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 and12.8% 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.
Highlights
Embryonic stem (ES) cells, a pluripotent cell population with the capacity of self-renewal and differentiation into all body cell types and lineages, have great potential for use in regenerative medicine, research, and production of transgenic animals for xenotransplantation, e.g. the α-gal knockout pig [1,2,3]
We previously reported that cloned porcine embryos treated with a histone deacetylation inhibitor (TSA) had enhanced histone acetylation and superior development compared to control embryos [14]
Cumulus-oocyte complexes (COCs), aspirated from abattoir-derived ovarian follicles, were in vitro matured (IVM) in a 100-μL droplet of maturation medium (TCM 199 supplemented with 10% porcine follicular fluid and 10% fetal bovine serum (FBS)) containing gonadotropins (10 IU/mL hCG and 10 IU/mL PMSG) at 39°C under 5% CO2
Summary
Embryonic stem (ES) cells, a pluripotent cell population with the capacity of self-renewal and differentiation into all body cell types and lineages, have great potential for use in regenerative medicine, research, and production of transgenic animals for xenotransplantation, e.g. the α-gal knockout pig [1,2,3]. Limited success of establishing porcine ntES cell lines is mainly attributed to the low efficiency of SCNT due to poor embryonic development, presumably as a result of incomplete cellular reprogramming and inadequate support from the in vitro culture system [10]. That the developmental potential of in vitro-derived blastocysts was lower than that of in vivo blastocysts [11,12], these cloned blastocysts had less total cell numbers and low ratio of inner cell mass (ICM) to trophectoderm (TE) cells than their in vivo counterparts [13]. It is well known that reconstructed porcine embryos treated with TSA have an altered acetylation status of histone proteins, leading to enhanced reprogramming of the somatic genome and improved cloning efficiency [15,16]
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