The Establishment of porcine embryonic stem (pES)-like cells : effects of embryonic sources and culture conditions

Abstract

EXP. I aims to examine the effects of parthenogenetic activation (PA) protocols, sperm:oocyte ratio at fertilization in vitro(IVF), and MEK and GSK-3 inhibitors (i) on the developmental competence and quality of embryo. Groups of MII oocytes were stimulated with three different PA protocols as: I) 1.36 kV/cm, 30 msec, 2 pulses; II) 1.50 kV/cm, 60 msec, 2 pulses; and III) 1.0 kV/cm, 80 msec, 3 pulses. Blastocyst development was significantly greater in protocol III-activated oocytes than in protocol I- and II-activated oocytes (p<0.05), however, no significant difference in the mean number of blastocyst cells among the groups. Groups of denuded mature oocytes were fertilized with three different sperm:oocyte ratios as 1000:1, 2000:1, and 4000:1 for 6 h. Sperm penetration rate significantly increased when the oocytes were fertilized with 2000 and 4000 sperm:oocyte (p<0.05), by contrast, insemination with 1000 sperm:oocyte gave a significant higher rate of monospermic zygotes than 2,000 and 4,000 sperm:oocyte (p<0.05). Blastocyst development increased significantly in the group fertilized with 1,000 sperm:oocyte compared to those of 4,000 sperm:oocyte (p<0.05). In vitro fertilized and activated oocytes were cultured in IVC medium supplemented with 0.5µM MEKi and/or 3.0µM GSK-3i for 6 days. Cleavage and blastocyst rates increased significantly in IVF embryos treated with MEKi alone (p<0.05), however, both IVF- and PA-derived blastocysts cultured in MEKi had a significant higher cells number of ICMs and ICM:TE ratio than other groups (p<0.05). Nanog expression significantly increased in ICM cells of blastocysts cultured in MEKi rather than other groups (p<0.05). In conclusion, embryo development was optimal when three 80-msec consecutive pulses of 1.0 kV/cm and the ratio of sperm per oocyte at 1000:1 were used for PA and IVF, respectively, and suppression of ERK signaling using MEKi improves the development and quality of porcine embryos, and can promote the pluripotency in the ICM cells. EXP. II aims to compare the quality of blastocysts produced from IVF, PA (by using the optimal protocol resulting from our previous study; Chapter II), and in vivo fertilization (IVV) on establishment of pES-like cells. Zona pellucida (ZP)-free blastocysts derived from IVF-, PA-, and IVV were cultured on STO feeder. The rate of blastocysts attachment was significantly greater in IVV embryos than those derived from in vitro (p<0.01). The primary colony formation was also significantly greater (p<0.01) in IVV than IVF blastocysts, on the contrary, none of the primary colony was formed in cultured PA blastocysts. After continuous culture, the greater number of putative ESC lines were significantly developed from IVV than IVF blastocysts (p<0.01). These colonies showed all characteristics of typically ES cells including positive AP activity and expressions of pluripotent markers and genes. In conclusion, embryos produced from IVF technique could be served as alternative source for porcine ES-like cell establishment as in vivo counterpart. EXP. III aims to examine the effect of FBS and KSR supplementations on the derivation of pES-like cells. In vivo hatched or ZP-free blastocysts were cultured in ES medium supplemented with different (volume/volume) ratios of FBS:KSR (20:0, 10:10, and 0:20). The attachment rate was significant higher (100%) in blastocysts cultured in media supplemented with 20% FBS and 10% FBS+10% KSR than those cultured in KSR alone (64.4%). Blastocysts cultured in a mixture of FBS and KSR showed a higher tendency rate of ICM outgrowths than other groups. The formation of primary ES-like colonies was significantly greater in blastocysts growing in 10% FBS+ 10% KSR than those cultured in 20% FBS and 20% KSR (64.4, 17.8 and 33.3%, respectively). Three (5.88%) and six (40%) putative ES cell lines were observed from groups cultured in 20% KSR and 10% FBS+ 10% KSR, respectively. These colonies showed all the characteristics of typically ES cells. In conclusion, the culture condition supplemented with equal ratio at 10% of FBS and KSR is more suitable for the isolation and culture of pES-like cells than that supplemented with FBS or KSR alone. EXP. IV aims to evaluate the efficiency of feeder cells on the isolation and culture of undifferentiated pES-like cells. In vivo hatched or ZP-free blastocysts were cultured on different feeders as PESF, PTSF, HFK, MEF, and STO. Attached blastocysts cultured on HFK and STO feeder layers showed a higher percentage of ICM outgrowths than those cultured on PESF (76.7, 72.9 and 38.9%, respectively; p<0.05) The rates of primary ES-like colony formation (30.6 vs 76.7%) and the number of putative ES cell lines (1 vs 7) were significantly decreased when ICM outgrowths were cultured on PESF compared to those cultured on HFK, respectively; p<0.05). Only colonies from one (25%) and three (50%) cell lines derived respectively on PTSF and STO feeder layers could be maintained in undifferentiated stage by presenting all the characteristics of typically ES cells. In conclusion, feeder type plays an important role in establishing ES cells, while PTSF and STO cells were the best in maintaining porcine ES-like cells in an undifferentiated stage.