Single-Cell Profiling of an In Vitro Model of Human Interneuron Development Reveals Temporal Dynamics of Cell Type Production and Maturation.

MGE-like progenitors and neurons are generated in vitro from hESCs(A) Summary of hESC differentiation procedure. (B) D10 immunostaining for SOX2 (red), and MKI67 (green) (C) FOXG1 (red) and NKX2-1(green) at D24. (D,E) GAD67 (green) and SST (red), at D54 and D100, respectively. (F) Percentage of FOXG1 and NKX2-1 expressing cells for 100 ng/ml rmShh + 1 μM purmorphamine (dark green bars) and 50 ng/ml Shh + 0.5 μM purmporphamine (light green bars). 86 ± 2.8% of cells expressed FOXG1 in high Shh and 85 ±1.0% in low Shh cultures. NKX2-1 was expressed in 74.5 ± 9.4% and 65.2 ± 6.2% of cells in high and low Shh-treated cultures, respectively. (G) Quantification of interneuron markers at D54 (dark blue bars): GAD67, 77.2 ± 4.5%; SST, 46.3 ± 6.1%; CALB2, 16.4 ± 2.9%; NR2F2, 37.3 ± 9.6%; 7.2 ± 3.3%. D100 (light blue bars): GAD67, 47.2 ± 7.5%; SST, 24.9 ± 4.5%; CALB2, 12.3 ± 1.7%; NR2F2, 23.4 ± 3.0%; TH, 5.3 ± 1.7%. (H) Percentage SST cells expressing marker at D54 (dark orange bars): CALB2, 21±4.7% ; NR2F2,35.6 ± 9.9%; TH, 5.8 ± 2.1%. D100 (light orange bars): CALB2, 10.3 ± 2.5% ; NR2F2, 29.9 ± 4.9% ; TH, 8.21 ± 3.41%. (I) SOX2 (red) and MKI67 (green) at D10 in DCX-citrine cells. (J) FOXG1 (red) and NKX2-1 (green) expression at D24 in DCX-citrine cells. (K) GAD67 (red) and Citrine (green). (L) MKI67 (red) and Citrine(green). (M) The percentage of citrine-positive cells observed during FACs analysis at each timepoint: D19, 8.7 ± 1.7%; D24, 22.1 ± 1.1% ; D54, 80.7 ± 1.2%; D100, 70.6 ± 6.0% ; D125, 40.8 ± 6.2%. (N) D24 quantification of NKX2-1 (76.2 ± 2.9%, dark grey bar) and FOXG1 (85.7 ± 0.3%, light grey bar) in DCX-citrine (O) Quantification of MAP2 (89 ± 2.3%, dark blue bar), GAD67 (80.5 ± 6.8 %, blue bar), and SST (32.6 ± 3.5%, light blue bar) in DCX-citrine cells at D54. Scale bars = 100 μm. Error bars = s.e.m.

In vitro and in vivo subpopulations are comparable10K cell Citrine− and Citrine+ populations were sorted at D24, D54, D100, and RNAseq was performed. N=3 experiments/timepoint. (A) Telencephalic atterning genes: OTX2, FOXG1, LHX2, SIX3, SOX2, PAX6 and EMX2. Midbrain/hindbrain markers: PAX2, IRX3, EN2 and GBX2. MGE transcription factors: NKX2-1, ASCL1, DLX1, DLX5, LHX8, LHX6, ZEB2, SOX6, abARX, and SATB1. (B) Migration genes: DCX, CXCR7, CXCR4, and ERBB4. Interneuron maturation genes: GAD1, SLC32A1, SST, NPY, CALB2, GRIA2, and GRIA4, CCK, RELN, NOS1, PVALB, and VIP. Values are expressed in Log10 (TPM+1). (C) Representative flow plot of mid-gestation human cortical cells stained for PAX6 and SOX2. Gated sub-populations are named and frequency averaged across four donors (96 to 115 dpc) is shown in parentheses. (D) Hierarchical clustering of marker genes for distinct cortical cell types generated from 100-cell sub-populations from the four cortical specimens as in panel B. (E) Principal component analysis (PCA) of Citrine+ and Citrine− 10,000-cell populations from indicated stages of differentiation and 100-cell populations of primary cortical interneurons (P9-green dots). Primary cortical interneurons are most similar to D54 Citrine+ cells (neurons) in PCA space. For Citrine+ vs Citrine − gene expression levels, *P<0.05, **P<0.01, ***P<0.001, Mann-Whitney (BH FDR correction).

