The work with hESCs was approved by the Partners ESCRO (Embryonic Stem Cell Research Oversight) Committee under protocol number 2006-04-001A. hESC lines H7 (WA-07, XX, passages P29–P35) and H9 (WA-09, XX, approximately passage 35) were cultured according to the guidelines established by the National Academy of Sciences. Cells were propagated on mitomycin-C (10 μg/ml for 150 minutes; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com)-inactivated human (D551; American Type Culture Collection, Manassas, VA, http://www.atcc.org) fibroblasts in serum replacement medium (SRM) (Dulbecco's modified Eagle's medium [DMEM], 15% knockout serum replacement; Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com). Differentiation of hESCs was adapted from previously published protocols . Briefly, neuroectodermal differentiation (“rosette formation”) of hESCs was induced by coculture on transgenic MS5-Wnt1 stromal feeder cells (Dr. L. Studer, Sloan-Kettering Institute, New York). hESCs were triturated and plated in a density of approximately 0.5–1 colonies per six-well plate on a confluent layer of mitotically inactivated MS5-Wnt1 cells using SRM for 14 days, followed by N2 medium (DMEM/F-12; Invitrogen Corporation) (N2-A; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). Neuroectodermal induction was achieved either without or with the addition of 300 ng/ml Noggin (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) for 7 (1 week) or 21 (3 weeks) days to the culture medium (Fig. 1A). Media were changed every 2 days, and cells were differentiated toward the DA phenotype in the presence of growth factor combinations as follows: 200 ng/ml sonic hedgehog (SHH), 100 ng/ml fibroblast growth factor 8 (FGF8) (both R&D Systems, Inc.), 20 ng/ml brain-derived neurotrophic factor (BDNF) (PeproTech EC Ltd., London, http://www.peprotechec.com), 20 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen Corporation), 1 ng/ml transforming growth factor type β3 (TGF-β3) (Calbiochem, San Diego, http://www.emdbiosciences.com), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF), 0.5 mM dibutyryl cAMP, and 0.2 mM ascorbic acid (AA) (all from Sigma-Aldrich). At day 21 of differentiation, rosettes were harvested mechanically from feeders and gently replated on 15 μg/ml polyornithine plus 1 μg/ml laminin-coated culture dishes in N2 medium supplemented with bFGF, BDNF, AA, SHH, and FGF8. After 9 days (day in vitro 30 [DIV30]) cells were passaged using 0.05% trypsin/EDTA (Invitrogen Corporation) and spun at 1,000 rpm for 5 minutes. Cells were resuspended in N2 medium and plated again at a density of approximately 50,000–100,000 cells per square centimeter on polyornithine/laminin-coated dishes in the absence of bFGF but in the presence of BDNF, AA, SHH, and FGF8. After an additional 7 days of culture (DIV37), cells were differentiated until DIV42 or DIV49 in the absence of SHH and FGF8 but in the presence of BDNF, AA, cAMP, GDNF, and TGF-β3.
Figure Figure 1.. Culture conditions and development of neuroectodermal cells. (A): Schematic representation of the protocol used to differentiate human embryonic stem cells (hESCs). hESCs were differentiated in a multistep protocol supporting dopamine neuronal cell development  and modified by adding 300 ng/ml Noggin during the stromal feeder cell-based neuroectodermal induction (DIV0–21) for either 1 week (DIV0–7) or 3 weeks (DIV 0–21). On DIV21, rosettes were selected and manually transferred to polyornithine/laminin-coated culture dishes, expanded, and further differentiated using the sequential addition of media supplements as indicated (see Material and Methods for further details). At DIV42, cells were harvested for transplantation into the striatum of 6-hydroxydopamine-treated rats. (B): The effects of Noggin on neuroectodermal cell development. Left panels: In the absence of Noggin, hESC colonies developed into typical crater-like structures with a single layer of homogeneous cells in the center and a rim with heterogeneous cellular morphologies. In these conditions, the colonies had very little to no rosettes. An overview of colonies at low magnification and a representation of four colonies at higher magnification are shown in the inset (×5). Right panels: Addition of Noggin for 3-weeks led to increased formation of rosette-forming ESC colonies. An overview of colonies from Noggin-treated cultures at low magnification, which macroscopically appear homogeneous with a high cell density, is shown. Bright-field image (×5) of the border of a single colony at DIV21 showing an accumulation of rosettes (arrows). Abbreviations: AA, ascorbic acid; BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; DIV, day in vitro; FGF8, fibroblast growth factor 8; SHH, sonic hedgehog; SRM, serum replacement medium; TGF-β3, transforming growth factor type β3.
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