Abstract
Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-588) contains supplementary material, which is available to authorized users.
Highlights
Rat embryonic stem cells (ESC) lines are a tool for gene targeting (Tong et al 2011; Yamamoto et al 2011), the generation of transgenic animals (Kawamata and Ochiya 2010, Kawamata and Ochiya 2011), and for in vitro differentiation approaches (Wang et al 2012; Peng et al 2013)
The paracrine factors leukemia inhibitory factor (LIF) and stem cell factor (SCF) were highly transcribed in Mouse embryonic fibroblasts (MEF) and NIH/3 T3 cells but not in the rat cells while inversely the transcription of bone morphogenetic protein 4 (BMP4) was significantly higher in rat fibroblasts than in MEFs or NIH/3 T3 cells (Figure 1e)
These fibroblast cell lines were used in comparison to epithelial cell lines derived from female genital tract of mouse, rat and human as well as chorion carcinoma cells to identify the optimal feeder cell type that provides culture conditions supporting the pluripotency of ESCs in vivo
Summary
Rat ESC lines are a tool for gene targeting (Tong et al 2011; Yamamoto et al 2011), the generation of transgenic animals (Kawamata and Ochiya 2010, Kawamata and Ochiya 2011), and for in vitro differentiation approaches (Wang et al 2012; Peng et al 2013). In 2008 the first pluripotent rat ESC lines derived from blastocysts of inbred Dark Agouti (DA) and outbreed Spargue-Dawley (SD) rats using 2i-LIF medium (Buehr et al 2008; Li et al 2008) were established. In this study we showed that the immortalized tumor rat fibroblast cell line TRF-O3 as innovative feeder cells supported the culture of rat pluripotent and germ-line transmissible ESCs. Usage of TRF-O3 feeder cells was a time saving, cost-effective approach to minimize animal usage by avoiding the repeated preparation of fresh embryonic fibroblasts
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