Establishment Of LIF Dependent Novel Mouse Embryonic Stem Cell Line

Embryonic Stem Cells(ESCs)are pluripotent and self-renewal stem cells which derived from the inner cell mass(ICM)of the blastocyst.Since the establishment of mouse ESCs in the 1981,its in vitro culture conditions have been one of the hot topics of scientific research.In 2008,researchers showed that na?ve pluripotency was maintained by the chemical defined medium-2i/LIF supplements(Inhibitors of Mek1/2 and Gsk3 b,known as 2i;LIF: Leukaemia inhibitory factor),which primarily affect canonical WNT,FGF/ERK,and JAK/STAT3 signaling.However,whether one of these tripartite supplements alone is sufficient to maintain na?ve self-renewal remain unclear.In here,we focus whether the application of LIF could establish L-ESCs cell lines and identify their characteristics.First,we culture na?ve 2i/LIF-ESCs in LIF alone medium and investage the LIF support pluripotency of ESCs or not.Second,LIF dependent novel ESCs were established,which was named L-ESCs,and then its pluripotent characteristics and developmental potential in vivo and in vitro were identified.The results showed in below:1.Establishment of L-ESCsThe LIF culture system was used to induce six kinds of 2i/ L-ESCs for total 16 times.The morphological characteristics and single-cell clone formation ability in L-ESCs were similar to 2i/L-ESCs.2.Pluripotency of L-ESCsThe results of a series of experiments including Real-time Quantitative PCR,Alkaline Phosphatase staining and immunofluorescence staining showed that L-ESCs had pluripotency similar to 2i/L-ESCs,Some L-ESCs cells retain the characteristics of the 2-cell embryonic period.Karyotype analysis revealed that L-ESCs had a more stable chromosome composition than 2i/ L-ESCs.3.Assessment of potential of L-ESCs in vitro and vivoL-ESCs have in vitro differentiation ability with expression of mesoderm,endoderm and ectoderm marker genes in N2B27 basic medium for 3-6 days.In vivo,L-ESCs were able to form teratomas and contribute to different tissue and the germlines of E13.5 chimeric embryos.In summary,we show that LIF alone is able to support mouse embryonic stem cells self-renew and pluripotency as well as developmental potency.This study and analysis of LIF alone dependent mouse ESCs reprogramming and epigenetic research in the future will provides theory and technological support for clinical application of ESCs.