ABSTRACT
The derivation of somatic cell types from pluripotent and self-renewing embryonic stem (ES) cells offers attractive prospects for basic research, compound development, and regenerative medicine. A key prerequisite for biomedical applications of ES cells is the ability to differentiate and isolate defined somatic cell populations at high purity. In this study, we explore the potential of the Talpha1- enhanced green fluorescent protein (EGFP) transgene and polysialic acid (PSA)-neural cell adhesion molecule (NCAM) as lineage selection markers for the derivation of ES cell-derived neurons. Upon controlled in vitro differentiation, ES cells engineered to express EGFP under control of the Talpha1-tubulin promoter exhibited exclusive transgene expression in neurons. Similarly, PSA-NCAM expression during the early stages of ES cell differentiation was restricted to neuronal progeny. Talpha1- EGFP- and PSA-NCAM-positive neurons comprised both inhibitory and excitatory phenotypes. Compared to Talpha1-EGFP, the expression of PSA-NCAM was initiated at slightly earlier stages of neural differentiation. FACSorting of Talpha1-EGFP-positive cells and immunopanning of PSA-NCAMexpressing cells yielded neuronal populations at purities up to 99.6% and 96.9%, respectively. These findings depict Talpha1-EGFP and PSA-NCAM as suitable markers for high-purity selection of early ES cell-derived neurons.
Subject(s)
Cell Lineage , Cell Separation/methods , Embryo, Mammalian/cytology , Neurons/cytology , Stem Cells/cytology , Animals , Cell Culture Techniques , Cell Differentiation , Cell Line , Cell Separation/standards , Flow Cytometry , Green Fluorescent Proteins/genetics , Mice , Neural Cell Adhesion Molecules/analysis , Phenotype , Sialic Acids/analysis , Tubulin/geneticsABSTRACT
Pluripotency and the capacity for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for cell-replacement strategies. A key prerequisite for a therapeutic application of ES cells is the generation of defined somatic cell populations. Here we demonstrate that a targeted insertion of the EGFP gene into the tau locus permits efficient fluorescence-activated cell sorting (FACS)-based lineage selection of ES cell-derived neurons. After in vitro differentiation of heterozygous tau EGFP ES cells into multipotent neural precursors, EGFP is selectively induced in postmitotic neurons of various neurotransmitter phenotypes. By using FACS, ES cell-derived neurons can be enriched to purities of more than 90%. Because neuron-specific EGFP fluorescence is also observed upon transplantation of ES cell-derived neural precursors, the tau EGFP mutant represents a useful tool for the in vivo analysis of grafted ES cell-derived neurons.
