Fig. 1: LTR5Hs activity contributes to the blastoid formation potential of hnPSCs.
From: A human-specific regulatory mechanism revealed in a pre-implantation model
a, Representative bright-field image of wild-type blastoids (n = 3 biological replicates). Scale bar, 400 µm. b, Representative confocal images of a wild-type blastoid immunostained with HERVK envelope (env) antibodies and DAPI (n = 4 biological replicates). Scale bar, 100 µm. c, Schematic of nontarg-CARGO and LTR5Hs-CARGO hnPSCs and the generation of blastoids. The cartoon summarizes how LTR5Hs-CARGO promotes H3K9me3 deposition and LTR5Hs repression (right). crRNA, CRISPR RNA; LPA, media containing 1-oleoyl lysophosphatidic acid sodium salt; PALLY, media containing PD0325901, A83-01, leukemia inhibitory factor, LPA and Y-27632; tracRNA, trans-activating crRNA. The silhouette of the human was created in BioRender. Fueyo, R. (2025) https://BioRender.com/tmgd0pn. d, Blastoid-forming potential of hnPSC depends on LTR5Hs activity (β-regression, two-sided log-likelihood test = 2.39 × 10−13). Blastoid formation was assessed in 23 LTR5Hs-CARGO (n = 3 biological replicates) and 24 nontarg-CARGO (n = 2 biological replicates) clonal cell lines. Plotted is the proportion of blastoids per well versus Taqman LTR5Hs RNA levels normalized to RPL13A RNA levels. The purple line represents the LTR5Hs-CARGO regression line. The purple circles with black outlines indicate clones used for scRNA-seq experiments. e, Bright-field images of structures collected upon LTR5Hs high repression (left; dark spheres), medium repression (middle; dark spheres/blastoid-like structures) and no LTR5Hs repression (right; blastoid-like structures). Images are representative of 23 LTR5Hs-CARGO (n = 3 biological replicates) and 24 nontarg-CARGO (n = 2 biological replicates) clonal cell lines. Scale bar, 400 µm. f, PCA of the bulk RNA-seq transcriptomes obtained from nontarg-CARGO and LTR5Hs-CARGO clonal cell lines with high or medium expression levels (total of 11 clonal cell lines in biological duplicates or triplicates). g, Representative images of blastoid (left; n = 11 from three biological replicates) or dark sphere (right; n = 13 from three biological replicates) immunostained with cleaved CASP3 (red) and DAPI (blue). Scale bars, 100 µm. h, Quantification of cleaved CASP3 immunostainings described in panel g. Red diamonds display the median values. Significance was determined by an unpaired, two-tailed Student’s t-test: P = 8.5 × 10−7.
