Fig 2: LIF signaling mediators are present in the dorsal, but not in the ventral, cortex, and LIF signaling perturbation impacts the ratio of oRGs and IPCs in cortical tissue(A) During GW17 and GW18, LIFR is present in the oSVZ on NESTIN+ RG in the dorsal neocortex, but not in the ventral GE. LIF co-labels DCX+ neurons in the CP, but not in the GE. LAMININ+ vascular cells are in both cortex and GE.(B) Reanalysis of human neural tissue2 identified highest LIFR expression in the neocortex.(C) LIFR is present in HOPX+ oRG cells in the oSVZ (white arrowheads) of GW19 cortical tissue.(D) LIF expression overlaps with NEUN+ excitatory neurons that are CTIP2+ (yellow arrowheads) or SATB2+ (white arrowheads) during GW15–19.(E) Cortical tissue was sliced, cultured at air liquid interface (ALI), and treated with LIF, SC144, or ruxolitinib for 1 week.(F) LIF inhibition decreases HOPX+ oRG cells, whereas LIF addition reduces EOMES+ IPCs (one-way ANOVA with multiple comparisons, HOPX: vehicle vs. SC144: *p < 0.018, vehicle vs. Rux: p = 0.0539; EOMES: vehicle vs. LIF: *p < 0.0252, vehicle vs. SC144: p = 0.6552, vehicle vs. Rux: p = 0.0576, n = 7 biological samples). Data are represented as mean ± SD.

Fig 3: CGE-like INs are present in dorsal forebrain organoids and oRG-derived cultures and increase after LIF treatment(A) Proportions of IN subtypes in forebrain organoids captured in scRNA-seq.(B) Correlations between 3 month organoid30 and primary GE datasets.4(C) Correlations between 6 month organoids30 and primary GE datasets.4(D) UMAP of IN subtypes and proportions in organoids treated with LIF during peak oRG expansion.(E) In situ hybridization of PCDH9 (unpaired t test, ****p < 0.0001, n = 3 biological samples), SCGN (unpaired t test, **p < 0.0014, n = 3 biological samples), and DLX5 (unpaired t test, p = 0.0624, n = 3 biological samples) in the oSVZ of GW18 and GW19 cortical slices treated with and without LIF (white arrowheads). Data are represented as mean ± SD.(F) UMAP of FACS-isolated RGs at day 0 and progeny after 4 weeks in culture.(G) Feature plots of IPC_IN marker BEST3+, IN marker DLX5+, and MGE markers LHX6 and NKX2–1.(H) Feature plots of CGE markers SCGN, PROX1, and SP8.(I) Proportions of IN subtypes derived from FACS-isolated oRG cells captured in scRNA-seq.(J) GW16 FACS-isolated oRG cells cultured under control and LIF-treated conditions (DLX5: ****p < 0.0001; NEUN ****p < 0.0001, n = 4 biological samples). Data are represented as mean ± SD.

Fig 4: LIF signaling increases the number of oRG cells in forebrain organoids(A) Protocol for dorsal forebrain organoid induction.(B) LIF treatment during weeks 6–8 increases the number of HOPX+ (unpaired t test: week 10: *p = 0.024, n = 5 differentiation batches from 3 lines) and GFAP+ (unpaired t test: *p < 0.04, n = 5 batches from 3 lines) oRG cells in week 10 organoids. No changes in SOX2+ or pHH3+ cells were observed (unpaired student t tests; p > 0.05 for all comparisons). Data are represented as mean ± SD.(C) Continuous LIF treatment increases SOX2+ (unpaired t test for control vs. LIF, week 10: *p < 0.0185, n = 3 batches from 3 lines; week 15: ****p < 0.0001,n = 4 batches from 3 lines), pHH3+ (unpaired t test, week 10: **p < 0.0034, n = 5 batches from 3 lines, week 15: **p < 0.0054, n = 2 batches from 2 lines), HOPX+ (unpaired t test: week 10: ****p < 0.0001, n = 5 batches from 3 lines; week 15: ****p < 0.0001, n = 3 batches from 3 lines) and GFAP+ oRG cells (unpaired t test, week 10: ****p < 0.0001, n = 3 batches from 3 lines; week 15: **p < 0.0018, n = 2 batches from 2 lines). Data are represented as mean ± SD.(D) Uniform Manifold Approximation and Projection (UMAP) plots separated by cluster and treatment group.(E) Feature plots showing the expression of cortical markers FOXG1, SOX2, HOPX, GFAP, EOMES, and NEUROD6.