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Mouse trophectoderm stem cells generated with morula signalling inducers capture an early trophectoderm state

Nature Cell Biology volume 27, pages 1572–1586 (2025)Cite this article

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Abstract

The first embryonic cell differentiation in mice segregates the trophectoderm and the inner cell mass. Successful derivation of mouse trophoblast stem cells (TSCs) and trophectoderm stem cells (TESCs) has greatly facilitated the understanding of trophoblast differentiation. However, our understanding of early trophectoderm differentiation remains incomplete. Here we report the establishment of a morula-derived trophectoderm stem cell (MTSC) line from 32-cell embryos that show enhanced and uniform trophoblast core gene expression. Importantly, distinct from TSCs or TESCs, MTSCs represent a much earlier trophectoderm state (E3.5) than that of TSCs (E5.5–6.5) and TESCs (E4.5–5.5). MTSCs can robustly integrate into all cell lineages of the placenta. Moreover, MTSCs can self-organize to form placenta organoids. When partially differentiated MTSCs aggregate with embryonic stem cells, they form blastoids that efficiently implant uteruses. Finally, MSTC medium can efficiently convert embryonic stem cells, TSCs and TESCs into MTSC-like cells. Thus, MTSCs capture an early blastocyst trophectoderm state and provide a research model for studying trophoblast development.

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Fig. 1: Specification of TE (32-TE) and ICM lineages occurs at the 32-cell stage, and the gene transcription profile of 32-TE is distinct from that of classic TSCs.
Fig. 2: Derivation of TESCs from 32-cell stage mouse embryos.
Fig. 3: The molecular properties of MTSCs differ from those of TSCs and TESCs.
Fig. 4: scRNA-seq analysis indicates that MTSCs represent an earlier TE state than TSCs and TESCs.
Fig. 5: MTSC medium promotes TSC core gene expression and supports feeder-free culture.
Fig. 6: MTSC inducers can reprogram ESCs into MTSC-like cells.
Fig. 7: MTSCs develop into terminally differentiated cells of placenta in vitro and in vivo.
Fig. 8: Progenies of MTSCs generate trophoblast organoids in vitro and initiate decidualization in vivo.

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Data availability

Sequencing data that support the findings of this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject ID: PRJNA955722, PRJNA1092867 and PRJNA1092531 (RNA-seq data of MTSCs, scRNA-seq data of cell lines and scRNA-seq data of organoids). Previously published data that were reanalysed here are available under the following accession codes from the Gene Expression Omnibus (GEO): GSE45719, GSE84892, GSE109071, GSE100597 and GSE123046 (developmental stage ranging from zygote to E6.54,50,51,52,53). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the Center for Animal Research and Resources, Yunnan University for providing animal care. We thank X. Zuo for embryo transplantation and F. Zhang for additional technical assistance. We thank M. Chen and Y. Tao for their support of the organoid culture. This work was supported by the Science and Technology Leading Talent Project Grant, Yunnan Province (202005AB160006 to J.Z.),the National Key Research Program Project Grant (2018YFC1003304 to J.Z.), the Basic Research Program of Yunnan Province Grant (202001BB050010 to S.W.) and the National Natural Science Foundation of China (22306154 to W. Guo).

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Author notes

  1. These authors contributed equally: Yake Gao, Mingying Li.

Authors and Affiliations

  1. Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China

    Yake Gao, Mingying Li, Wei Guo, Shu Wei, Fang Yan, Wenrui Han, Tong Yin, Yunkun Dang & Jian Zhang

  2. Reproductive Medicine Center, Chengdu Shuangnan Hospital, Chengdu Medical College, Chengdu, China

    Yake Gao

  3. Department of Reproductive Medicine, The First People’s Hospital of Yunnan Province, Kunming, China

    Mingying Li

  4. The Affiliated Hospital of Kunming University of Science and Technology, Kunming, China

    Mingying Li

  5. Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou, China

    Wei Guan, Huanhuan Li & José C. R. Silva

  6. Center for Animal Research and Resources, Yunnan University, Kunming, China

    Xueting Zhang & Jian Zhang

  7. Southwestern United Graduate School, Kunming, China

    Jian Zhang

  8. School of Life Sciences, Fudan University, Shanghai, China

    Jian Zhang

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Contributions

J.Z. and Y.G. conceived and designed the study. Y.G. and M.L. performed the bulk of the experiments and analysed the data. W. Guan analysed the single-cell sequencing data. W. Guan, W. Guo, S.W., F.Y., W.H., X.Z., T.Y, Y.D., H.L., J.C.R.S. and J.Z. provided additional technical support and data analysis. Y.G. and J.Z. wrote the manuscript. All authors approved the manuscript.

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Correspondence to Jian Zhang.

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Extended data

Extended Data Fig. 1 Specification of trophectoderm (32-TE) and inner cell mass lineages occurs at the 32-cell stage and the gene transcription profile of 32-TE is distinct from that of classic trophoblast stem cells.

