AN IN VITRO PATHWAY FROM ES CELLS TO NEURONS AND GLIA

Mouse embryonic stem (ES) cells can be induced to differentiate into neurons and glia in vitro. Induction protocols are straightforward and involve culture in the presence of retinoic acid. They result in an efficient conversion of undifferentiated ES cells to neural cells. Mature neurons produced have the key physiological, morphological and molecular properties of primary cultured neurons derived from the central nervous system. Most significantly, they form functional chemical synapses that utilize either glutamate, GABA or glycine as neurotransmitters. ES cellderived glial cells also correspond well with their normal counterparts. During induction, ES cells undergo a series of developmental steps that resemble key stages in the early mouse embryo. This supports the hypothesis that the in vitro pathway is a valid model of the normal developmental pathway leading to neurons and glia. The in vitro system combines 3 experimental strengths. It is suitable for genetic manipulation, affords large numbers of cells and allows precise manipulation of the culture environment. It is thus suitable for a wide variety of mechanistic studies in the areas of neural development and cell biology. Introduction Mouse embryonic stem (ES) cells strongly resemble cells of the inner cell mass and primitive ectoderm, which are transient structures found in the embryonic day 4-6 (E 4 –6 day) embryo. The primitive ectoderm, which is the source of all cells of the body, gives rise to the neural plate by E8. The neural plate is quickly transformed into the neural tube. At early stages, the neural tube consists predominantly of rapidly dividing stem cells. Stem cells later exit the cell cycle to give rise to the neurons and glia of the nervous system. ES cells have the inherent capacity to differentiate into all cell types of the nervous system as demonstrated by formation of chimeric mice from embryos in which ES cells are transplanted into the inner cell mass. At first glance, it would appear unlikely that the developmental lineage extending from ES cells to mature neurons and glia could be recapitulated in cell culture because the processes which give rise to the vertebrate nervous system, while not understood in detail, appear to be highly complex. Contrary to this expectation, it has proved possible to reconstitute, at least partially, this pathway in vitro starting with ES cells and ending with differentiated neurons and glia. The result is an experimental system combining 3 strengths. Genetics is available because the ES cell is the leading model for targeted modification of the genome. Since ES cells are a large-scale source of cells, biochemical investigation is feasible. Finally, tissue culture makes it ES cell-derived neurons 2 possible to alter the environment of cells in precise ways. Thus studies with the system have the potential for making important contributions to understanding neuronal and glial development and function. This review covers the literature in which ES cells have been induced to differentiate along a neural lineage in cell culture. ES cells are maintained in vitro as totipotent stem cells by culture in the presence of the cytokine leukemia inhibitory factor (LIF). Most studies of in vitro differentiation of ES cells begin by culturing ES cells as small aggregates called embryoid bodies (EBs). EBs are formed by withdrawing LIF and culturing cells in dishes with a non-adhesive substratum. Standard EB cultures are composed of a wide variety of cell types, including a small proportion with a neuronal appearance (see for example Martin, 1981). Three papers published in 1995 demonstrated that more efficient in vitro differentiation of ES cells into neural cells is possible. All utilized retinoic acid (RA) but differ in the details of culture method. In our laboratories (Bain et.al, 1995), ES cells were cultured as EBs in the absence of LIF for 4 days. Culture was then continued for 4 additional days in the presence of RA. Next, aggregates were dissociated with trypsin and plated as monolayer cultures on an adhesive substrate. Within several days, large numbers of neuron-like cells appeared. The phenotype of these cells (reviewed below) justifies designating them as neurons. About 40% of the cells in the cultures were neurons. These grew on top of a layer of flat cells adhering to the substrate. Many of the flat cells were glia, but the exact percentage has not been determined. A study by Strubing et.al (1995) used a similar approach. EBs were formed by the 'hanging drop' culture method. These EBs were exposed to RA for the first 2 days of culture and then cultured in suspension for an additional 2 days in the absence of RA. Next, the EBs were cultured on an adhesive substrate; neurons appeared about 2 days after plating on the adhesive substrate. RA had a dramatic effect. All EBs exposed to RA gave rise to neurons whereas only 15% of control EBs not exposed to RA had neurons. In the study of Fraichard et.al (1995), EBs were formed in mass culture and exposed to RA for the first 2 days. They were then plated onto an adhesive substrate in the absence of RA. After 4-5 days, neuronal cells appeared. Thus 3 related protocols involving culture of EBs in the presence of RA result in extensive neuronal differentiation. Another approach to obtaining neurons from ES cells has been described (Okabe et.al, 1996). In this case, ES cells were cultured as EBs for 4 days in standard medium in the absence of added RA. Next the EBs were transferred to an adhesive substrate, to which they attach, and the standard medium replaced by a serum-free medium. Over the next several days, many cells die. Many of the surviving cells are tightly packed epithelial cells that are nestin positive. In the presence of bFGF these cells proliferate and go on to differentiate into neurons