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{"project":"2_test","denotations":[{"id":"17967047-16352714-85283482","span":{"begin":120,"end":122},"obj":"16352714"},{"id":"17967047-11121071-85283483","span":{"begin":2719,"end":2721},"obj":"11121071"},{"id":"T60257","span":{"begin":120,"end":122},"obj":"16352714"},{"id":"T41656","span":{"begin":2719,"end":2721},"obj":"11121071"}],"text":"Maintenance and differentiation of hESCs and hCNS-SCns.\nhESC line Cy203 (Cythera) was cultured as previously described [12]. To differentiate into neuroepithelial precursor cells, colonies were manually isolated from mouse embryonic fibroblasts (MEFs) and cut in small pieces. These pieces were transferred to a T75 flask with hESCs differentiation media (same hESC medium but 10% KSR and no FGF-2). Medium was changed the next day by transferring the floating hESC aggregates to a new flask. After culturing for a week, the hESC cell aggregates formed mature embroid bodies (EBs; ∼10 um round clusters with dark centers). EBs were plated on a coated 10-cm dish in hESC differentiation media. The next day, the medium was changed to DMEM/F12 supplemented with ITS and fibronectin. Medium was changed every other day for a week or until the cells formed rosette-like columnar structures that were isolated manually. These structures were then transferred to coated dishes in neural induction medium (DMEM/F12 supplemented with N2 and FGF-2) for a week. Elongated single cells were separated from leftover aggregates using non-enzymatic dissociation. After one to two passages, the cells formed a monolayer of homogeneous NPs (negative for Sox1 immunostaining). Upon confluence, cells will form neurospheres that can also be isolated from the neuroepithelial precursor cells (positive for Sox1 immunostaining). At any of these two stages, pan-neuronal differentiation can be achieved after three to four weeks. hESC line HUES6 was cultured on MEF feeders as previously described (http://www.mcb.harvard.edu/melton/hues/) or on GFR matrigel coated plates. Cells grown on matrigel were grown in MEF-conditioned medium and FGF-2 was used at 20 ng/mL instead of 10 ng/mL for cells grown on MEFs. To differentiate neuroepithelial precursors, colonies were removed by treatment with collagenase IV (Sigma) and washed three times in growth media. The pieces of colonies were resuspended in HUES growth media without FGF2 in an uncoated bacterial Petri dish to form EBs. After one week, EBs were plated on polyornathine/laminin coated plates in DMEM/F12 supplemented with N2 and FGF2. Rosette structures were manually collected and enzymatically dissociated with TryPLE (Invitrogen), plated on polyornathine/laminin coated plates, and grown in DMEM/F12 supplemented with N2 and B27-RA and 20 ng/mL FGF-2. Cells could be grown as a monolayer for up to at least ten passages. Cells were Sox1 and nestin positive and readily differentiated into neurons upon withdrawal of FGF-2. Human central nervous system stem cell line FBR1664 (StemCells) which is referred to as hCNS-SCns in the main text was cultured as previously described [23]. The cells were cultured in medium consisting of Ex Vivo 15 (BioWhittaker) medium with N2 supplement (GIBCO), FGF2 (20 ng/mL), epidermal growth factor (20 ng/mL), lymphocyte inhibitory factor (10 ng/mL), 0.2 mg/ml heparin, and 60 ug/mL N-acetylcysteine. Cultures were fed weekly and passaged at ∼two to three weeks using collagenases (Roche). The following antibodies and corresponding dilutions were utilized for the immunohistochemical analysis of marker genes in Cyt-ES and HUES6-ES: Sox2 (Chemicon, 1:500), Oct4 (Santa Cruz, 1:500), Sox1 (Chemicon, 1:500), Nestin (Pharmingen, 1:250); hCNS-SCns: Sox2 (Chemicon, 1:200), Nestin (Chemicon, 1:200)."}

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