Abstract
The differentiation and screening methodology proposed here is an efficient in vitro system to screen and study effects of small molecules and bioagents and is an alternative to studies that use live animals and embryos. The method is based on engineering a stable murine embryonic stem (ES) cell line expressing lineage-specific promoters that drive selection and reporter genes. Additionally, uniform embryoid bodies (EBs) are used for differentiation studies that allow synchronous differentiation. The reporter and selection marker genes are expressed only in lineages where the promoter is functional. The differentiated cell type can be identified by reporter gene expression and the selection marker can be used for selective enrichment of that particular cell population. The method described here is useful in screening small molecules or bioagents that can differentiate stem cells into particular lineages or cell types. Identified compounds are useful in areas such as stem cell-based regenerative medicine and therapeutics. The method described here has been applied to neuronal cell differentiation.
Highlights
Embryonic stem (ES) cells are pluripotent and derived from an early embryonic stage of development
We describe a method to screen small molecules/bioagents that differentiate embryonic stem (ES) cells to a particular lineage and enrich for a differentiated cell type
To verify that necdin promoter drove the beta-galactosidase reporter in neuronal cells, the retinoic acid-differentiated cells grown on laminin-coated coverslips on day 12 were fixed and immunostained for the neuronal marker, low-affinity nerve growth receptor (LNGFR) and for beta-galactosidase (Figure 2(A))
Summary
Embryonic stem (ES) cells are pluripotent and derived from an early embryonic stage of development. When cultured in the presence of leukocyte inhibitory factor (LIF), ES cells remain undifferentiated. Removal of LIF and regulation of culture conditions lead to ES cell differentiation into specific cell types [1]. Differentiation of ES cells into different cell lineages is of great importance in regenerative medicine. Differentiation into specific cell type(s) and enrichment of the differentiated cell type(s) are critical for therapeutic application purposes. We describe a method to screen small molecules/bioagents that differentiate ES cells to a particular lineage and enrich for a differentiated cell type
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