Transient expression of Ascl1 and Dlx2 (AD) generates GABAergic iN cells from human ES cells. (a) Omission of Myt1l slows the formation of neuronal morphologies, but exogenous Myt1l is not necessary for neuronal induction mediated by Ascl1 and Dlx2. (b) Endogenous MYT1 family members are induced in AD-transduced human ES cells (qRT-PCR results expressed as log2 fold change between AD-transduced cells and human ES cells. Mean ± s.e.m., n = 3 biological replicates). (c) MYT1L protein is present in AD-iN cells 5 weeks postinfection. (d) Lentiviral vectors and timeline for GABAergic iN cell induction. Cells are transduced with three viruses expressing rtTA, a fused Ascl1-T2A-puromycin resistance gene, and a fused Dlx2-hygromycin resistance gene. (e) AD-iN cells express telencephalic marker FOXG1 and GABAergic neuron markers (GABA, DLX proteins, GAD1/2 (GAD67/65)). (f) MAP2-positive cells coexpress GABA, DLX proteins or GAD1/2 (GAD65/67) after 5 weeks of conversion. (g) qRT-PCR analysis in AMD and AD-iN cells (5 WPI) cultured on mouse glia (three biological replicates each), mouse glia, and Ngn2-iN cells (i) or EGFP-sorted AD-iN cells (ii and iii) after coinfection of hES cells with a constitutive EGFP virus. (h) AD- iN cells cocultured with mouse glia for 4 weeks show highly branched MAP2-positive neurons that coimmunostain for CB, CR and SST. Expression of CB and SST or CR and SST is largely nonoverlapping. (i) Quantification of marker overlap. (mean ± s.e.m., n = 3 biological replicates). (j) PV-expressing AD-iN cells were detected. (k) Single-cell qRT-PCR analysis of 64 AD-iN cells for genes indicated on the left. Ct, crossing threshold (g,k). Scale bars, 50um (a,e,h,j).
