mTOR activity paces human blastocyst stage developmental progression

Abstract

Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.

Keywords: blastoid; development; diapause; dormancy; human; mTOR; pluripotent stem cells.

Graphic abstract

Figure 1. Similar pattern and sensing of mTOR pathway activity in human and mouse pre-implantation embryos

(A) Immunofluorescence (IF) staining of a human blastocyst at E6 for the mTOR downstream target phospho-S6, the EPI marker NANOG, and the TE marker CDX2. Scale bars, 50 μm. (B) IF staining of a mouse blastocyst at E4.5 for pS6 and the ICM marker OCT4. Mouse and human embryos display a similar pattern of pS6 staining. Scale bars, 50 μm. (C) Human pre-implantation embryos cultured in 1.7 nM IGF from 2-cell until blastocyst stage yield a significantly higher percentage of blastocysts compared with control embryos. As control, data from all thawed blastocysts in the assay year were used. (D) IGF treatment increases the number of ICM cells in human pre-implantation embryos. n = 6 embryos were used. Solid lines indicate median, and dashed lines mark interquartile range. (E) IGF treatment increases the number of ICM cells in mouse pre-implantation embryos (at 1.7 nM). n = 6, 10, and 8 for control, 1.7, and 17 nM, respectively. Solid lines indicate median, and dashed lines mark interquartile range. Results in (D) and (E) are not statistically significant per one-way ANOVA and show tendency. See also Figure S1.

Figure 2. mTOR activity regulates TE development

(A) IF staining for the EPI (SOX2), HYPO (GATA4), and TE markers (GATA3) on control blastoids (day 4) and mTORi-treated (from day 2 to day 4) blastoids. TE, trophectoderm; EPI, epiblast; HYPO, hypoblast. (B) Bright-field images of control and mTORi blastoids. Scale bars, 200 μm. (C) Quantification of structure sizes of control and mTORi blastoids. Dashed lines indicate median, and dotted lines mark interquartile range. Statistical test is unpaired t test. (D) Fluorescence-activated cell sorting (FACS) plots of CCR7 expression (pTE marker) in control and mTORi blastoids. (E) Mean intensity quantifications of NR2F2 staining of control and mTORiblastoids on indicated days. Horizontal lines indicate median, boxes span first to third quartile, and error bars span min-to-max values. Statistical test is one-way ANOVA with multiple testing correction. (F) Quantification of attachment capability of control and mTORi blastoids onto endometrial cells. Column heights show mean, and error bars indicate standard deviation. Statistical test is one-way ANOVA with multiple testing correction. See also Figure S2.

Figure 3. Developmental delay of human blastoids in extended pre-implantation culture

(A) Bright-field images of blastoids cultured in control conditions or treated with the mTOR inhibitor RapaLink-1. Indicated time points (days 1–8) denote culture time after blastoid formation (starting at day 4 of PSC aggregation). (B) Bright-field images of control and mTORi-treated human blastoids in comparison with an untreated blastoid in extended culture. Right panel shows quantification of cell number in the ICM and TE based on IF stainings. Dashed lines indicate median, and dotted lines mark interquartile range. (C) Bright-field images of an mTORi-treated mouse blastocyst and an in vivo-diapaused mouse blastocyst in comparison with an untreated blastocyst in extended culture. Right panel shows quantification of cell number in the ICM and TE based on IF stainings. Dashed lines indicate median, and dotted lines mark interquartile range. Scale bar, 100 μm. (D) Longitudinal morphological scoring of control and mTORi-treated blastoids at the indicated RapaLink-1 concentrations. n, number of blastoids. (E) IF staining of the EPI markers OCT4 and SOX2, TE marker GATA3, and HYPO markers GATA4 and SOX17. Scale bar, 100 μm. (F) Gene set enrichment analysis (GSEA) of mTORi-treated human blastoids and mouse blastocysts. The protein set: diapause comprises 179 proteins that are significantly upregulated in in vivo mouse diapause. (G) Heatmap of 113 proteins upregulated in diapause and detected in proteomes of mTORi-treated mouse blastocysts and human blastoids. 41 proteins are commonly upregulated in all three dormancy conditions. See also Figures S2 and S3.

