Optimization Of In Vitro Culture System Of Porcine Early Embryo And Its Influence On The Establishment Of Embryonic Stem Cell Lines

The culture environment during in vitro culture was the basic condition to ensure the normal development of porcine embryos,and the composition of related medium was the key factor to determine the quality of in vitro embryos.Currently commonly used,such as Whitten’s medium,North Carolina State University(NCSU)-23 medium,modified Chatot Ziomek Bavister(CZB)medium,Beltsville embryo medium(BECM)-3 and porcine zygote medium 3(PZM-3)all supported the limited development of porcine embryos.The chemically defined medium generally lacks proteins,growth factors and other natural components,and the in vitro embryos cultured from the culture medium may eventually affect the establishment of embryonic stem cell lines(ESCs)due to problems in development quality and germ layer differentiation.Based on single-cell transcriptome sequencing analysis of porcine preimplantation embryos,the researchers explored the molecular basis of lineage differentiation and pluripotency changes during pig embryonic development,and identified pluripotency-related signaling pathways at different stages of embryonic development.On the basis of this,the combination of small molecule inhibitors was screened to achieve the establishment of porcine ESCs and the construction of a culture system,but porcine Na(?)ve state ESCs with germline chimeric ability have not been obtained.Previous studies focused on the exploration of the culture system of ESCs derived from embryos,but often ignored the importance of embryo culture system.The initial quality of blastocysts is crucial for the establishment of ESCs from inner cell mass(ICM)and hatched blastocysts.Due to the uneven quality of embryos,researchers often need to screen embryos with good development status for a large number of inoculation in order to improve the efficiency of line construction.However,due to the limitation of cost and culture conditions,the number of embryos in vivo and the quality of embryos in vitro are limited,which results in the low efficiency of line construction of porcine embryo-derived pluripotent stem cells for a long time.Based on the above problems,this study tries to optimize the embryo quality at the embryo culture level,and optimize the embryo into high-quality embryos suitable for the derivation of ESCs by changing the culture system.In the related research on the in vitro culture system of pig embryos,researchers generally choose to add nutrients or growth factors to the commonly used culture systems to simulate the maternal in vivo physiological environment.A culture system that properly subtracts,replaces or adds certain components in a chemically defined medium could also further improve the quality of in vitro embryos,increase blastocyst rate,implantation rate and overcome the effect of developmental retardation,making in vitro embryos more closed to the natural state in vivo.However,there are many factors that affect embryonic cell proliferation,apoptosis,oxidative metabolism and other developmental processes.Adding individual chemical factors or nutrients alone can only improve individual regulatory pathways or their targets,but has limited impact on embryonic development and cannot improve overall embryo quality.Therefore,screening suitable embryo culture systems in vitro has an important impact on improving embryo quality and improving the efficiency of ESCs establishment.Stem cell culture medium usually contains nutrient substrates,antioxidants,growth factors and cytokines required for embryonic development.The above components can provide necessary nutrients during embryonic development,and can also promote cell proliferation and maintain the self-renewal capacity of cells.In addition,different combinations of small molecules are often added to the stem cell culture medium due to the cell requirements of the derived target state,and these small molecules are often key regulators to maintain the stem cell state.The use of stem cell culture medium or some of its components in modified embryo culture medium can promote the improvement of embryo quality.This study drew on the screening idea of ESCs culture system,combined with the transcriptomic sequencing data of early embryos and the research results of existing stem cell culture system,and analyzed the effects of different types of stem cell culture medium and each component in the culture medium on in vitro embryo development by using simple and efficient porcine parthenogenetic embryos as the research object.After identifying the key components in the stem cell culture medium that are beneficial to