Investigation Of The Differentiation Of Murine Embryonic Stem Cells Into Neural Or Muscle Fate.

Mouse embryonic stem cells (ES cells) are derived from inner cell mass. It candifferentiate into variety cell types of three-germ laryer in vitro or in vivo and ispotentially used to therapy certain diseases in the future. Bear it in mind, it is veryimportant to explore the mechanisms and signaling pathways that are involved indifferentiation of ES cell into specific cell lineage, especially those in muscle orneural fate determination. To investigate whether LIF and p53 exert a role indifferentiation of ES cells into neural or muscle fate, we used different methods toexplore their function during ES cell differentiation in the following two parts,respectively.Part ILeukemia inhibitory factor (LIF), a pleiotropic cytokine, is extensively used inmaintaining embryonic stem cell pluripotency. However, in a serum-and feeder-freemedium, LIF alone is insufficient to sustain ES cell self-renewal and to blockneuronal differentiation. In a low-density culture condition, it also claimed ES cellsform neurospheres as a default manner and LIF might require to neuronaldifferentiation for cell survival. This work aimed to investigate whether LIFperforms a role in mediating differentiation of ES cells into neuronal fate. We reporthere that LIF increases the production of neuronal cells under various cultureconditions. Inhibition of LIF downstream JAK/STAT3 leads to ES cellsdifferentiation into glial fate, and blocking of LIF downstream MEK pathwaysupports differentiation of ES cell toward neuronal lineage. Furthermore, LIFinhibits cell apoptosis and promotes cell proliferation in the process. Meanwhile, thecommitment to form both mesoderm and extraembryonic endoderm is inhibited andthe determination to form neural progenitor is enhanced when LIF was included inthe culture medium. We thus show that LIF facilitates ES cell differentiation intoneuronal cells.Part IIThe p53 gene is widely expressed in embryo, tissues and tumors, and itsdeficiency can rescue embryonic defects in certain genes null embryos. However, itis still poorly understood whether p53 is involved in myoblast and neuronal fatedetermination during embryogenesis. We established the ES cell clone in which p53protein was persistently suppressed by stable expression of p53 RNAi, and GFP wasexpressed in a p53 RNAi transcription independent manner. With the classicalprotocol in which the differentiation of ES cells into neural or muscle cell isspecifically modulated by retinoic acid (RA), we evaluated the function of p53during myoblast and neuronal commitment. With RA treatment, silencing of p53 byRNAi in ES cells leads to dominant muscle cell production but lack of neuronal cell,indicating that p53 indeed plays a role during muscle and neuronal fate commitment.It thus provides a good model for investigating cross-talk between RA and p53pathways during myogenesis and neurogenesis from ES cells.