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Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

Siriboon C, Lin YH, Kere M, Chen CD, Chen LR, Chen CH, Tu CF, Lo NW, Ju JC - PLoS ONE (2015)

Bottom Line: We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines.Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium.In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC; Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.

ABSTRACT
We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

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Immunofluorescence staining of pluripotency markers in ntES cell colonies derived from aggregated cloned embryos.Expressions of pluripotency markers (Oct4, Nanog and Sox2) are shown (red) in PES3 at passage 15 (left panel) and PES1 at passage 10 (right panel). Nuclei are stained with DAPI (blue). Scale bars = 100 μm.
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pone.0118165.g004: Immunofluorescence staining of pluripotency markers in ntES cell colonies derived from aggregated cloned embryos.Expressions of pluripotency markers (Oct4, Nanog and Sox2) are shown (red) in PES3 at passage 15 (left panel) and PES1 at passage 10 (right panel). Nuclei are stained with DAPI (blue). Scale bars = 100 μm.

Mentions: The ES-like cell colonies also had typical ES cell morphology including densely packed, small and round nuclei with well-defined boundaries and expression of AP activity (Fig. 3). Based on immunofluorescence staining and RT-PCR analysis, our ES-like cell colonies consisted entirely of those cells expressing Oct4, Nanog, Sox2, and Rex01 (Figs. 4 and 5).


Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

Siriboon C, Lin YH, Kere M, Chen CD, Chen LR, Chen CH, Tu CF, Lo NW, Ju JC - PLoS ONE (2015)

Immunofluorescence staining of pluripotency markers in ntES cell colonies derived from aggregated cloned embryos.Expressions of pluripotency markers (Oct4, Nanog and Sox2) are shown (red) in PES3 at passage 15 (left panel) and PES1 at passage 10 (right panel). Nuclei are stained with DAPI (blue). Scale bars = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4334543&req=5

pone.0118165.g004: Immunofluorescence staining of pluripotency markers in ntES cell colonies derived from aggregated cloned embryos.Expressions of pluripotency markers (Oct4, Nanog and Sox2) are shown (red) in PES3 at passage 15 (left panel) and PES1 at passage 10 (right panel). Nuclei are stained with DAPI (blue). Scale bars = 100 μm.
Mentions: The ES-like cell colonies also had typical ES cell morphology including densely packed, small and round nuclei with well-defined boundaries and expression of AP activity (Fig. 3). Based on immunofluorescence staining and RT-PCR analysis, our ES-like cell colonies consisted entirely of those cells expressing Oct4, Nanog, Sox2, and Rex01 (Figs. 4 and 5).

Bottom Line: We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines.Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium.In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC; Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC.

ABSTRACT
We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Show MeSH