+ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca2+. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.

Isolation of a Germline-Transmissible Embryonic Stem (ES) Cell Line from C3H/He Mice

Abstract We have isolated three embryonic stem (ES) cell lines from C3H/He mice using mouse STO cells as a feeder layer. One ES cell line (H-1) was male, and two (H-2 and H-3) were female, as determined by polymerase chain reaction, in situ hybridization, and karyotype analyses. All were immunocytochemically reactive with a C3H strain-specific antibody. Injection of cells from the female ES H-3 line into C57BL/6 blastocysts yielded four chimeras with slight coat color chimerism. All chimeras were male, and as expected, no germline-transmission was observed. By contrast, when male ES H-1 cells were injected into the perivitelline space of 8-cell C57BL/6 embryos, one male mouse with overt coat color chimerism was recovered, and it produced ES H-1-derived offspring exclusively. This germline-transmissible C3H/He cell line represents a novel addition to those ES lines currently employed for gene manipulation studies of development.

Self-renewal and differentiation of a basic fibroblast growth factor-dependent multipotent hematopoietic cell line derived from embryonic stem cells.

Despite the accumulation of information on the origin of hematopoietic stem cells, it is still unclear how these cells are generated in ontogeny. Isolation of cell lines equivalent to early embryonic hematopoietic progenitor cells can be helpful. A multipotent hematopoietic progenitor cell line, A‐6, was isolated from H‐1 embryonic stem (ES) cells. The self‐renewal of A‐6 cells was supported by basic‐fibroblast growth factor (b‐FGF) and their differentiation into definitive erythroid cells, granulocytes and macrophages was induced after co‐culture with ST‐2 stromal cells. A‐6 cells were positive for the surface markers of hematopoietic stem cell, c‐kit, CD31, CD34, Flt3/Flk2, PgP‐1, and HSA, but were negative for that of the differentiated cells. Reverse transcription‐polymerase chain reaction analysis showed that A‐6 cells produced mRNA from SCL/tal‐1 and GATA‐2 genes. Among various cytokines examined, only stem cell factor (SCF) and Flt3/Flk2 ligand (FL) supported the proliferation of A‐6 cells instead of b‐FGF. The FL, as well as b‐FGF, supported the self‐renewal of A‐6 cells, whereas SCF induced differentiation into myeloid cells. A‐6 cells will be useful for the characterization of hematopoietic progenitor cells derived from ES cells and provide a model system to realize the control mechanisms between self‐renewal and differentiation of hematopoietic stem cells.

CELL ASSOCIATION PATTERN IN AGGREGATES CONTROLLED BY MULTIPLE CELL-CELL ADHESION MECHANISMS

We have previously assumed the presence of two mechanisms for the aggregation of Chinese hamster V79 cells, the Ca2+‐dependent one and the Ca2+‐independent one. In order to examine if each of these mechanisms contributed differently to the various aspects of cell aggregation, the morphology of V79 cell aggregates, pretreated so that they were provided with only one of the two adhesion mechanisms, was compared by light and electron microscopy. The adhesion among cells with only the Ca2+‐dependent mechanism was very tight, with the formation of gap and intermediate junctions. Cells were arranged in a rod or dendric shape in aggregates. In aggregates of cells with only the Ca2+‐independent mechanism, cells were loosely attached to each other without the formation of specialized junctions and the aggregates were of globular shape. In aggregates of cells with both mechanisms, both characteristics of the above two aggregates were found. Four clones of V79 cells, which formed colonies with different morphology when they were grown in soft agar, were isolated. It was found that such differences were due to the different activity of the Ca2+‐independent mechanism among these clones. These results suggested that the two adhesion mechanisms play different roles in the cell arrangement in aggregates.

Efficiency of neural differentiation of mouse P19 embryonal carcinoma cells is dependent on the seeding density

Serum-free culture conditions for retinoic acid-induced neural differentiation of mouse P19 embryonal carcinoma cells were determined for future ex vivo retroviral gene transfer and brain transplantation studies. Neural differentiation of P19 cells was dependent on the seeding densities, and both neurons and astroglia differentiated efficiently at high seeding densities (2 x 10(4) and 5 x 10(4) cells/cm2) but not at low seeding density (1 x 10(4) cells/cm2). In addition, P19 cells cultured at 5 x 10(4) cells/cm2 showed neural differentiated whether or not they were infected with Friend leukemia virus FrC6-V, which inhibited neural differentiation at 2 x 10(4) cells/cm2. These results indicate that FrC6-V-infected P19 embryonal carcinoma cells should be seeded at high density to achieve efficient neural differentiation in vitro for ex vivo gene transfer with a FrC6-V-derived retroviral vector system.

Monolayer culture conditions suitable for primitive erythroid cell differentiation from embryonic stem cells in vitro

Embryonic stem (ES) cells effectively differentiated into primitive erythroid/mesodermal cells when grown in the absence of both a feeder layer and leukemia inhibitory factor (LIF). The formation of a three‐dimensional structure, exogenous mesoderm induction factors and exogenous hematopoietic growth factors were not essential for their differentiation. Primitive erythroid cells were first detected on day 5 in the differentiation‐permissive cultures. Differentiation into other mesodermal cells was always preceded by that into primitive erythroid cells. Precursor cells of erythroid cells but of other hematoid cells were also detected in this system. This model system is useful for studying the early steps of mesoderm formation in mouse embryogenesis.

The Activin A‐Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum‐Free Medium

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum‐free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum‐free conditions examined. P19 and AT805 EC cells grew even in serum‐free medium without activin A but their growth rates were slightly facilitated by its addition. F9 EC cells also grew in the medium without activin A and its addition somewhat inhibited their growth rate. Three independently isolated ES cell lines and feeder‐dependent PSA‐1 EC cells also grew in serum‐free medium without activin A if leukemia inhibitory factor (LIF) was supplemented. The addition of activin A had little effect on their growth rates. These findings suggest that PCC3 EC cells are a sort of nutritional mutant requiring activin A, thus making them useful in stidies on the growth regulatory mechanisms of EC/ES cells and/or the action of activin on EC/ES cells.