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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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Morphologies of aggregated cloned and PA blastocyst embryos.(a) Day-7 cloned porcine blastocysts produced by OBCT and embryo aggregation. 1×: single cloned embryo; 2×: two cloned embryos were aggregated in a well, and 3×: three cloned embryos were aggregated in a well. (b) Day-7 3× aggregated porcine parthenogenetic embryos. Scale bar = 100 μm.
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pone.0118165.g001: Morphologies of aggregated cloned and PA blastocyst embryos.(a) Day-7 cloned porcine blastocysts produced by OBCT and embryo aggregation. 1×: single cloned embryo; 2×: two cloned embryos were aggregated in a well, and 3×: three cloned embryos were aggregated in a well. (b) Day-7 3× aggregated porcine parthenogenetic embryos. Scale bar = 100 μm.

Mentions: Day-7 aggregated embryos derived by OBCT and PA processes (Fig. 1A-B) were tested for their ability to form outgrowths and primary ES cell colonies. Attached embryos in 2× and 3× aggregated groups (62.8 and 76.2%, respectively) performed better (P < 0.05) than those of 1× and 3×PA group (41.6 and 44.3%, respectively; Table 2). Outgrowths and primary colonies were detected 5–8 days after initial seeding. Similarly, more outgrowths were formed in 2× and 3× aggregated (42.6 and 55.2%, respectively) groups than those in 1× and 3×PA groups (23.4 and 25.3%; P < 0.05). In addition, 12.8 to 26.2% of primary colonies were observed in 2× and 3× groups, higher than those from 1× group (3.9%), but similar to that of 3×PA group (13.9%). The putative porcine ES cells had typical ES cell morphology, with compact colonies and distinct borders (Fig. 2B-C). However, most of the colonies only maintained typical ES morphology for one or two passages and then either differentiated or degenerated. However, five ES cell lines derived from the 3× group maintained proliferation without differentiation beyond three passages (Table 2).


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)

Morphologies of aggregated cloned and PA blastocyst embryos.(a) Day-7 cloned porcine blastocysts produced by OBCT and embryo aggregation. 1×: single cloned embryo; 2×: two cloned embryos were aggregated in a well, and 3×: three cloned embryos were aggregated in a well. (b) Day-7 3× aggregated porcine parthenogenetic embryos. Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118165.g001: Morphologies of aggregated cloned and PA blastocyst embryos.(a) Day-7 cloned porcine blastocysts produced by OBCT and embryo aggregation. 1×: single cloned embryo; 2×: two cloned embryos were aggregated in a well, and 3×: three cloned embryos were aggregated in a well. (b) Day-7 3× aggregated porcine parthenogenetic embryos. Scale bar = 100 μm.
Mentions: Day-7 aggregated embryos derived by OBCT and PA processes (Fig. 1A-B) were tested for their ability to form outgrowths and primary ES cell colonies. Attached embryos in 2× and 3× aggregated groups (62.8 and 76.2%, respectively) performed better (P < 0.05) than those of 1× and 3×PA group (41.6 and 44.3%, respectively; Table 2). Outgrowths and primary colonies were detected 5–8 days after initial seeding. Similarly, more outgrowths were formed in 2× and 3× aggregated (42.6 and 55.2%, respectively) groups than those in 1× and 3×PA groups (23.4 and 25.3%; P < 0.05). In addition, 12.8 to 26.2% of primary colonies were observed in 2× and 3× groups, higher than those from 1× group (3.9%), but similar to that of 3×PA group (13.9%). The putative porcine ES cells had typical ES cell morphology, with compact colonies and distinct borders (Fig. 2B-C). However, most of the colonies only maintained typical ES morphology for one or two passages and then either differentiated or degenerated. However, five ES cell lines derived from the 3× group maintained proliferation without differentiation beyond three passages (Table 2).

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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