Compound e

PSCs were first differentiated to neuroepithelia (NE) in a neural medium consisting of DMEM/F12, N2 supplement, and non-essential amino acids in the presence of SB431542 (2 μM), DMH1 (2 μM), and CHIR99021 (3 μM, all from Stemgent) for 7 days. At d8, NE were treated with the addition of retinoic acid (RA, 0.1 μM) and purmorphamine (Pur, 0.5 μM) for 7 days for MN induction. At d14, MN progenitors were isolated and expanded as floating clusters in suspension in the same medium but without SB431542, DMH1 and CHIR99021 for an additional 7 days before plating on the laminin substrate for generating mature neurons. To generate synchronized post-mitotic neurons, the cultures were treated from d18-21 with compound E (0.1 μM, from Enzo) to block cell proliferation.

Leukemic cells from Vav1^–/– mice were cocultured with feeder layers of OP9 stromal cells overexpressing either GFP (OP9-GFP) or the Delta-like 1 protein (OP9-DL1) in MEMα supplemented with 20% fetal calf serum. OP9 cells were provided by Dr. M.L. Toribio. When indicated, cells were treated with either Compound E (200 nM, Enzo) or vehicle alone (DMSO) and collected at the indicated time points. For in vivo experiments, 500,000 cells were intravenously injected into recipient WT mice.

Control hiPSC motor neuron and cortical neuron progenitors were derived as described [71 (link)] from multiple donors (Table 2). These cells were cultured on Matrigel (Corning) coated plates in base medium, comprised of 50% NeuroBasal (Gibco), 50% advanced DMEM (Gibco), supplemented with B27 and N2 (gibco), 100 μg/ml Pen-Strep (Gibco), and 2 mM L-alanyl-L-glutamine dipeptide (Gibco). For expansion of progenitors, FGF (20 ng/ml) (Gibco) was added to base medium. Differentiation of MN progenitors was achieved using base medium with compound E (Enzo) (0.1 μm) and the growth factors BDNF (10 ng/ml) and GDNF (10 ng/ml) unless stated. Astrocytes were generated from iPSC using a modified protocol described in Hall and colleagues [71 (link)]. Derived astrocyte progenitors were cultured in neuronal base medium with FGF (20 ng/ml) (Gibco) and EGF (20 ng/ml) (Thermo). For differentiation, astrocytes were cultured in base medium without growth factor supplements. All cells were cultured with 5% CO2 in humidified atmosphere at 37°C (Table 3).

iPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA (Life Technologies, 0.5mM). All cell cultures were maintained at 37°C and 5% carbon dioxide. MN differentiation was performed using an adapted version of a previously published protocol (Hall et al., 2017). Briefly, iPSCs were first differentiated from neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 Glutamax, Neurobasal, L- Glutamine, N2 supplement, non-essential amino acids, B27 supplement, β-mercaptoethanol (all from Life Technologies) and insulin (Sigma). Treatment with small molecules from day 0 to day 7 was as follows: 1 μM Dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience), and 3 μM CHIR99021 (Miltenyi Biotec). On day 8, the neuroepithelial layer was enzymatically dissociated using dispase (GIBCO, 1 mg/mL), plated onto laminin coated plates and next patterned for 7 days with 0.5 μM retinoic acid and 1μM Purmorphamine. At day 14, MN precursors were treated with 0.1μM Purmorphamine for a further 4 days before being terminally differentiated in 0.1 μM Compound E (Enzo Life Sciences) to promote cell cycle exit.

MN differentiation was carried out using an adapted version of a previously published protocol^{19 (link)}. Briefly, iPSCs were first differentiated to neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 Glutamax, Neurobasal, L-Glutamine, N2 supplement, non-essential amino acids, B27 supplement, β-mercaptoethanol (all from Life Technologies) and insulin (Sigma). Treatment with small molecules from day 0–7 was as follows: 1 µM Dorsomorphin (Millipore), 2 µM SB431542 (Tocris Bioscience), and 3.3 µM CHIR99021 (Miltenyi Biotec). At day 8, the neuroepithelial layer was enzymatically dissociated using dispase (GIBCO, 1 mg ml⁻¹), plated onto laminin coated plates and next patterned for 7 days with 0.5 µM retinoic acid and 1 µM Purmorphamine. At day 14 spinal cord MN precursors were treated with 0.1 µM Purmorphamine for a further 4 days before being terminally differentiated for >10 days in 0.1 µM Compound E (Enzo Life Sciences) to promote cell-cycle exit. At relevant timepoints, cells were harvested for RNA extraction or fixed in 4% paraformaldehyde for immunolabelling.