In this study we have successfully

In this study we have successfully created 3-D SFEBs from undifferentiated induced pluripotent stem (iPS) alzheimer\'s disease that accurately recapitulates aspects of the development of the cortex and cortical networks. In order to facilitate the creation of SFEBs that approximate an in vivo frontal cortex and that can be used for imaging and electrophysiological applications, we have developed a technique that is a variation of the Stoppini method used in organotypic preparations (Stoppini et al., 1991).
Material/methods For this approach, undifferentiated human iPS cells (human iPSC line 8446B, male 34years old, made from fibroblasts obtained from the Coriell Institute) were maintained on gamma-radiated mouse embryonic feeders (MEFs, GlobalStem), in KO-DMEM/20% KSR (Invitrogen) containing: Pen-Step (1/100), Glutamax (2mM), Non-Essential Amino Acids (0.1mM), β-mercaptoethanol (0.1mM) and b-FGF (10ng/mL; R&D Systems). To differentiate iPS cells, colonies were manually cleaned to remove spontaneously differentiated cells, brought to single cells enzymatically using Accutase (Invitrogen) and re-suspended in media containing 10μM of the Rho-kinase inhibitor Y-27632 (ROCKi) (Stemgent), to minimize apoptosis. Cultures were plated for 1h on gelatin-coated plastic to remove MEFs, and the supernatant was subsequently removed and used for differentiation. Cells were plated in a 96-well V-bottom plate (Oregon Scientific) at a density of 9000 cells per well in DMEM/F12 containing the dual-SMAD inhibitors SB431542 (10μM) and LDN193189 (250nM) (Stemgent) to inhibit the Activin/Nodal and BMP branches of the TGFβ pathway respectively. Recombinant Dickkopf-1 (Dkk-1) (R&D) (200ng/mL) was also added to the culture solution. Using dual-SMAD inhibition and Dkk-1 allowed the iPS cells to be driven towards an anterior forebrain neuronal fate (Chambers et al., 2009; Hansen et al., 2011). At day 14 the SFEBs were transferred by pipetting with wide-orifice tips onto Millipore mesh inserts (MI) (0.4μM pore size) inserted into 6-well plates containing the previously described medium. The SFEBs rest on the surface of the Millipore insert with the medium underneath. The insert contains the mesh attached to the outer plastic rim on which the SFEB rests. The medium was replaced every other day until day 18, when a second differentiation medium containing DMEM/F12, Glutamax (2mM), N2 Supplement (0.1mM), and Pen-Strep (1/100) (all Invitrogen) was added to the well containing SFEBs on inserts and dual-SMAD inhibition and Dkk-1 was gradually withdrawn by partial media changes. At day 24, the DMEM/F12 solution was replaced with a final differentiation supplement containing Neurobasal medium, B-27 supplement without retinoic acid, Glutamax (2mM) and Pen–Strep (1/100) until day 30 when the experiments were performed. During the incubation period on the mesh inserts, the SFEBs began to thin considerably from ~400μM to ~100μM, optimal thickness for both imaging and electrophysiological studies at day 30 (Figs. 1A–C). SFEBs on MIs were imaged after being placed in an imaging chamber on a Zeiss LSM 510 upright confocal microscope using a 40× water-dipping objective (1.2 N.A.). Maximal projections were made from image stacks obtained through the z-dimension at an average interval of 1.5μm, made from 60 to 80 images. After cryosectioning, free-floating SFEBs were placed on glass slides and imaged using a Nikon TS100F inverted epifluorescence microscope with a 20× (0.4 N.A.) objective. To obtain quantification of expression, 518×518 pixel confocal images of SFEBs were analyzed by loading into Volocity (Perkin Elmer). Two areas of 200×200 pixels were randomly selected at the edge and center of the SFEBs. Within these areas, cells were counted according to a staining profile and outputted to excel for analysis. The data for both ROIs at the center and the edge of the SFEB was averaged together and percentages reflect this across N=4 SFEBs unless otherwise stated.