High-purity lineage selection of embryonic stem cell-derived neurons

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-NCAM-expressing 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.