Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained in vitro for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Animals    Cell Preparation    Suspension Culture by Aggregate Formation    Adherent Culture of Aggregates after Suspension Culture    Karyotyping    Short-Tandom Repeat Microsatellite Analysis    Characterization of ES Cells by Immunostaining    Analysis of DNA Content by Propidium Iodide (PI) Staining   RESULTS    The Effects of Suspension Culture under Various Conditionson the Formation Of Tetraploid ES Cells    Identification of Cell Origin    Characterization and Ploidy Shift of Tetraploid Es Cells   DISCUSSION   REFERENCES

• Jae Hee Lee(Laboratory of Stem Cell and Bioevaluation, WCU Biomodulation Program, Seoul National University, Seoul 151-742, Korea, Department of Obstetrics, Gynecology & Reproductive Biology, Michigan State University College of Human Medicine, Grand Rapids, MI, USA)
• Seung Pyo Gong(Department of Marine Biomaterials and Aquaculture, Pukyong National University, Busan 608-737, Korea)
• Jeong Mook Lim(Laboratory of Stem Cell and Bioevaluation, WCU Biomodulation Program, Seoul National University, Seoul 151-742, Korea, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea, Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-744, Korea)
• Seung Tae Lee(Department of Animal Biotechnology, Kangwon National University, Chuncheon 200-701, Korea) Corresponding author