Fig 2: Chimpanzee cerebral organoids recapitulate cortex development.(A) Bright-field image showing a representative chimpanzee organoid (Sandra A, left) and a cryosection from a chimpanzee organoid (PR818-5) immunostained for PAX6 (magenta) and Ctip2 (green) combined with DAPI staining (blue) (right) at day 52. Scale bars, 200 µm. (B, C) Cryosections of cortical regions from chimpanzee (Sandra A) and human (SC102A-1) organoids at day 52 immunostained for PAX6 (magenta), Ctip2 (B, green) and TBR2 (C, green), without (B) and with (C) DAPI staining (blue). Asterisks, ventricular lumen; scale bars, 50 µm. (D) Cartoon showing NSPC types (APs, BPs) and neurons enriched in zones within the neocortex at mid-neurogenesis. CP, cortical plate; N, neuron. (E) Heatmap showing normalized correlation (Z-score) of single-cell transcriptomes from chimpanzee cerebral organoid cortex with bulk RNA-seq data from laser-microdissected zones (Fietz et al., 2012) from 13 wpc human neocortex. CP, cortical plate. (F) Scatterplot showing NSPC and neuronal signature scores derived from analysis of fetal cerebral cortex single-cell transcriptomes (Figure 1—figure supplement 1) calculated for each chimpanzee cerebral organoid cortical cell. (G) Heatmap showing expression of AP, BP, and neuron (N) marker genes. Each column represents a single cell, each row a gene. Cell type and maximum correlation to bulk RNA-seq data from cortical zones are shown in the top sidebar. APs and BPs were sub-classified based on G1 (light grey) or S-G2-M (dark grey) phases of the cell cycle. (H) Lineage network based on pairwise correlations between chimpanzee cerebral organoid cortical cells reveals a structured topology where VZ-APs connect to cortical plate (CP) neurons (N) through SVZ-BPs. Cells are coloured based on cortical zone (top left) or cell type assignment (bottom left). APs, BPs, and neurons were classified based on maximum correlation with single-cell transcriptomes from the human fetal neocortex. Expression of markers PAX6, TBR2, and MYT1L are shown to the right.DOI: http://dx.doi.org/10.7554/eLife.18683.00310.7554/eLife.18683.004Figure 1—source data 1.Processed single-cell RNA-seq data for chimpanzee cells.*.txt file containing processed chimpanzee single-cell RNA-seq data (344 single cells) in log2(FPKM) with genes in columns and cells in rows. The first 7 columns contain metadata for each cell: cortex: assignment of cell to cortex (1) or to other regions within organoid(0); tSNE_1: tSNE1 loading for each cell; tSNE_2: tSNE2 loading for each cell; PC1: PC1 loading for each cell; PC2: PC2 loading for each cell; species: species of origin for each cell; cell_id: unique ID for each cell, with information about the experiment and the age of the organoid of origin for each cell.DOI: http://dx.doi.org/10.7554/eLife.18683.00410.7554/eLife.18683.005Figure 1—source data 2.Genes describing cell populations in the chimpanzee organoids.List of genes identified by PCA on all chimpanzee organoid single-cell transcriptomes as being most informative for defining cell populations.DOI: http://dx.doi.org/10.7554/eLife.18683.005

