Cell Cycle Synchronization of Porcine Fetal Fibroblasts: Effects of Serum Deprivation and Reversible Cell Cycle Inhibitors1

Abstract

The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6-73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24-72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9-80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 +/- 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 +/- 5.8% of the cells at the end of G1 stage and 37.0 +/- 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

“…In the present study it is shown that only serum starved and fully confluent states had synchronized more cells at G 0 / G 1 phases which were significantly different than that of cycling state (Table 1). Our results differ with the findings of other authors (Kubota et al, 2000;Kues et al, 2000;Gibbons et al, 2002;Zou et al, 2002;Gomez et al, 2003;Han et al, 2003;Liu et al, 2004;Hayes et al, 2005). Among them, Liu et al (2004) stated that chemical synchronization of the cell cycle stage of rabbit fetal fibroblasts to G 0 /G 1 (25-50%) was less effective as compared to serum deprivation.…”

“…Fluorescence-activated cell sorting (FACS) has been proven an efficient tool to analyze the cell cycle of tumor cells and normally growing cells (Alessi et al, 1998;Boquest et al, 1999;Kues et al, 2000). Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC) is particularly useful to distinguish G 0 phase from G 1 phase (Boquest et al, 1999).…”

“…Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC) is particularly useful to distinguish G 0 phase from G 1 phase (Boquest et al, 1999). There are many studies which have been done on the synchronization of cell cycles using different chemicals and methods (Blow, 1993;Kubota and Takisawa, 1993;Fenteany et al, 1994;Mayer and Noble, 1994;Vesley et al, 1994;Ferrari et al, 1995;Yan et al, 1995;Cotgreave, 1997;Hitomi and Staney, 1999;Kubota et al, 2000;Kues et al, 2000;Gibbons et al, 2002;Lee and Piedrahita, 2002;Zou et al, 2002;Gomez et al, 2003;Han et al, 2003;Gongalez-Cabrera et al, 2004;Liu et al, 2004;Hayes et al, 2005).…”

Disabled-2 Protein Facilitates Assembly Polypeptide-2-independent Recruitment of Cystic Fibrosis Transmembrane Conductance Regulator to Endocytic Vesicles in Polarized Human Airway Epithelial Cells

“…(14). CFBE41o-cells transfected with plasmid DNA were seeded on collagen-coated plastic tissue culture plates (Corning Corp.) and cultured for 72 h. FBS and the selection antibiotic were removed from the media 18 h before experiments to augment cell polarization and cell cycle synchronization (25,26). The parental CFBE41o-cells were maintained under the same culture conditions but without puromycin (12).…”

Possibility to Establish Chicken Stem Cell from Non-germline Tissue; Detection of Colony-forming Cells after Chicken Fibroblast Culture and Subsequent Stem Cell Characterization

“…Various cells mediating structural and functional roles, such as collagen network formation and wound healing, are derived from mesenchymal tissue, leading to cell differentiation and transformation (Moulin et al, 1999;Kues et al, 2000). We therefore presumed that fibroblasts or fibroblast-derived cells in developing embryos might to maintain their pluripotent activity.…”