By 35?days in culture, these hiPSC aggregates had begun to differentiate into cells representative of the two cerebellar germinal layers, suggesting the presence of both GABAergic and glutamatergic progenitors, which could, in theory, both be further expanded and matured . For our purposes, we focused on the further maturation of Purkinje cells, demonstrating the presence of human calbindin-positive cells after 15?days of co-culture with mouse cerebellar progenitors. representative of the two cerebellar CHMFL-BTK-01 germinal zones, the rhombic lip (Atoh1-positive) and ventricular zone (Ptf1a-positive), could be identified, with the latter giving rise to cells positive for Purkinje cell progenitor-specific markers, including Lhx5, Kirrel2, Olig2 and Skor2. Further maturation was observed following dissociation and co-culture of these cerebellar progenitors with mouse cerebellar cells, with 10% of human cells staining positive for the Purkinje cell marker calbindin by day 70 of differentiation. This protocol, which incorporates modifications designed to enhance cell survival and maturation and improve the ease of handling, should serve to make existing models more CHMFL-BTK-01 accessible, in order to enable future advances in the field. Electronic supplementary material The online version of this article (10.1007/s12311-017-0913-2) contains supplementary material, which is available to authorized users. (1000?rpm). Following centrifugation, the cell pellet was gently resuspended to give a single cell suspension, in induction medium, consisting of Iscoves modified Dulbeccos medium/Hams F12 1:1, insulin (7?g/ml, Sigma-Aldrich, St. Louis, CHMFL-BTK-01 MO, USA), crystallisation-purified bovine serum albumin (BSA, 5?mg/ml, Sigma-Aldrich), chemically defined lipid concentrate (1%), monothioglycerol (450?M, Sigma-Aldrich), apo-transferrin (15?g/ml, Sigma-Aldrich) and penicillin/streptomycin (1%). At this stage, induction medium was additionally supplemented with 50?M Y-27632 (Sigma-Aldrich) and 10?M SB431542 (Tocris, Bristol, UK). To allow for reaggregation, 12,000 cells/well were transferred to three to four low-adhesion V-bottomed 96-well PrimeSurface culture plates (Sumitomo Bakelite, Tokyo, Japan) in this supplemented induction medium, and incubated for 48?h at 37?C, 5% CO2. Recombinant fibroblast growth factor 2 (FGF2, 50?ng/ml, R&D Systems, Minneapolis, MN, USA) was added to the culture on day 2. A one-third volume replacement was performed on day 7, using fresh induction medium without Y-27632 or 10?M SB431542, and a further full volume medium replacement was performed on day 14. On day 21, cell aggregates were transferred from 96-well plates to low-adhesion 24-well plates (Corning). A micropipette with a cut-off tip was used to transfer approximately four aggregates into each well, and aggregates were incubated for 14?days in differentiation medium, consisting of neurobasal medium supplemented with GlutaMAX (1%), N2 (1%) and penicillin/streptomycin (1%). A full-volume replacement with fresh differentiation medium was performed on day 28. Dissociation and Co-Culture of Purkinje Cells On day 35 of suspension culture, cell aggregates were transferred into microcentrifuge tubes (approximately 10 aggregates per tube) using a micropipette with a cut-off tip. Aggregates were washed twice with HHGN, consisting of 1 HBSS supplemented with 2.5?mM HEPES pH 7.3C7.5, 35?mM glucose and 4?mM NaHCO3. This was followed by incubation in Neuronal Isolation Enzyme with Papain (200?l per 10 aggregates) for 20C30?min at 37?C, with periodic agitation. After careful removal of the enzyme solution, aggregates were washed gently three times with HHGN. Dissociation to a single cell suspension was performed by trituration 20C25 times in 500?l seeding medium, consisting of DMEM/F12 with L-glutamine, supplemented with N2 Rabbit Polyclonal to CSTF2T (1%), 1.4?mM additional CHMFL-BTK-01 L-glutamine, 5?g/ml additional insulin (Sigma-Aldrich), penicillin/streptomycin (1%) and 10% HyClone US-defined heat-inactivated foetal bovine serum (FBS, GE Healthcare Life Sciences, Little Chalfont, UK), taking care to avoid air bubble formation. Cells were then pooled, and seeding medium added to 5?ml, before centrifugation at 185(900?rpm) for 5?min. Following centrifugation, the supernatant was removed and the pellet resuspended in 250?l seeding medium. Cells were counted, and the concentration adjusted to 8??106 cells/ml in seeding medium. In parallel, mouse cerebellar cells were prepared. Pregnant dams (C57BL/6) at 18?days of gestation were sacrificed, and cerebella were dissected from the CHMFL-BTK-01 pups. Approximately one litter was used for each iPSC line. Cerebella were washed twice with HHGN, and incubated in TrypLE Express for 10?min at 37?C, with periodic agitation. This was followed by a further three washes with HHGN, before dissociation by trituration in seeding medium, centrifugation and resuspension at 8??106 cells/ml, as described above. Human and mouse cells were mixed at a ratio of 1 1:10, and 55?l of the mixed cell suspension was seeded as a small bubble on Cell Desk LF plastic coverslips (Sumitomo Bakelite), previously coated with poly-L-ornithine (0.5?mg/ml, Sigma-Aldrich) and natural mouse laminin (10?mg/ml). After incubation for 3?h at 37?C, 5% CO2, 500?l of culture medium, consisting of DMEM/F12 with L-glutamine, supplemented with N2 (1%), 1.4?mM additional L-glutamine, 5?g/ml additional insulin (Sigma-Aldrich), penicillin/streptomycin (1), 0.5?ng/ml tri-iodothyronine (T3, Sigma-Aldrich), 100?g/ml BSA (Sigma-Aldrich), 50?ng/ml recombinant human brain-derived neurotrophic factor (BDNF, R&D Systems) and 50?ng/ml recombinant human neurotrophin 3 (NT3, R&D Systems), was gently added to each well to reduce the final serum.