Fig 3: EOMES protein is not detectable during several stages of murine fibroblast reprogramming. (A) Schematic illustration of the Eomes alleles used in (B–H). MEFs carry a GFP knock-in at the Eomes GFP/+ locus (upper and middle panel) allowing for quantification of Eomes GFP/+ positive cells by putative GFP expression19. A lentiviral 3-factor (OKS) reprogramming construct includes a tdTomato reporter to track expression of pluripotency markers during reprograming20. (B) FACS-based quantification of GFP- and tdTomato-positive cells during reprogramming at the indicated days. GFP-positive cells arise only late, after the reprogramming process indicating differentiation of formed iPSCs (day 21). (C) Corresponding phase contrast images (upper panel) and fluorescence images of the MEF cultures during reprogramming (red, middel panel) and the Eomes GFP/+ reporter signal (green, lower panel). (D) Scheme for spontaneous in vitro differentiation of WT-iPSCs towards embryoid bodies representing early germ layer formation mirrored in the three colors. (E) Comparison of marker gene expression for mesendoderm expression peaks during reprogramming of murine fibroblasts in comparison to iPSCs and cells differentiated in EBs. All mRNA levels are expressed relative to the housekeeping gene Hmbs and values have been normalized to day 10 reprogramming cultures, which have been set to 1 to illustrate fold induction. (F) Scheme for in vitro differentiation of Eomes GFP/+ reporter iPSCs isolated from (A) in embryoid bodies toward mesendoderm using high doses of Activin A. Germ layer formation is mirrored in the three colors, while high doses of Activin A favor endoderm formation (green). (G) Eomes GFP/+ reporter iPSCs are differentiated towards mesendoderm. Expression of GFP validates the functionality of the Eomes GFP/+ reporter. (H) FACS-based quantification of independent experiments from (G).

Fig 4: EGFR+ tRGs generate EGFR+ PyN-IPCs and EGFR+ bMIPCs. A, F Coronal brain sections (60 µm thick) stained for DAPI. B EOMES, EGFR and OLIG2 triple immunostained sections. Note EOMES+ cells only in the cortex whereas EGFR+ cells in both the cortex and LGE. C Higher magnification images showed EGFR+EOMES+ PyN-IPCs in the cortical ISVZ and EGFR+OLIG2+ bMIPCs (arrows) in the cortical IFL. D, E Numbers of EGFR+EOMES+ cells and EGFR+OLIG2+ cells in the VZ, ISVZ, IFL and OSVZ of the human cortex at GW18 (D) and GW23 (E). G Some EGFR+EOMES+ PyN-IPCs in the cortical ISVZ and a large number of EGFR+OLIG2+ bMIPCs with a large soma in the cortical IFL were shown. Note that EOMES+ cells did not express OLIG2. Also note that EOMES was expressed in some EGFR+ SNP like cells (had an apical process extended to the ventricular surface, arrowheads in C and G). H Schematic of human cortical tRGs and their direct progeny. Initially, EGFR+ASCL1+ tRGs produce EGFR+ASCL1+EOMES+ PyN-IPCs, but later they produce bMIPCs that express higher levels of EGFR, ASCL1 and OLIG2. We propose that EGFR+ PyN-IPCs differentiate into upper layer PyNs.

Fig 5: Upregulation of primitive streak and mesendoderm markers during reprogramming of human somatic cells of fibroblast and keratinocyte origin, but absence of EOMES protein. (A) Schematic overview of the reprogramming experiment for the somatic cells of ectodermal –keratinocytes, and mesodermal origin – fibroblasts. (B) Expression patterns of indicated genes during reprogramming of human keratinocytes (upper row) and human fibroblasts (lower row). All mRNA levels are expressed relative to the housekeeping gene HMBS and values have been normalized to iPSCs, which have been set to 1 to illustrate fold induction. (C) Schematic overview of the experimental setup of mesendoderm differentiation. (D) Comparison of marker gene expression for definitive endoderm (CER1) and mesendoderm/primitive streak (LHX1, EOMES) are lower in magnitude for the reprogrammed cells compared to the differentiated cells. (E,F) Protein expression of EOMES and NANOG during the time course of human keratinocytes (E) and human fibroblasts (F) reprogramming. The scale bar represents 100 µm for all images. (G) Western Blot analysis EOMES and NANOG protein expression during reprogramming of fibroblasts. Actin was used as loading control. Lane 1: human foreskin fibroblasts (HFFs) protein lysate, lane 2–6 and 9–15: consecutive days of HFFs reprogramming at indicated time points (d = day), lane 7: fibroblast derived iPSCs, lane 8: mesendodermal differentiation as a positive EOMES control. (H) Mesendodermal differentiation of iPSCs shows a positive signal for EOMES protein.