Antibodies to the region-specific transcription factors Bf1, Dlx, En1, and Pax6 were used to explore whether functional donor cell integration depends on the acquisition of a regional phenotype. Our data show that incorporated neurons frequently exhibit a lacking or ectopic expression of these transcription factors. Thus, the lack of an appropriate regional "code" does not preclude morphological and synaptic integration of ES cell-derived neurons.

Culture and labeling of ESGPsMouse ES cells (line J1) (Li et al., 1992) were aggregated to embryoid bodies and subsequently plated in ITSFn medium (Okabe et al., 1996). After 5 days, cells were trypsinized and propagated for 5 days in polyornithine-coated dishes in a DMEM/F12-based medium supplemented with 10 ng ml -1 FGF2. They were then harvested and replated in medium supplemented with FGF2 and EGF (20 ng ml -1 ). In some experiments in vitro, the cells were propagated through an additional passage in medium supplemented with FGF2 and PDGF-AA (10 ng ml -1 , see Results). In vitro differentiation into astrocytes and oligodendrocytes was induced by growth-factor withdrawal as Supplemental data available online

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.

Pluripotency, virtually unlimited self-renewal and amenability to genetic modification make embryonic stem (ES) cells an attractive donor source for cell-mediated gene therapy. In this proof of concept study, we explore whether glial precursors derived from murine ES cells (ESGPs) and engineered to overexpress human arylsulfatase A (hASA) can cross-correct the metabolic defect in an animal model of metachromatic leukodystrophy (MLD). Transfected ES cells showed an up to 30-fold increase in ASA activity. Following in vitro differentiation, high expression of ASA was found in all stages of neural and glial differentiation. hASA-overexpressing ESGPs maintained their ability to differentiate into astrocytes and oligodendrocytes in vitro and in vivo. After transplantation into the brain of neonatal ASA-deficient mice, hASA-overexpressing ESGPs were found to incorporate into a variety of host brain regions. Four weeks after engraftment, immunofluorescence analyses with an antibody to sulfatide revealed a 46.774.0% reduction of immunoreactive sulfatide deposits in the vicinity of the hASA-positive engrafted cells, thereby significantly extending the rate of sulfatide reduction achieved by the endogenous ASA activity of non-hASA-transfected control cells (21.175.8%). These findings provide first in vivo evidence that ES cells may serve as a potential donor source for cell-mediated enzyme delivery in storage disorders such as MLD.