Optimization Of Porcine Early Embryo And Embryonic Stem Cell Culture System

There have been many studies on porcine pluripotent stem cells(pPSCs)culture,however,there were limited pPSCs passaged stably and contributed to chimeras.Embryo qualities and culture systems are of great significance during embryonic stem cells(ESCs)establish.Core transcription factors and signaling pathways are play key roles in early embryonic development.The process of establishing ESCs is equivalent to combining chemical compounds and cytokines in vitro to maintain the proliferation and pluripotency of inner cell mass(ICM).Therefore,we added cytokines which promoted ICM formation during early embryonic development and ESCs culture respectively based on embryos sequencing data,and further tested their effects on embryos and ESCs pluripotency.The main works and results are as follows:1.We analysed the transcriptome data of pig in vivo embryos at different stages and enriched the signal pathways required to ICM regulation.The results showed that WNT signaling pathway,cAMP signaling pathway,PI3K-AKT signaling pathway,MAPK signaling pathway and JAK-STAT signaling pathway participated in the formation of ICM.Based on the above results,activators enriched in these signaling pathway were added during porcine parthenogenetic embryos culture,the results showed that,CHIR99021 and rpIL6 promoted blastocyst formation.2.1 ?M CHIR99021 and 10 ng/mL rpIL6 were added to detect their effects on parthenogenetic blastocyst qualities.The results showed that combination of 1 ?M CHIR99021 and 10 ng/mL rpIL6 increased blastocyst percentage(49.23±8.40% v.s.32.34±4.15%,P < 0.05),blastocyst hatching percentage(16.19±1.96% v.s.10.25±1.12%,P< 0.05),ICM cell number(7.72±2.30 v.s.4.28±1.60,P<0.01),expression of pluripotency genes,such as OCT4(P<0.01),SOX2(P<0.01)and NANOG(P<0.05),and genes in WNT signaling pathway and JAK-STAT signaling pathway.In addition,combination of 1 ?M CHIR99021 and 10 ng/mL rpIL6 increased ESC establishment efficiency(KOFL system: 34.70±4.77% vs 25.12±2.10%,P<0.05;LCDMIV system: 37.82±2.56% vs 25.26±3.18%,P<0.01).3.Verified the effects of 1 ?M CHIR99021 and 10 ng/mL rpIL6 by in vitro fertilized embryos development.The results showed that combination of 1 ?M CHIR99021 and 10 ng/mL rpIL6 promoted in vitro fertilized blastocyst formation(28.47±2.16% v.s.19.65±4.02%,P<0.05),ICM cell number(6.60±1.50 v.s.4.50±0.89,P<0.01),blastocyst hatching percentage(12.03±1.73% v.s.7.27±0.79%,P<0.05),and the expression of pluripotency genes OCT4(P<0.01),SOX2(P<0.05)and NANOG(P<0.01).4.Selected PSCs culture systems which could promote cell pluripotency based on KOFL system.The results showed that both LCDM system and LCDM supplied with rpIL6 system could support the pPSCs maintain,up-regulated the expressions of pluripotency genes OCT4?SOX2 and REX1;Supplied SB431542,CHIR99021 and Forskolin in KOFL system both could support pPSCs dissociated to single cells during passage,increased cell proliferation abilities and up-regulated the expressions of pluripotency genes OCT4?SOX2 and KLF4.5.Established cell lines in the selected mediums and further optimized the culture systems.The results showed that pPSCs-LCDMIV could be dissociated to single cells during passage,had higher expressions of pluripotency genes OCT4(P<0.05),SOX2(P<0.01)and NANOG(P<0.01)compared to pPSCs-KOFL.The cell proliferated more than 15 passages;pEPSCs were established with in vivo blastocysts from Bama pig based on pEPSC system.The pEPSCs could maintain stable passage for a long time,remain much higher pluripotent,and sustain the potential differentiate into three germ layers;Added SB431542,CHIR99021 and Forskolin in KOFL system,maintained high hLIF concentration and deleted bFGF defined a LSCFors system,or maintained high bFGF concentration and low hLIF concentration defined a F-LSCFors system.These two systems could support the pPSCs de novo derivation,and improve the expressions of pluripotency genes SOX2,NANOG and KLF4,but the expression of OCT4 was limited.The pluripotency of the pPSCs were improved partially.6.Enriched signaling pathways in pPSCS-LSCFors and pPSCS-F-LSCFors,the results showed that cAMP signaling pathway,PI3K-AKT signaling pathway,JAK-STAT signaling pathway and MAPK signaling pathway were up-regulated in the LSCFors system,and cAMP signaling pathway and PI3K-AKT signaling pathway,JAK-STAT signaling pathway and WNT signaling pathway were up-regulated in FLSCFors system,which participated in the regulation of pluripotency.The above results indicated that WNT signaling pathway,JAK-STAT signaling pathway,cAMP signaling pathway and PI3K-AKT signaling pathway were important in porcine early embryonic development and pPSCs pluripotency.The activation of WNT signaling pathway and JAK-STAT signaling pathway promoted blastocyst formation and blastocyst quality.Adding cytokines or chemical compounds to activate cAMP signaling pathway,PI3K-AKT signaling pathway,JAK-STAT signaling pathway and WNT signaling pathway during pESCs culture were of great significance to improve pESCs pluripotency and cell proliferation.This study optimized the porcine early embryo and embryonic stem cell culture systems,which conducived to obtaining more high-quality embryos and pluripotency improved embryonic stem cells.