L-WNT3A Cells And The Conditional Medium Support The Proliferation And Differentiation Of Mouse Embryonic Stem Cells

Mouse embryonic fibroblast cells (MEF) are widely used as the feeder cells during the culture of embryonic stem cells (ESCs). However, the major problems of MEFs as feeder cells are the times of cell passage and lots of killings of mice. L Wnt3a cell line is from the genetically modified subcutaneous connective tissue cells, which persistent secret activated Wnt-3A protein. In present study, the L-Wnt3a cells were treated with mitomycin C, and the growth state of the treated cells were analysized by MTT and obtained the best suitable concentration and processing time in mitomycin C. The treated cells or the cell conditioned medium were then used to culture mouse ESCs. The results showed that the mitomycin C at10μg/ml concentration and treated the cells for4h could significantly inhibit the proliferation of the L-Wnt3a cells but did not affect their viability. When the mouse blastocysts were incubated on L-Wnt3a feeder layer, primary colonies formed and one stem cell line was established. When culture of the ESCs in feeder-free, the conditioned medium from L-Wnt3a cells robustly improved ESCs proliferation without differentiation. The ESCs in L-Wnt3a conditioned medium were ALP positive, expressing pluripotent markers, Oct4, Sox2, Nanog, E-cadherin and SSEA-1. In vivo and in vitro differentiation analyses showed that the ESCs formed embryoid body and teratoma, and differentiated into three germinal layers. When injected the ESCs into blastocoele, the chimeric offsprings were produced. In conclusion, L-Wnt3a cells and their conditional medium can support the prolifereation and differentiation of mouse ES cells.