Enitors is provided in Standard Protocol 3.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptCurr Protoc Hum Genet. Author manuscript; offered in PMC 2017 July 01.Wang et al.PageBasic Protocol 1: Preparation of hES or iPS cells for neuron differentiationhES or iPS cells (hPS cells) can be cultured on a layer of feeder cells or inside a feeder-free technique(Kime et al., 2015; Mallon et al., 2006; Thomson et al., 1998). hPS cells cultured on feeder cells or extracellular matrix (e.g. matrigel) show no obvious variations in hypothalamic differentiation with this protocol. Inside the protocol described right here, we sustain and expand hPS cells cultures in 6-well plates with mouse embryonic fibroblasts (MEF). We then seed hPS cells on matrigel-coated plate (feeder-free) to initiate neuron differentiation once every single nicely reaches 9500 confluence. The hPS cells needs to be in uniform shape and active proliferation prior to beginning differentiation. Components (Industrial information for all reagents are supplied in Appendix Tables 1 and two.) hES or iPS cell line Mouse embryonic fibroblasts (MEF) Matrigel DMEM:F12 medium hES medium (see Reagents and Options) mTeSRTM1 total kit TrypLE Express Enzyme Rock inhibitor (Y27632)Author Manuscript Author Manuscript Author Manuscript Author Manuscript0.four Trypan blue Centrifuge (for 15ml tubes) 6-well culture plate (NuncTM Cell-Culture Treated Multidishes) 15ml Falcon tubes (ThermoFisher) 50ml Falcon tubes (ThermoFisher) Falcon5ml round bottom polystyrene tube with cell strainer cap (ThermoFisher) 1.5ml micro centrifuge tubes five CO2, 37 humidified incubator Cell counting chambers (ThermoFisher) Inverted microscope (Olympus) Countless automated cell counter (ThermoFisher) 1. The day just before thawing or splitting hES or iPS cell lines, thaw matrigel (store at -80 ) overnight at 4 .Curr Protoc Hum Genet. Author manuscript; offered in PMC 2017 July 01.Wang et al.Page2.Dilute matrigel in cold DMEM/F12 medium (v/v, 1:50) and maintain the mixture on ice. Then add 1ml diluted matrigel into every properly on 6-well plate and place the plate in 37 incubator. Preserve matrigel and DMEM/F12 medium on ice when preparing the diluted matrigel. We recommend cooling the pipet (10 ml) and recommendations (1ml) at -20 ahead of use.Author Manuscript3.two hours later, the matrigel plate is ready for use.L-selectin/CD62L Protein Storage & Stability Making use of a 20objective lens, gel-like structure ought to be visible on the plate. Matrigel must be evenly distributed around the plate. Prolonged incubation may possibly trigger uneven distribution with the gel (thicker periphery, thinner center).IL-4 Protein Formulation Author Manuscript Author Manuscript Author Manuscript4.PMID:23880095 hES or iPS cells are maintained and expanded in hES medium on MEF plates. When the hES or iPS colonies have reached 9500 confluency on MEF plates (Figure 1A), detach cells by treating with TrypLE express enzyme (0.five ml/well) for four min in 37 incubator, or until morphological changes as shown in Figure 1B occur. For hPS cells cultured on matrigel plates in mTeSR medium, the procedures at this step and following methods will be the same. Plates should be inspected microscopically to assess integrity of person hPSC contacts with neighboring cells.five.Harvest cells by adding three ml hES medium to every single nicely and detach all cells from the plate by pipeting up and down. Transfer cell suspension to 15 ml falcon tube. Spin down cells at 800 rpm for 4 min at RT. Aspirate supernatant and resuspend cell pellet with 2ml hES medium with ten M Rock inhibitor. Use P1000 Gilson pipet to d.
