Establishment of a zebrafish embryonic stem cell line using a feeder-free culture method and in vitro pluripotency characterization
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Date
2014
Authors
Sing Yee, Ho
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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.