Single cells are distinguishable by transcriptomic signatures(A) PCA of single cells based on high variance genes separates neurons from progenitors along PC1 (Differentiation stage), and separates cells based on their maturation along PC2 (Pseudotime). Top genes loading on PCs 1 and 2 are shown as heat maps along the top and right edges of the PCA plot, respectively. Each box shows the average expression of a particular gene for PCs 1 or 2 for all cells within 1 of 40 evenly spaced bins in PC space. For both D and E, white represents minimal log-transformed expression while red represents maximal expression of a particular gene. P < 10−100 for all genes (B) Example genes showing extensive changes in expression with pseudotime, differentiation stage, or both. Grey dots indicate a cell with FPKM=0.

Single cell transcriptome analysis reveals progenitor and neuron diversity dynamics(A) Iterative WGCNA reveals distinct transcriptomic groups at each time point. Gene expression levels represent centered cluster averages of log2(tpm+1) values. (B-G) Immunofluorescence was used to visualize cell types present in some of the clusters identified. B): ASCL1(red) and NKX2-1(green)-positive cells at D24; C) SP8 (red) and CITRINE (green)-positive cells at D24; D) OLIG2 (red) and ASCL1 (green)-positive cells at D54; E) CORT (red) and CITRINE (green)-positive cells at D54; F) HOPX (red) and CRYAB (green)-positive cells at D100; G) RBFOX3 (red) and CITRINE (green)-positive cells at D100. Arrowheads indicate double-positive cells, Scale bar = 50 μm.

Gene expression differences within cell type groups(A) Assignment to cell type groups based on gene expression: Citrine− cells were assessed based on Z-scores for canonical transcription factors. Z > 0 = positive expression. PAX6+/NKX2-1− cells were designated pTEL (light blue), NKX2-1+/PAX6− cells were either designated as pMGE (ASCL1−, dark blue) or ipMGE (ASCL1+, pink), NKX2-1−/PAX6− cells were designated as progenitors (p, green) or intermediate progenitors (ip, ASCL1+, periwinkle). Citrine+ cells were assigned identity based on gene expression. PAX6+/SP8+ cells were designated nLGE (yellow), ERBB4− neurons were designated nMGE (light orange), SST+/ERBB4+ cells were designated nCTX (dark orange), and OLIG1/OLIG2/PDGFRα+ cells were designated oMGE (maroon). B) Cell type groups identified and classified using WGCNA emerged in distinct temporal patterns. Citrine− cell types: pTEL (light blue), pMGE (dark blue), ipMGE (pink), p (green), and ip (periwinkle). Citrine+ cell types: nLGE (yellow), nMGE (light orange), nCTX (dark orange) and oMGE (maroon). Large circle: >75 cells, Medium circle: 26–75 cells, Small circle: 1–25 cells. Violin plots show gene expression distribution in log2 (tpm+1) for each cluster, scaled relative to the maximal value. (C) Temporal transcriptomic changes within Citrine− cell types. (D) Temporal transcriptomic changes within Citrine+ cell types.

Differentially expressed genes during SST neuron differentiation(A) Analysis of genes differentially expressed between D54 SST clusters reveal the top 21 genes highly enriched in the more immature CORT− clusters, and the top 29 genes enriched in CORT+ clusters are shown. B) Mouse brain expression of select genes found to be differentially regulated during SST cell differentiation.