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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

Gender determination of pluripotency rat ES cells. a. SRY-PCR demonstrated that 8 male ESC lines out of 43 were established in two separate experiments. The rat ESC line ES21 forms undifferentiated pluripotent colonies demonstrated by b. phase contrast, c. alkaline phosphates expression, and positive stainings for d. Nanog, e. Oct4, f. SSEA-1 and g. SSEA-3 (10×); scale bar is 100 μm.
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Fig2: Gender determination of pluripotency rat ES cells. a. SRY-PCR demonstrated that 8 male ESC lines out of 43 were established in two separate experiments. The rat ESC line ES21 forms undifferentiated pluripotent colonies demonstrated by b. phase contrast, c. alkaline phosphates expression, and positive stainings for d. Nanog, e. Oct4, f. SSEA-1 and g. SSEA-3 (10×); scale bar is 100 μm.

Mentions: ICMs from 4.5 dpc blastocysts were isolated through immunosurgery and seeded on mitotic-inactivated TRF-O3 cells as feeders in 2i-LIF medium on 96 well plates in two different experiments. In the first round 23 ICMs out of 46 showed clonal growth representing a 50% plating efficiency. 6 of these established ESC lines were identified as male (26.1%). A plating efficiency of 71% was achieved in the second round with 20 ESC lines derived from 28 ICMs containing 2 male ESC lines (10.0%) (Figure 2a). The ESC clones appeared to be round-shaped colonies with smooth surface and sharp edge (Figure 2b) consisting of alkaline phosphatase positive cells (Figure 2c). Immunocytofluorescent staining revealed the expression of the pluripotency markers SSEA-1, SSEA-3 (Northrup et al.2011; Fernandez et al.2011), Nanog and Oct4 (Figure 2d-2g) as well as the primordial germ cells - specific gene DDX4/MVH (data not shown) in the ESC lines.Figure 2


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)

Gender determination of pluripotency rat ES cells. a. SRY-PCR demonstrated that 8 male ESC lines out of 43 were established in two separate experiments. The rat ESC line ES21 forms undifferentiated pluripotent colonies demonstrated by b. phase contrast, c. alkaline phosphates expression, and positive stainings for d. Nanog, e. Oct4, f. SSEA-1 and g. SSEA-3 (10×); scale bar is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Gender determination of pluripotency rat ES cells. a. SRY-PCR demonstrated that 8 male ESC lines out of 43 were established in two separate experiments. The rat ESC line ES21 forms undifferentiated pluripotent colonies demonstrated by b. phase contrast, c. alkaline phosphates expression, and positive stainings for d. Nanog, e. Oct4, f. SSEA-1 and g. SSEA-3 (10×); scale bar is 100 μm.
Mentions: ICMs from 4.5 dpc blastocysts were isolated through immunosurgery and seeded on mitotic-inactivated TRF-O3 cells as feeders in 2i-LIF medium on 96 well plates in two different experiments. In the first round 23 ICMs out of 46 showed clonal growth representing a 50% plating efficiency. 6 of these established ESC lines were identified as male (26.1%). A plating efficiency of 71% was achieved in the second round with 20 ESC lines derived from 28 ICMs containing 2 male ESC lines (10.0%) (Figure 2a). The ESC clones appeared to be round-shaped colonies with smooth surface and sharp edge (Figure 2b) consisting of alkaline phosphatase positive cells (Figure 2c). Immunocytofluorescent staining revealed the expression of the pluripotency markers SSEA-1, SSEA-3 (Northrup et al.2011; Fernandez et al.2011), Nanog and Oct4 (Figure 2d-2g) as well as the primordial germ cells - specific gene DDX4/MVH (data not shown) in the ESC lines.Figure 2

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