High-Purity Lineage Selection of Embryonic Stem Cell-derived Neurons

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.

Klf5 is involved in self-renewal of mouse embryonic stem cells

“…The α1-tubulin-EGFP vector was derived from pEGFP (Clontech), containing the G418 resistance gene, by replacing the CMV promoter with the rat α1-tubulin promoter from nucleotide -1050 to +5 (Schmandt et al, 2005). Klf5 cDNA (NIH Mammalian Gene Collection, Invitrogen) was sub-cloned into p3ϫFLAG-CMV7.1 vector (Sigma-Aldrich).…”

Electrophysiological evaluation of engrafted stem cell-derived neurons

“…The third technology additionally serves as a useful tool for studying the maturation and integration of donor cells in controlled, real-time scenarios. Crucial for studying vital donor cells in recipient tissues is the availability of genetically labeled donor cells that are readily identifiable in situ by expression of fluorescent indicator proteins (e.g., enhanced green fluorescent protein (EGFP)), under the control of either cell-type specific or constitutively active promoters [25][26][27][28][29][30] .…”

Embryonic Stem Cells Overexpressing the Recognition Molecules L1 and Tenascin-R Enhance Regeneration in Mouse Models of Acute and Chronic Neurological Disorders

“…By applying this method involving L1 antibody-coated surfaces, mouse ES cell-derived neurons have been isolated at high purity, which formed excitatory and inhibitory synapses and were electrically excitable after replating (Jungling et al, 2003). Similarly, ES cell-derived neural precursor cells have been efficiently purified after immunopanning for PSA-NCAM (Schmandt et al, 2005). MACS purification for cell surface molecules has been applied on both, mouse (David et al, 2005) and human ES cell-derived cells and an enrichment for labelled cells was described in these studies.…”