small molecule differentiated mESC replicate 1
Motor neuron differentiation for smMN cells was performed as previously described [Wichterle 2002; Crawford & Roelink 2007; Wichterle & Peljto 2008; Tan et al. 2016]. On Day 0 mESCs (C57/Bl6 carrying Hb9::GFP transgene) were dissociated into single cells with 0.05% trypsin (Invitrogen) and seeded at 5  104 cells per ml in ANDFK medium (Advanced DMEM/F12:Neurobasal (1:1), 10% Knockout Serum Replacement (vol/vol), Pen/Strep, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol) to initiate formation of embryoid bodies EBs. Medium was exchanged on day 2 of differentiation and motor neuron identity was induced by supplementing media with 1 M all-trans retinoic acid (RA) for caudlization and 500nM Sonic Hedgehog agonist (SAG) for ventralization to induce rostal spinal motor neuron progenitor identity. On day 4 of differentiation media was exchanged again and DAPT was added to induce synchronized exit of motor neuron progenitors from the cell cycle to become postmitotic motor neurons on Day 6. On Day 6 of differentiation, EBs were dissociated into single cells with trypsin and GFP positive motor neurons were sorted by FACS. A minimum of 100k neurons were used for each experiment. All differentiations were performed in triplicate from the same original clone of mESCs. DNA sequencing was performed on mESC cells and smMN cells that were temporally matched.
SUB15293812
smES1