Human cortical progenitors are diverse and intermixed during development. (a) Model of the progenitor compartment shows a mixture of ventricular radial glial cells (vRG-light blue), outer RGs (oRGs-purple), intermediate progenitors (IPCs-orange) and other mixed cortical cell types (gray). Known markers for each cell type are shown below. Note RGs identified by antibody staining are called SP (SOX⁺PAX⁺EOMES⁻ -dark blue nuclei), and IPCs are called SPE (SOX⁺PAX⁺EOMES⁺ -dark orange nuclei). (b) Immunocytochemistry images of 19 PCW germinal zones. Top left: Low magnification images stitched together (left), or individual micrograph (right) of the VZ, iSZ, and oSZ stained by DAPI, EOMES, PAX6, and SOX2 with the colors indicated. Scale bars are 100 µm. High magnification micrograph of oSZ region (middle) and VZ/iSZ region (bottom) show SP (arrow with filled arrowhead) and SPE (arrowhead) cells. Note many cells the VZ, iSZ, and oSZ lack progenitor markers and are unknown cell types (arrows with open arrowhead). Scale bars are 50 µm. Tables describing human germinal zone regions and symbols (c), and common human cortical progenitors, their germinal zone locations, and their flow cytometry (FACS) phenotypes (d).

High quality and yield RNA from fixed and sorted hESCs. (a,b) Quantification of Bioanalyzer RNA integrity numbers (RINs) and total recovered RNA purified from live or fixed cells, and RNA harvested using either the Qiagen microRNeasy Kit (RLT), or using a modified fixed-cell method with protease lysis and reverse crosslinking (PLRC). Sample colors are indicated in b. (a) RINs from 10,000, 1,000, and 100 sorted cells from 2–3 independent experiments. The biological replicates are indicated by the numbers above the bars. (b) Yield from column purified total RNA from 10,000 and 1,000 H1 hESCs. We diluted or concentrated samples such that 66 cell equivalents were loaded per lane of the Bioanalyzer. Data are from 12 biological replicates, except for the 1,000-cell PLRC samples which have 10 replicates, processed over three independent experiments. Barplots in a and b show mean ± SD. (c) Comparison of expression level of 82 genes by qRT-PCR of 10,000 live or fixed hESC samples processed with PLRC. Data are from n = 7–8 biological replicates across two independent experiments, and only genes that were detected in at least 3 replicates from both conditions were included.

FRISCR allows purification and amplification of mRNA from single fixed hESCs that is similar to live cells. (a) Schematic description of steps in the FRISCR method. Asterisks mark steps with stopping points. (b–f) We processed live or fixed single sorted H1 hESCs by standard Triton-X100 lysis (TL) or FRISCR, and sequenced after making SmartSeq2 cDNA libraries. (b) Representative Bioanalyzer traces of SmartSeq2 amplified cDNA are shown from the four experimental conditions indicated. (c) Alignment statistics for HiSeq RNA-Seq data from cells processed by the four experimental conditions. * P < 0.05 and ** P < 0.001 by one-way ANOVA followed by Tukey’s post-hoc test, and plots show mean ± SD. Data in (d–f) are from n = 7–15 biological replicates as shown, processed over two independent days of experiments. Analysis carried out in (d–f) is on libraries subsampled to 5 million reads. (d) Normalized average read counts per experimental preparation are shown across the percentile predicted transcript length (3′ to 5′). Only transcripts not alternatively spliced, and with TPM > 1 in at least 30 samples were selected. Solid line is average and shading illustrates SD. (e) Heatmap with hierarchical clustering for single hES cells showing pairwise Spearman correlation of all expressed genes. Color bars indicate experimental replicate and experimental condition. (f) Table showing the number of differentially expressed genes between hESCs from the different conditions calculated by DESeq with adjusted P < .01.

FRISCR allows profiling of primary human cortical progenitors. Color scheme throughout figure is shown in b. (a) FACS plots show gating strategy and frequency of gated cells compared to total cortical cells from three independent experiments. Left plot includes only G₀-G₁ singlet cortical cells (gated with DAPI) and right plot shows only the SOX2⁺PAX6⁺ cells gated on the left plot. (b) Barplots show the frequency of indicated genes detected per cell. Only cells with >10,000 mapped reads, >20% mRNA mapping, and GAPDH expression (n = 157 SP and n = 29 SPE cells) were included in this analysis.

Identification of human cortical progenitor cell types with FRISCR. (a) Module eigengene values derived from WGCNA analysis enrichment in cells. (b) Heatmap showing the Spearman correlation from individual cells by genes identified from the 5 WGCNA modules. (c) Heatmap shows correlation of single cell-derived WGCNA module eigengenes with 15 PCW (left) and 21 PCW (right) gene expression data from the BrainSpan Atlas of the Developing Human Brain. Abbreviations are: subgranular zone (SG), subplate (SP), intermediate zone (IZ), outer subventricular zone (oSZ), inner subventricular zone (iSZ), and VZ (ventricular zone). (d) Heatmaps showing genes both differentially expressed between cell clusters C and D (oRGs) versus cluster E (vRGs) from the FRISCR-prepared single cells (data shown on left), as well as differentially expressed between oSZ and VZ regions of 21 PCW human tissues (data shown on right). Common progenitor markers are included at the top of the heatmap for reference. All heat maps show normalized expression with red indicating high expression, blue indicating low expression; colorbars for a, b and the FRISCR single cell plot in d are indicated in the bottom of a.

Confirmation of vRG and oRG markers. (a) Grayscale (left) or merged (right) micrographs of germinal zone showing co-staining of SOX2 (magenta) and ANXA1, CRYAB or HOPX (green). Tissue is 16 PCW cortex, scales are 100 µm. Abbreviations: marginal zone (MZ), cortical plate (CP), subplate/intermediate zone (SP/IZ), outer subventricular zone (oSZ), inner subventricular zone (iSZ), and ventricular zone (VZ). (b) HOPX expression in the VZ; scale is 50 µm. Co-expression with SOX2 (arrowhead), SOX2-only cells (arrow), and HOPX⁺ apical endfoot are indicated (asterisk). (c) A mitotic cell co-stained with SOX2, HOPX and phospho-VIM shows basal cell process (asterisk) and cell body (arrowhead); scale is 20 µm. (d) Full cortical thickness (left), and insets (right) from germinal zone showing co-staining of SOX2 (magenta) and HOPX (green). Scales are 100 µm. (e) Quantitation of SOX2⁺HOPX⁺ or SOX2⁺HOPX⁻ cells in oSZ that are Ki67⁺. (f) Quantification of SOX2⁺ANXA1⁺ or SOX2⁺ANXA1⁻ cells in VZ that are Ki67⁺. Data is mean ± SD for (e) five or (f) six human tissues at 14–19 PCW; ** P < 0.01 by Wilcoxon rank sum test. Diagrams show the percentage overlap of SOX2 and HOPX in oSZ cells (g), and of SOX2 and ANXA1 in VZ cells (h). Data in g and h from n = 9 and n = 10 human tissues between 14 and 19 PCW, respectively. 200 to 500 cells evaluated per brain. (i) Summary of oRG and vRG cell type markers identified in this study and overlap with existing RG and IPC markers.