embryonic development,the above-mentioned key components in the stem cell culture medium are combined with the regulators of the signaling pathways related to cell proliferation and ICM development in the porcine early blastocyst to screen out suitable ones.Combination of small molecules and culture medium components to construct a new optimized system for in vitro culture of pig early embryos.Ultimately,a significant improvement in embryo quality(cell number,ICM ratio,and hatching rate)during in vitro culture of pig parthenogenetic embryos is achieved,and the embryonic cells are maintained in a state conducive to the derivation of stem cell lines.The establishment efficiency of pig parthenogenetic embryonic stem cells was significantly improved,and a solid foundation was laid for the establishment of Na(?)ve state embryonic stem cells.The main results were as follows:(1)KOFL-M,LCDM-M,and EPSC-M could promote the development rate of in vitro D5.5-D7.5 parthenogenetic embryos,increase the blastocyst diameter and hatching rate,and reduce the apoptosis of embryonic cells.The above three stem cell culture medium could promote the total cell numbers of in vitro D7.5 parthenogenetic embryos,among which EPSCM and EPSC-M(-)could promote the proliferation of ICM and increase the proportion of ICM in the total cell numbers,while KOFL-M and LCDM-M could promote the proliferation of ICM and trophectoderm(TE)cells together.The basic medium of EPSC embryonic stem cells,EPSC-M(-),still showed a positive effect on the development of parthenogenetic embryos after the removal of complex small molecular components,but its maintenance effect on ICM cells was weakened.Different small molecules have different effects on embryonic development,and there is the redundant and unreasonable collocation of small molecules in the existing stem cell culture medium.The composition of different stem cell culture medium was different,and the mechanism affecting embryo development was different.(2)The effects of various components in stem cell culture medium on embryonic development showed that fetal bovine serum(FBS)or serum substitute N2B27 and antioxidant components(Vc and β-ME)in stem cell culture medium could improve the development ability of parthenogenetic embryos.The basic media DMEM/F12 supported the growth of parthenogenetic blastocysts.The specific concentration of small molecules in each stem cell culture medium can promote the development of parthenogenetic blastocyst,and due to different small molecule regulatory pathways,the mechanism of affecting the development of parthenogenetic embryos were also different,and the synergistic effect among small molecules could improve the quality of parthenogenetic embryos in multiple dimensions.(3)The newly constructed five-factor optimized system DNB(+3i/LA),which was composed of five small molecules XAV939,CHIR99021,Activin A,PD0325901 and human LIF(h LIF)commonly used in porcine stem cell culture medium,could promote the development of parthenogenetic embryos and increase the number of ICM cells.In addition,the seven-factor optimized DNB(+3i/LA6V)system supplemented with IL6 and VEGF based on DNB(+3i/LA)system could further improve the development ability of parthenogenetic embryos,increase the number of ICM cells and reduce cell apoptosis.XAV939 and CHIR99021 caused β-CATENIN to accumulate in the cytoplasm and inhibited the WNT signaling pathway controlling cell differentiation.IL6 and h LIF jointly activated the downstream JAK-STAT3 signaling pathway to maintain cell pluripotency.PD0325901 and VEGF promoted cell proliferation by regulating MAPK or PI3 K.High concentration of Activin A inhibited TGFβsignaling pathway and prevented TE cells proliferation.(4)DNB(+3i/LA6V)optimization system could improve the efficiency of establishing pg ESCs in different stem cell culture systems.The expression of pluripotency marker genes in the obtained pg ESCs lines were up-regulated,and the expression of Na?ve state-related genes were significantly up-regulated,while the expression of some Primed state-related genes was down-regulated..The pluripotency of pg ESCs remained stable during long-term passage in vitro.In this paper,we analyzed the effect of porcine stem cell culture mediums on embryo development in vitro,and elucidated the mechanism of effect of various components in porcine stem cell culture medium on embryo development.A general optimized system for in vitro embryo culture,DNB(+3i/LA6V),was screened out,which can promote the establishment of different types of pg ESCs,laying a foundation for improving the quality of various types of embryos in vitro and promoting the establishment of porcine Na?ve state ESCs lines.