원문정보
초록
영어
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.
목차
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