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Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm.

Zschemisch NH, Eisenblätter R, Rudolph C, Glage S, Dorsch M - Springerplus (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Characterization of murine fibroblasts. Phase contrast images (20×) of a. rat embryonic fibroblasts (REF), b. mouse embryonic fibroblasts (MEF), c. female tumor rat fibroblasts (TRF-O3) and d. male tumor rat fibroblasts (TRF-T2) e. RT-PCR analysis of the expression patterns of fibroblast specific genes and important paracrine factors. GAPDH was used as endogenous control.
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Fig1: Characterization of murine fibroblasts. Phase contrast images (20×) of a. rat embryonic fibroblasts (REF), b. mouse embryonic fibroblasts (MEF), c. female tumor rat fibroblasts (TRF-O3) and d. male tumor rat fibroblasts (TRF-T2) e. RT-PCR analysis of the expression patterns of fibroblast specific genes and important paracrine factors. GAPDH was used as endogenous control.

Mentions: Growth factors, cytokines, and extracellular matrix components secreted from feeder cells contribute to the maintenance of ESC pluripotency in vitro. Therefore, we cultured and characterized REFs derived from the strain WKY/Ztm as well as MEFs from the NMRI strain. Furthermore, TRF cell lines were isolated from male and female teratomas caused by Dnd1 deficiency in WKY/Ztm rats. Morphological evaluation of the cells in vitro confirmed a polygonal shape with long filopodia typical for embryonic fibroblasts from rat (Figure 1a) and mouse (Figure 1b) while the female TRF-O3 (Figure 1c) and male TRF-T2 (Figure 1d) cells were more spindle-shaped and elongated forming a dense confluent cell layer. TRFs were maintained for more than 50 passages without morphological signs of senescence or reduced proliferation capacity in contrast to rapid loss of viability in MEFs. To differentiate between normal fibroblast and carcinoma-associated fibroblasts (CAF) the expression of fibroblast specific genes, and of Acta2 as marker for myofibroblast were performed (Egeblad et al.2005; Kalluri and Zeisberg2006). RT-PCR analysis revealed high expression of Col1a2 in REFs, TRF-O3, and TRF-T2 cells but showed only sparse Col1a2 mRNAs in the murine cell lines. Reciprocally, vimentin showed much higher transcription rates in MEFs and NIH/3 T3 cells derived from mouse embryos in contrast to the weak activity in rat fibroblasts (Figure 1e). As expected, the key enzyme of the collagen synthesis process P4ha2 was detected at the same transcription level in all fibroblast cell lines tested, while slight expression of the S100A4 gene (also known as fibroblast specific protein 1 in mouse and human) was found in MEFs and TRFs. Only REFs and NIH/3 T3 cells showed higher transcription rates of S100A4. Acta2 was expressed equally in all cell lines investigated. The paracrine factors LIF and SCF were highly transcribed in MEFs and NIH/3 T3 cells but not in the rat cells while inversely the transcription of 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.Figure 1


Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm.

Zschemisch NH, Eisenblätter R, Rudolph C, Glage S, Dorsch M - Springerplus (2014)

Characterization of murine fibroblasts. Phase contrast images (20×) of a. rat embryonic fibroblasts (REF), b. mouse embryonic fibroblasts (MEF), c. female tumor rat fibroblasts (TRF-O3) and d. male tumor rat fibroblasts (TRF-T2) e. RT-PCR analysis of the expression patterns of fibroblast specific genes and important paracrine factors. GAPDH was used as endogenous control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197200&req=5

Fig1: Characterization of murine fibroblasts. Phase contrast images (20×) of a. rat embryonic fibroblasts (REF), b. mouse embryonic fibroblasts (MEF), c. female tumor rat fibroblasts (TRF-O3) and d. male tumor rat fibroblasts (TRF-T2) e. RT-PCR analysis of the expression patterns of fibroblast specific genes and important paracrine factors. GAPDH was used as endogenous control.
Mentions: Growth factors, cytokines, and extracellular matrix components secreted from feeder cells contribute to the maintenance of ESC pluripotency in vitro. Therefore, we cultured and characterized REFs derived from the strain WKY/Ztm as well as MEFs from the NMRI strain. Furthermore, TRF cell lines were isolated from male and female teratomas caused by Dnd1 deficiency in WKY/Ztm rats. Morphological evaluation of the cells in vitro confirmed a polygonal shape with long filopodia typical for embryonic fibroblasts from rat (Figure 1a) and mouse (Figure 1b) while the female TRF-O3 (Figure 1c) and male TRF-T2 (Figure 1d) cells were more spindle-shaped and elongated forming a dense confluent cell layer. TRFs were maintained for more than 50 passages without morphological signs of senescence or reduced proliferation capacity in contrast to rapid loss of viability in MEFs. To differentiate between normal fibroblast and carcinoma-associated fibroblasts (CAF) the expression of fibroblast specific genes, and of Acta2 as marker for myofibroblast were performed (Egeblad et al.2005; Kalluri and Zeisberg2006). RT-PCR analysis revealed high expression of Col1a2 in REFs, TRF-O3, and TRF-T2 cells but showed only sparse Col1a2 mRNAs in the murine cell lines. Reciprocally, vimentin showed much higher transcription rates in MEFs and NIH/3 T3 cells derived from mouse embryos in contrast to the weak activity in rat fibroblasts (Figure 1e). As expected, the key enzyme of the collagen synthesis process P4ha2 was detected at the same transcription level in all fibroblast cell lines tested, while slight expression of the S100A4 gene (also known as fibroblast specific protein 1 in mouse and human) was found in MEFs and TRFs. Only REFs and NIH/3 T3 cells showed higher transcription rates of S100A4. Acta2 was expressed equally in all cell lines investigated. The paracrine factors LIF and SCF were highly transcribed in MEFs and NIH/3 T3 cells but not in the rat cells while inversely the transcription of 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.Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl-Neuberg-Str.1, 30625 Hannover, Germany.

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.

No MeSH data available.


Related in: MedlinePlus