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Background Embryonic stem cells (ESCs) have the potential to form teratomas when implanted into immunodeficient mice, but data in immunocompetent mice are limited. We therefore investigated teratoma formation after implantation of three different mouse ESC (mESC) lines into immunocompetent mice. Materials and methods BALB/c mice were injected with three highly germline competent mESCs (129Sv, BALB/c and C57BL/6) subcutaneously or under the kidney capsule. After 4 weeks, mice were euthanized and examined histologically for teratoma development. The incidence, size and composition of teratomas were compared using Pearson Chi-square, t-test for dependent variables, one-way analysis of variance and the nonparametric Kruskal- Wallis analysis of variance and median test. Results Teratomas developed from all three cell lines. The incidence of formation was significantly higher under the kidney capsule compared to subcutaneous site and occurred in both allogeneic and syngeneic mice. Overall, the size of teratoma was largest with the 129Sv cell line and under the kidney capsule. Diverse embryonic stem cell-derived tissues, belonging to the three embryonic germ layers, were encountered, reflecting the pluripotency of embryonic stem cells. Most commonly represented tissues were nervous tissue, keratinizing stratified squamous epithelium (ectoderm), smooth muscle, striated muscle, cartilage, bone (mesoderm), and glandular tissue in the form of gut- and respiratory-like epithelia (endoderm). Conclusions ESCs can form teratomas in immunocompetent mice and, therefore, removal of undifferentiated ESC is a pre-requisite for a safe use of ESC in cell-based therapies. In addition the genetic relationship of the origin of the cell lines to the ability to transplant plays a major role.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. These cells are appropriate for creation of animal models of human genetic diseases, the study of gene function in vivo and differentiation into specific types as potential therapeutic agents for several human diseases. We describe here, the production of new ES cell lines from blastocysts recovered from the C57BL/6 and BALB/c mouse strains by changing the concentration of leukemia inhibitory factor (LIF) and primary culture conditions. The established cell lines were analyzed by simple karyotype, C banding, alkaline phosphatase activity, and Oct-4 expression as well as for the presence of the SRY gene. Two ES cell lines from C57BL/6 and three from the BALB/c were produced. The two C57BL/6 ES cell lines were established with either 1000 or 5000 IU LIF, whereas the BALB/c ES cell lines required 5000 IU LIF. Four of the ES cell lines had a normal karyotype. C banding and sex-determining region of Y chromosome-polymerase chain reaction showed that all cell lines had an XY sex chromosome composition. All five of the cell lines expressed alkaline phosphatase activity and Oct-4. One of the BALB/c ES cell lines, when injected into C57BL/6 blastocysts, produced high rates of chimerism as assessed by coat color, and the male chimera produced germ-line offspring when mated with BALB/c females. These results indicate that ES cells from inbred strains can be isolated using commercially available reagents and that the establishment of BALB/c ES cell lines may require different culture conditions to the 129 or C57BL/6 strains.