Suspension Culture-Mediated Tetraploid Formation in Mouse Embryonic Stem Cells

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 36권 / 1호
ㆍ저자명 : Jae Hee Lee, Seung Pyo Gong, Jeong Mook Lim, Seung Tae Lee

목차

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

영어 초록

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.

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