Establishment And Biological Characteristics Research Of Noval Embryonic Stem Cell Derived From Different Mouse Development Stage Embryos

Mouse embryonic stem cells(ESCs)were originally derived from the inner cell mass of mouse blastocysts.The advanced stem cells(ASCs)derived from the ESCs were cultured in "Activin A + BMP4 + CHIR99021 + LIF"(ABC/L)medium,had high methylation,was with hypermethylation,and contributed to full-term chimeras,as well as generated mice by tetraploid complementation.In here,we first time used the ABC/L medium to establish embryonic stem cell lines derived from different development embryonic stages,including 2-cell,4-cell,8-cell,morula,blastocyst,and E 5.5 gastrula.Furthermore,we tested the pluripotency of the ABC/L-ESCs.Finally,we observed the damage resistance of the ABC/L-ESCs which derived from blastocyst by paraformaldehyde damage.The experimental results were showed in follows:1.Established ABC/L-ESC lines from different developmental stages embryos in ABC/L mediumWe established ABC/L-ESC lines from different developmental stages embryos in ABC/L medium,including 2-cell,4-cell,8-cell,morula,blastocyst,E 5.5,and the establishment rates of the ABC/L-ESCs were 12.5%,25%,10.9%,28.6%,10% and6.67%,respectivily.The results of AP staining showed that all ABC/L-ESCs groups were positive.Compared to control group(2i/L-ESCs),ABC/L-ESCs lines derived from 2-cell,4-cell and 8-cell had higher growth rate and single cell cloning efficiency.2.Biological characteristics of ABC/L-ESCsKaryotype analysis showed that the proportion of ABC/L-ESCs derived from 2-cell with 40 chromosomes was 77.22%.The expression of the pluripotency genes Oct4 and Sox2 in all ABC/L-ESCs was significantly higher than that of the control 2i/LESCs in RNA expression level.Among them,the mesoderm genes T expression levels of different ABC/L-ESCs lines had the following characteristics: up-down-up-down in different ABC/L-ESCs lines derived from 2-cell,4-cell,8-cell and morula,respectivital.The results indicated the genes T may play different roles in different embryonic stages.Also,some ABC/L-ESCs lines(derived from 2-cell,8-cell and morula)expressed highly DNA methylation-related genes,such as Dnmt3 a,Dnmt3b,Dnmt3 L.Immunofluorescence showed that pluripotency proteins OCT4 and SOX2 were also expressed in all ABC/L-ESCs lines,and some ABC/L-ESCs lines expressed mesodermal marker protein T,which was consistent with the results of RT-q PCR.In vitro differentiation experiments,ABC/L-ESCs derived from 2-cell stage had revealed the ability to differentiate into three germ layers,and can be chimerized into mouse fetus and yolk sac.3.The anti-damage ability of ABC/L cell linesThe same cells number of ABC/L-ESCs and 2i/L-ESCs derived from blastocyst stage were placed in different concentrations of paraformaldehyde to test the damage of cells.We found the number surviving of ABC/L-ESCs was significantly more than that of 2i/L-ESCs after 4th days.After damage at the same concentration and at the same time,Western Blotting proved that ABC/L-ESCs expressed low levels of pH2AX(DNA damage marker protein)compared with 2i/L-ESCs.The recovery time and the response time to damage in ABC/L-ESCs are completely different compared with 2i/L-ESCs.The experimental results showed that of ABC/L-ESCs is more sensitive and had a stronger ability to resist damage than 2i/L-ESCs.In summary,the ABC/L medium can maintain that derived from different embryonic stages stem cells,self-renewal,pluripotency and in vitro differentiation.ABC/L-ESCs can be more resistant to damage than 2i/L-ESCs.This study provides a new platform to study on maintaining stem cells self-renewal and pluripotency,and open one way for stem cells to treat diseases in the future.