Figure 4. mTORi-treated blastoids maintain the cell lineages of the blastocyst

(A) UMAP plot generated from scRNA-seq data. Cells are color-coded based on their respective sample and lineage. Plots on the right show the expression levels of the indicated lineage markers. TE, trophectoderm; EPI, epiblast; HYPO, hypoblast. (B) Left, UMAP of integrated human embryonic reference data, sourced from seven human embryonic datasets ranging from pre-implantation to post-implantation stages (see STAR Methods). Colors represent cell type annotations as outlined in the publications. Right, data points are colored based on the developmental stage. (C) Projection of control (left) and mTORi blastoid cells (right) onto reference datasets. (D) Percentages of the indicated cell types in control and mTORi (day 3) blastoids. (E) Dot plot showing the expression of lineage-specific marker genes in control and mTORi-treated (day 3) blastoids. Dot size corresponds to the percentage of cells in the sample expressing the gene, while color intensity reflects the scaled average expression level. See also Figures S4 and S5.

Figure 5. mTORi places human peri-implantation PSCs in a reversible dormant state

(A) Representative bright-field images of naive, naive-primed intermediate, and primed pluripotent human cells before, during, and after mTORi treatment as compared with mouse ESCs. Scale bar, 500 μm. (B) Percentage of annexin V-positive apoptotic cells during the course of mTORi treatment in RSeT and PXGL culture. Statistical test is one-way ANOVA with multiple testing correction. (C) Proliferation curves of human ESCs in PXGL culture treated with the catalytic mTOR inhibitors RapaLink-1 and INK128. Proliferation rate of mouse ESCs cultured in INK128 is shown in comparison. (D) Cell-cycle distribution of mTORi-treated human and mouse cells, as determined by EdU incorporation and DNA content. Differences are non-significant per two-way ANOVA. Schematic at the bottom shows the relative durations of the cell cycle and each phase, based on measurements of proliferation and EdU integration (Figures 3C and 3D). (E and F) Principal component analysis of top 500 variable proteins in the indicated conditions in PXGL (E) and RSeT (F) culture. (G and H) Gene ontology analysis of differentially expressed proteins in mTORi-treated and released PXGL (G) and RSeT (H) cells. Unique terms are shown to reduce redundancy. Complete lists are provided in Table S3. (I and J) K-means clustering of downregulated (left) or upregulated (right) genes during pausing shows reversibility of expression pattern in PXGL (I) and RSeT (J) cells. Scaled log₂(LFQ+1.1) values are shown. Top two most enriched clusters for PXGL and RseT conditions are shown. LFQ, label-free quantification. See also Figure S6.

Figure 6. Sustained differentiation potential in post-implantation culture of mTORi-treated blastoids

(A) Reactivation and extended post-implantation culture after dormancy. Blastoids were treated with mTORi for 3 days, then mTORi was withdrawn and blastoids were cultured on matrigel-coated plates. After 4 days, cells were stained for the EPImarker SOX2, the TE marker GATA3, and the trophoblast differentiation marker CGB. Scale bars, 100 μm. (B) Pregnancy test strips detecting the secretion of hCG into the medium of control and reactivated blastoids (3 days) cultured on matrigel-coated plates for 4 days. hCG, human chorionic gonadotropin. (C) UMAP plot of scRNA-seq data comparing post-implantation culture of control and reactivated blastoids. Cells are color-coded based on their respective sample and lineage. (D) UMAP plots showing the expression levels of the indicated lineage markers. Left, UMAP clusters showing subpopulations of each lineage. Right, expression levels of lineage-specific marker genes within each cluster and sample. Dot size corresponds to the percentage of cells expressing the gene, and color intensity reflects the scaled average expression level. (E) Left, in vivo reference atlas of early human development. Control (middle) and reactivated (right) cells were projected onto this reference atlas. (F) Percentages of the indicated cell types in control and reactivated blastoids. See also Figure S7.

Figure 7. Regulatory pathways in human and mouse dormancy

(A) Density plots showing the levels of pathway expression in human and mouse cells (top) or embryos/blastoids (bottom) in mTORi relative to controls. Pathway and protein alterations in response to mTORi show a statistically significant correlation between species. ESCs were collected after reaching a stably dormant state (mouse day 4, human day 8). R, Spearman’s rho correlation coefficient. (B) Common and species-enriched pathways in mTORi-treated human and mouse ESCs and blastocysts/blastoids. Complete list of pathways is available in Table S4. (C) IF stainings of hPSCs subjected to the indicated treatments. H3S10p, mitosis marker; pS6, mTOR target; OCT4, pluripotency marker. Scale bar, 20 μm. (D) Percentage of dividing (H3S10p⁺) cells in each condition. Statistical test in one-way ANOVA with multiple testing correction. (E) Proliferation curves of naive hPSCs subjected to the indicated treatments. (F) Working model summarizing the dormancy response in mouse and human.