Embryonic stem cell differentiation in defined medium

Embryonic stem (ES) cells which are derived from the epiblast in the preimplantation embryo can be propagated indefinitely in an undifferentiated state in culture and have an ability to give rise to various types of cells including neural cells. Most previously published protocols for mouse ES cell neural differentiation involve induction by retinoic acid (RA) or by exposure to mitogens, or medium containing proprietary supplements or conditioned by other cell types. In this thesis, we developed completely defined serum-free and retinoid-free differentiation (SFD) medium, and compared physiological, molecular and phenotypic characteristics of neurons differentiated in SFD medium with or without transient exposure to RA. Mouse ES cells induced in SFD medium alone efficiently differentiated into neurons and glia. However, RA treatment increased the number of cells with neuronal properties and allowed neurons to rapidly establish axon/dendritic polarity. Moreover, neurons in cultures induced with RA express functional AMPA, NMDA, GABA and glycine receptor and glutamatergic neurons are more frequent. Whereas, neurons in cultures differentiated with SFD medium alone failed to express functional glycine receptors and a large proportion of the cells are GABAergic. BrdU analysis and overall gene expression profiles of cell populations induced in SFD medium with or without RA showed that cultures induced in SFD medium contain a higher number of proliferating cells. In addition, cultures induced with SFD medium alone generated more diverse early progenitor cells and a significant number of cells expressing forebrain markers, whereas the majority of cells differentiated with RA expressed markers of interneuron subtypes in the spinal cord. These results suggest that cultures induced in SFD medium include a large proportion of cells that are relatively immature and acquire anterior neural fate, whereas cells differentiated with RA are relatively more mature and adopt posterior neural fate. In addition, we demonstrate that human ES cells express a number of connexin genes and exhibit extensive dye and electrical coupling via gap junction channels similar to early embryos. These data suggest that human ES cells can be a good model system to study the role of gap junctions in early development.