After 10 days in culture post-differentiation (Day 25; Fig. 1b), MNACs contained ~ 5% neurons expressing tubulin beta-3 chain (TUBB3; Supplementary Fig. 3a and b) and ~ 95% astrocytes expressing astrocyte markers (unpublished data). A large proportion of neurons (78–84%) co-expressed the motor neuron markers ISL LIM homeobox 1/2 (ISL1/2) and non-phosphorylated neurofilament-H (SMI32), representing a limb innervating subtype that are highly susceptible to ALS [2]. Here, ISL1 is linked to amyotrophic lateral sclerosis.