Extended Data Fig. 6: Bulk and single-cell RNA sequencing of blastoids and blastocysts.

a, b, Bulk RNA sequencing. a, Transcription factor expression distance map. Differentially expressed transcription factors between the ES cells, TS cells, blastoids, virtual blastoids (see Methods), E3.25 and E3.5 blastocysts (P < 0.05, DESeq negative binomial distribution) were used to generate a non-supervised distance map of transcription factors selected according to the Riken transcription factor database. The scale is log₂. b, Gene ontology (GO) and KEGG pathways. Genes differentially regulated (P < 0.05, DESeq negative binomial distribution) were analysed using DAVID³⁸ and corresponding GO and KEGG pathways are presented. The full lists of genes related to the depicted GO terms are in Supplementary Table 1 sheet 1. c–e, Single-cell sequencing. c, Schematic depicting the origin of the single cells. t-SNE maps of single cells from ES cells, TS cells, blastoids, trophospheres and embryoid bodies. The colours represent the origin of cells assessed by FACS sorting indexes (Supplementary Table 1 sheet 8). d, Clusters of similar cells, as generated by the RACE-ID protocol³⁶. The heat map on the right is the distance map of single cells. The clusters from the t-SNE map and from the heat map are identified using the same colour code. Cells were processed as described in the Methods. e, The Krt18 and Oct4 genes are markers of the blastocyst trophectoderm and ICM compartments, respectively⁴¹, which we confirmed are also valid to mark the compartments of blastoids using immunohistochemistry (KRT8/18 and OCT4) and H2B-RFP⁺ ES cells. This image is representative of three independent blastoid experiments. f, t-SNE map representation of key genes for the trophectoderm, trophoblast differentiation and placental cell types. g, Heat map showing expression of genes of interest: markers of the trophectoderm and ICM compartments by Krt18 and Oct4, as identified in e; markers of pluripotency and differentiation, previously identified in ES cells and blastocyst cells via single-cell sequencing⁴²; genes previously identified as markers of WNT/β-catenin targets regulating a naive state of pluripotency⁴³; transcription factors upregulated in totipotent cells⁴⁴; genes previously identified as differentiation markers in ES cells via single-cell sequencing⁴²; genes previously identified as markers differentiating ES cells from epiblast stem cells³⁰; markers of trophoblast differentiation; and markers of trophectoderm morphogenesis. The scale is log₁₀.