Citation Context

... serum and penicillin/streptomycin at 37º, 5% CO 2. ES1 cells were differentiated into pan-neural precursor cells through embryoid body formation for 4 days and selection in ITSFn media for 5–7 days (=-=Okabe et al., 1996-=-), and they were maintained in FGF2 and EGF2 (both from R&D Systems) containing chemically defined media as described (Conti et al., 2005). These cells uniformly express nestin and Sox2 and upon growt...

Citation Context

...lity to differentiate into any of the several different cell types of the adult animal, e.g., adipocytes (Dani et al., 1997), oocytes (Hubner et al., 2003), hepatocytes (Jones et al., 2002), neurons (=-=Okabe et al., 1996-=-), muscle cells (Rohwedel et al., 1994), cardiomyocytes (Wobus et al., 1991), and hematopoietic cells (Wiles and Keller, 1991; Nishikawa, 1997). Transplantation of cells differentiated from ES cells h...

Citation Context

...po-transferrin (Intergene), 30 nM sodium selenite (Sigma), and 2.5 µg/ml fibronectin (Life Technologies, Karlsruhe, Germany). This medium has been shown to favor the survival of neuroepithelial cells =-=(28)-=-. After 5 days, cells were triturated to a single cell suspension and further propagated on polyornithine-coated dishes (15 µg/ml, Sigma) in the presence of FGF-2 (10 ng/ml), which permits the prolife...

Citation Context

...to expand and differentiate. A second source of stem cells is from embryonic stem (ES) cell lines. ES cells are pluripotent and immortal (Bain et al., 1995; Brustle et al., 1999; Deacon et al., 1998; =-=Okabe et al., 1996-=-). ES cells have been successfully induced to express neural phenotypes (Lee et al., 2000; Spenger et al., 1994, 1995; Studer et al., 1995, 1996; Yan et al., 2001). Several potential problems exist wi...

Citation Context

...al feeders were prepared as described previously (Kawasaki et al. 2000). The PA6-SHH stable cell line has been established by viral transduction using a retroviral construct expressing human SHH N-terminus (generous gift from Dr Suh-Kim at Ajou University, Korea) followed by blasticidin (5 lg/ mL, Invitrogen) selection. Undifferentiated hES colonies were detached from MEF feeders by incubation with 200 U/mL collagenase IV (Invitrogen) for 60 min at 37C, followed by gentle dissociation into small clusters and then cells were resuspended in serum-free insulin/transferrin/selenium (ITS) medium (Okabe et al. 1996) with 0.2 mM ascorbic acid (AA, Sigma-Aldrich). Clusters were plated on intact culture dish surface (for the direct differentiation method) or on a layer of PA6 stromal cell feeder (for the co-culture method). In the direct differentiation protocol without feeder, rosettelike structures were formed by 2–3 weeks of culture in ITS + AA. Only the clusters of rosette structures were mechanically dissected and were grown as floating spheres in N2 (Johe et al. 1996) supplemented with bFGF (20 ng/mL) and AA (0.2 mM). After 1 week in culture, intact spheres were transferred onto coverslips (Bellco, Vi...

Citation Context

...derm differentiation. The various approaches include (1) treatment of serum-stimulated EBs with retinoic acid (Bain et al. 1995), (2) sequential culture of EBs in serum followed by serum-free medium (=-=Okabe et al. 1996-=-), (3) differentiation of ES cells as a monolayer in serum-free medium (Tropepe et al. 2001; Ying et al. 2003b), and (4) differentiation of ES cells directly on stromal cells in the absence of serum (...

Citation Context

...lopment of nerve cells. Pluripotent ES cells can differentiate in vitro into neurons, astrocytes, and oligodendrocytes (Fraichard et al., 1995), following the same order in which they appear in vivo (=-=Okabe et al., 1996-=-). Recent studies have shown that ES-derived neural precursors cells can be grown in free-floating aggregate cultures similar to neurospheres. These cells can be clonally expanded in defined condition...

Citation Context

...lopment of nerve cells. Pluripotent ES cells can differentiate in vitro into neurons, astrocytes, and oligodendrocytes (Fraichard et al., 1995), following the same order in which they appear in vivo (=-=Okabe et al., 1996-=-). Recent studies have shown that ES-derived neural precursors cells can be grown in free-floating aggregate cultures similar to neurospheres. These cells can be clonally expanded in defined condition...