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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

Expression patterns of ESC compared to TRF-O3 feeder cells in 2i and YPAC medium. Transcription levels of pluripotency and differentiation markers were compared in ES21 from passage 7 and passage 15 cultured of TRF-O3 as feeder cells in 2i-LIF and YPAC medium performing RT-PCR. + positive control: pooled cDNAs from pluripotent rat embryonic germ cell lines (Northrup et al.2011). GAPDH was used as endogenous control.
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Fig4: Expression patterns of ESC compared to TRF-O3 feeder cells in 2i and YPAC medium. Transcription levels of pluripotency and differentiation markers were compared in ES21 from passage 7 and passage 15 cultured of TRF-O3 as feeder cells in 2i-LIF and YPAC medium performing RT-PCR. + positive control: pooled cDNAs from pluripotent rat embryonic germ cell lines (Northrup et al.2011). GAPDH was used as endogenous control.

Mentions: Only the microinjection in blastocysts of the female cell lines ES1 and ES10 and male line ES21 resulted in chimeric rats, while ES9 cells did not integrate into the embryos. Two highly chimeric females (out of 3 male and 2 female chimeric offspring) (Figure 3a) that originated from ES21 showed germ line transmission (Figure 3b). Therefore, the ESC line ES21 was analyzed in more detail. Chromosome counting revealed a diploid karyotype of ES21 in more than 80% of the metaphases (Figure 3c). Subcutaneous injection of ES21 cells into immunodeficient mice led to the formation of teratomas containing tissues derived from all three germ layers demonstrating pluripotency (Figure 3d). The expression pattern of ES21 was characterized through the transcription of the pluripotency markers c-kit, Klf4, Nanog, Rex1, Oct4 and Sox2 as well as the proto-oncogene c-myc (Figure 4). In contrast to the ESCs the underlying feeder cells showed weak expressions of Rex1, Oct4 and Sox2. Only the Klf4 activity was on the same level in the tumor rat fibroblasts and in the ES cells (Figure 4). Moreover, RT-PCR amplification revealed no expression of the entodermal marker AFP, a slight transcription of nestin as an early ectoderm marker, and an elevated expression of T-Brachyury as a marker for the early mesodermal cell lineage in ES21 cells (Figure 4).Figure 3


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)

Expression patterns of ESC compared to TRF-O3 feeder cells in 2i and YPAC medium. Transcription levels of pluripotency and differentiation markers were compared in ES21 from passage 7 and passage 15 cultured of TRF-O3 as feeder cells in 2i-LIF and YPAC medium performing RT-PCR. + positive control: pooled cDNAs from pluripotent rat embryonic germ cell lines (Northrup et al.2011). GAPDH was used as endogenous control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Expression patterns of ESC compared to TRF-O3 feeder cells in 2i and YPAC medium. Transcription levels of pluripotency and differentiation markers were compared in ES21 from passage 7 and passage 15 cultured of TRF-O3 as feeder cells in 2i-LIF and YPAC medium performing RT-PCR. + positive control: pooled cDNAs from pluripotent rat embryonic germ cell lines (Northrup et al.2011). GAPDH was used as endogenous control.
Mentions: Only the microinjection in blastocysts of the female cell lines ES1 and ES10 and male line ES21 resulted in chimeric rats, while ES9 cells did not integrate into the embryos. Two highly chimeric females (out of 3 male and 2 female chimeric offspring) (Figure 3a) that originated from ES21 showed germ line transmission (Figure 3b). Therefore, the ESC line ES21 was analyzed in more detail. Chromosome counting revealed a diploid karyotype of ES21 in more than 80% of the metaphases (Figure 3c). Subcutaneous injection of ES21 cells into immunodeficient mice led to the formation of teratomas containing tissues derived from all three germ layers demonstrating pluripotency (Figure 3d). The expression pattern of ES21 was characterized through the transcription of the pluripotency markers c-kit, Klf4, Nanog, Rex1, Oct4 and Sox2 as well as the proto-oncogene c-myc (Figure 4). In contrast to the ESCs the underlying feeder cells showed weak expressions of Rex1, Oct4 and Sox2. Only the Klf4 activity was on the same level in the tumor rat fibroblasts and in the ES cells (Figure 4). Moreover, RT-PCR amplification revealed no expression of the entodermal marker AFP, a slight transcription of nestin as an early ectoderm marker, and an elevated expression of T-Brachyury as a marker for the early mesodermal cell lineage in ES21 cells (Figure 4).Figure 3

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