Extended Data Fig. 7: Derivation of stem cells from human blastoids.
From: Blastocyst-like structures generated from human pluripotent stem cells
a, Derivation efficiency for blastoid naive ES cells, blastoid nEND, and blastoid TSCs. Thirty blastoids were used for each. b, Growth curves of blastoid naive ES cells, blastoid nEND and blastoid TSCs (n = 3 biological replicates; mean ± s.d.). c, Representative immunofluorescence images of blastoid naive ES cells (OCT4, KLF17), blastoid nEND (SOX17, FOXA2) and blastoid TSCs (CDH1, TEAD4). Scale bars, 100 μm. d, RT–qPCR results showing the relative expression levels of epiblast (POU5F1, SOX2, KLF17), hypoblast (GATA4, GATA6, SOX17) and trophectoderm (GATA3, TFAP2C) markers in blastoid naive ES cells, blastoid nEND and blastoid TSCs (n = 3 biological replicates; mean ± s.d.). e, t-SNE of naive ES cells, blastoid naive ES cells, blastoid nEND and blastoid TSCs. Plots are coloured according to sample (top left), SOX2 expression (top right), GATA6 expression (bottom left) and GATA3 expression (bottom right). f, Correlation analysis of blastoid naive ES cells, blastoid nEND and blastoid TSCs compared to published transcriptomes19,20,29,31,32,33,34 from established human ES cells, human TSCs and nEND. Each indicated sample represents merged cells (scRNA-seq datasets) or replicates (bulk RNA-seq datasets). The gene sets used in this analysis were defined in Zhou et al.31. g, Left, representative phase-contrast images showing manual cutting (red dashed line) and outgrowths (days 1 and 7) during the derivation of blastoid TSCs. Right, a representative phase-contrast image showing the morphology of blastoid TSCs (passage 5) derived from manually cut TLCs. Scale bars, 100 μm. h, A representative immunofluorescence image showing that human TSCs derived from cut TLCs express GATA3. Scale bar, 100 μm. i, Schematic showing the strategy to derive blastoid nEND and blastoid TSCs from OCT4-GFP− and SUSD2− double-negative single cells (DNSCs) isolated from human blastoids. j, FACS gating and sorting of OCT4-GFP− and SUSD2− DNSCs from dissociated human blastoids. k, l, Representative phase-contrast and immunofluorescence images showing that blastoid TSCs (k) and blastoid nEND (l) derived from FACS-sorted DNSCs express GATA3 and GATA6, respectively. Scale bars, 100 μm. m–o, Representative phase-contrast (left) and immunofluorescence (right) images showing that monomeric Kusabira-Orange (mKO)-labelled blastoid TSCs (m), blastoid nEND (n) and blastoid naive ES cells (o) contribute to the extraembryonic ectoderm, visceral endoderm and epiblast compartments of day-6 IVC mouse embryos after blastocyst injections. Higher-magnification images of the boxed areas are shown on the right. Scale bars, 100 μm. Images in c, g, h, k–o are representative of at least three independent experiments.
