The Effects Of Heparin And LIF In Culturing Mouse Embryonic Stem Cells Under The Self-made Serum-free Replacer

Objective:This study was designed to investigate the effects of heparin and leukemiainhibitory factor (LIF) in culturing mouse embryonic stem cells under the self-madeserum-free replacer.Methods：mESCs were most commonly maintained on mouse inactivated embryonicfibroblast feeders in medium supplemented with FBS, or proprietary replacements（such as knockout serum-replacement）together with LIF.According to the differenteffects of LIF, heparin, and LIF+heparin, this study was divided into three parts.The first: The effects of different concentrations of heparin on mouseembryonic stem cells were analyzed by observing cell growth.The second: The effects of different concentrations of LIF on mouse embryonicstem cells were analyzed by observing cell growth. The phenotypes of mESCs inundifferentiated state were analyzed by AKP staining, immunofluorescence andFACS analysis.The third: The effects of different concentrations of heparin+LIF on mouseembryonic stem cells were analyzed by observing cell growth. AKP staining was usedto detect the mouse embryonic stem cells which retained stable expression ofundifferentiated markers of mESCs, and a capacity to differentiate. The expression ofSSEA-1and SSES-3were also detected by flow cytometry, and Oct-4was inspectedby immunofluorescence.Results:Cultures grown in the self-made serum-free replacer failed. It may be related tolack of attachment, cell death, or overt cell differentiation. Instead, heparin couldpromote the adherent growth and play a valuable role in the dose-dependent in mouseembryonic stem cells. The cells held best grow station in the medium with100ng/ml heparin, but this medium only supported maintenance of the cells for six passages.LIF could significantly promote the adherent growth of mouse embryonic stemcells. When the concentration of LIF reached to10ng/ml, the cells could be seriallypropagated and retain their undifferentiated state in vitro for more than6passages inour serum-free, chemically defined media. We detected the mouse embryonic stemcells: the expression rate of SSEA-1，the specificity surface antigen of embryonicstem cell, was64.05%,and SSEA-3was1.0%; the results of the AKP staining andcell immunofluorescence detection of Oct-4were positive.Mouse embryonic stem cells cultured in the medium with10ng/ml LIF and100ng/ml heparin get the best growth state. The cells can be continuously passedwithout differentiation. We detected the mouse embryonic stem cells (passage3): theexpression rate of SSEA-1was82.49%, the expression rate of SSEA-3was2.15%. Inaddition, the detection of the AKP staining and cell immunofluorescence of Oct-4were strongly positive.Conclusion:The stem cells cultured in self-made serum-free replacer, added with heparin,were benefited to adhere and maintain the growth without differentiation. The concentration ofheparin is closely related to the growth capacity of cells. After several passages, the cellswere easier to differentiate.The mES cells cultured in self-made serum-free replacer, added with LIF, werebenefited to adhere and maintain an undifferentiated phenotype. The concentration ofLIF is a positive correlation in the growth capacity of the cells, when it was lowerthan100ng/ml.100ng/ml LIF largely inhibited the growth of mESCs.The mES cells cultured in self-made serum-free replace, added with LIF andheparin, were benefited to adhere and maintain an undifferentiated phenotype. Theconcentration of heparin is a key factor in the growth capacity of the cells. ThemESCs grew best at a concentration of heparin of100ng/ml. Undifferentiatedanalysis show that the mouse embryonic stem cells grew in the self-made basalmedium of serum replacer reached the standard of undifferentiated, which demon- strated that the self-made basal medium of serum replacer was enough to maintain thepluripotency of mouse embryonic stem cells.