Derivation And Characterization Of Mouse And Goat Embryonic Stem Cells In31Condition

Embryonic stem cells （ESCs） have the ability to maintain theirselves self-renewal andpluripotence, without additional growth factors, only by inhibition of signal pathway whichcan induce ESCs differentiation, chimera competent ESCs can be derivated. Rencently, anovel derivation system （"3i"）, including MEK inhibitor, ALK5inhibitor and GSK3βinhibitor, was successfully used for isolating high germLine competent ESCs from NODmouse and rat, which prevalently were regarded as difficulty in derivating ESCs. This studywas conducted to derive and characterize mouse ESCs and goat embryonic stem cell-likecells. The results are as follows:(1)In present study, we derived two novel ESCs lines from IVF blastocyst of Kunmingmice in a feeder-free and serum-free system containing2.5μM BIO. In this system, expandedIVF blastocysts could spontaneously hatch from zonae pellucidae and attached ongelatin-coated bottom of dishes. ESCs-like outgrowths could be observed without overfulltrophoblast cells. After further propagation, two Kunming mice ESC lines, designated asKMES1and KMES2, were obtained. These two novel ESCs shared common morphologicalproperties with other rodent ESCs, showed strong alkaline phosphatase activity and expressedpluripotent markers, including Oct-4, Nanog and SSEA-1. Embryoid body （EB） andteratoma test indicated that these ESCs could spontaneously differentiate into cell lineagesrepresentative of all three embryonic germ layers.(2)We used the embryos that came from the superovulation on118Guanzhong dairygoat with a result of total1204blastocysts to isolate goat embryonic stem cell-like cells.From the present study, it is evident that temperature has an effect on superovulation onGuanzhong dairy goat, and the temperature dropping abruptly lead to the low rate ofblastocysts received. All of the blastocysts were used for derivving and characterizzing goatembryonic stem cell-like cells in “3i” condition containing2.5μM BIO、0.5μM PD184352、0.5μM SB431542with feeder and serum. One goat embryonic stem cell-like cell line wasestablished. The goat cell-like cell colonies showed typical ESCs morphology and expressedESCs specific markers such as alkaline phosphatase, Oct-4and Nanog. The embryonic stemcell-like cells produced embryoid bodies and following prolonged culture differentiated intoneuron like cells. All of these results demonstrated the feasibility to isolate and establish mouse ESCs andgoat ES cell-like cell lines in “3i” system, which provide an important tool for studyingepigenetic effects in ESCs and developmental biology as well as gene targeting to producegenetically modified livestock.