Establishment of a zebrafish embryonic stem cell line using a feeder-free culture method and in vitro pluripotency characterization
| dc.contributor.author | Sing Yee, Ho | |
| dc.date.accessioned | 2015-09-03T07:42:31Z | |
| dc.date.available | 2015-09-03T07:42:31Z | |
| dc.date.issued | 2014 | |
| dc.description.abstract | Feeder layers are commonly used in zebrafish embryonic stem (ES) cell cultures to enable proliferation and prevent differentiation of the ES cells. In this study, a culture method using feeder-free was employed to derive and establish longterm zebrafish ES cell line from zebrafish blastula stage embryos. This cell line was designated as ZES1. It had been maintained for over 800 days in basal medium, DMEM, supplemented with fetal bovine serum, zebrafish embryo extract, trout serum and human basic fibroblast growth factor. ZES1 cells were round or polygonal in shape with large nuclei and sparse cytoplasm. Majority of ZES1 cells also displayed a diploid karyotype, typical of ES cells. ZES1 was able to form individual colonies of tightly packed cells that were positively stained for alkaline phosphatase and formed embryoid bodies when cultured in suspension. Pluripotency of ZES1 was confirmed when they were induced to differentiate in vitro at different seeding densities into several cell types from the three germ layers. ZES1 cells treated with all-trans-retinoic acid (RA) were also induced to differentiate into primarily neuronal cells. All the differentiated cell types obtained from high density differentiation as well as RA treatment were further identified through immunocytochemistry with cell lineage-specific protein markers. Immunocytochemistry using pluripotency-related protein markers showed that ZES1 cells were positively stained for SOX2 and POU5F1 that played critical roles during early embryogenesis, even though POU5F1 was also detected in differentiated cells. In conclusion, ZES1 cells possessed in vitro pluripotency characteristics which can be readily derived and maintained for longterm under feeder-free culture conditions. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/1161 | |
| dc.language.iso | en | en_US |
| dc.title | Establishment of a zebrafish embryonic stem cell line using a feeder-free culture method and in vitro pluripotency characterization | en_US |
| dc.type | Thesis | en_US |
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