(a) 16-cell and 32-cell stage embryos stained with PKH26 followed by dissociation. Manually selected dissociated outer blastomeres are shown. (b) ESCs (GFP positive) integrate into the ICM of blastocysts upon aggregation or injection. (c) Aggregation of 32-cell outer blastomeres (10 or 20) with 5 ESCs resulted in the formation of blastoids. (d) Degenerated decidua induced by blastoids after implantation on the 6th day. (e) Percentage of aggregates formed by different combinations of inner and outer blastomeres in 32-cell stage embryos (related to Fig. 1c). (f) Percentage of live births after blastocyst (control) or aggregates transferred to the mouse uterus (related to Fig. 1c). (g) Percentage of decidualization and live births after blastocyst (control) or blastoids transferred to the mouse uterus. (h) Heatmap analysis of differentially expressed genes between 32-TE cells and TSCs. 32-TE, outer cells of 32-cell stage embryos. (i) Heatmap analysis of differentially expressed genes between 32-TE, E3.5, TESCs, and TSCs. Differentially expressed genes were those with Padj<0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. Experiments were repeated three times (a-d) with similar results. Data are shown as mean ± SEM from three independent experiments (e-g). Numerical data are available as source data. Scale bars: 50 μm in a-c; 5 mm in d.

Extended Data Fig. 2 Morula trophectoderm stem cells are efficiently derived from 32-cell embryos by the inducers.

(a) Immunostaining for CDX2 and OCT4 of blastocysts developed from 32-cell embryos cultured with different concentrations of Y27632 for 24 h. (b) qRT-PCR analysis of mRNA expression of Oct4, Sox2, Cdx2, Tfap2c, Gata3, Gata6, and Sox17 of 32-cell embryos cultured with different concentrations of Y27632 for 24 h. (c) Blastocoel formation for 32-cell embryos treated with Y27632. (d) Blastocyst morphology after treatment of 32-cell embryos in different concentrations of LATS-IN-1 for 24 h. Control: untreated embryos (E4.0). (e) Immunostaining of CDX2 and OCT4 in blastocysts developed from 32-cell embryos cultured with different compounds for 24 h. (f) qRT-PCR analysis of mRNA expression of Cdx2, Gata3, Oct4, Sox2, Gata6, and Sox17 of 32-cell embryos cultured with different compounds for 24 h. (g) Cell outgrowth from 16-cell, 32-cell, E3.5, and E4.0 embryos cultured in the MTSC medium for 5 days. (h) Stem cell derivation efficiency with the TSC or the MTSC medium from 16-cell, 32-cell, E3.5, and E4.0 stage embryos. The data are detailed in Table S4. (i) Immunostaining of CDX2 and GATA6 for cells derived from 32-cell embryos cultured in the TSC medium for 6 days. XENCs (red arrow); TSCs (green arrow). (j) Immunostaining of CDX2 and GATA6 for cells derived from 32-cell embryos cultured in the TSC medium for 6 days. (k) Immunostaining of CDX2 and GATA6 of cells derived from 32-cell embryos cultured in the MTSC medium for 2 days (Noted co-localization of GATA6 and CDX2). Experiments were repeated three times (a, d, e, g, i-k) with similar results. Data are shown as mean ± SEM from three independent experiments (b, c, f, h), analyzed by two-tailed t-test without adjustment (c, h). Numerical data are available as source data. Scale bars: 50 μm in a, d, g, and k; 20 μm in e, i, and j.

Extended Data Fig. 3 MTSCs show different molecular properties compared to TSCs and FAXY-TSCs.

(a) Immunostaining for CDX2 and GATA6 proteins of 8-cell, 16-cell, 32-cell, E3.5, and E4.5 stage embryos and outgrowth cells from 32-cell stage embryos cultured in MTSC induction medium for 5 days. (b) Immunostaining for CDX2, OCT4, and GATA6 proteins of outgrowth cells from 32-cell stage embryos cultured in MTSC induction medium for 2 or 5 days. The CDX2 and GATA6 expression inserts from boxed regions, respectively. (c) Proliferated cells from outer blastomeres of 32-cell embryos (PKH26 labeled) cultured in the MTSC induction medium for 5 days. (d) Immunostaining for CDX2 and YAP1 of the outgrowth cultured with MTSC induction medium for 5 days from 32-cell stage outer blastomeres. The bottom enlarged region is indicated by white boxes. (e) Morphology of MTSCs at the indicated cell passage. (f) Immunostaining for CDX2 and SOX2 of TSCs and MTSCs. (g) Immunostaining for TFAP2C, KRT8, and E-CAD of MTSCs. (h) Proliferated cells from 32-cell stage embryo cultured in the FAXY-TSC induction system for 6 days. (i) qRT-PCR analysis of mRNA expression for Cdx2, Klf5, Eomes, Tfap2c, Krt8, and Gcm1 of MTSCs and FAXY-TSCs. Data are shown as mean ± SEM from three independent experiments, analyzed by two-tailed t-test without adjustment, nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. (j) Immunostaining for CDX2 and YAP1 of FAXY-TSCs and MTSCs. Experiments were repeated three times (a-h, j) with similar results. Numerical data and exact p values are available as source data. Scale bars: 50 μm in b-c, e, and h; 20 μm in a, d, f-g, and j.

Extended Data Fig. 4 MTSCs share part of cellular properties of morula trophectoderm.

(a) Immunostaining of CDX2 and KRT18 in 32-cell, E3.5, and E4.5 embryos. 32-cell embryos cultured with 4 μM Lats-in-1 for 36 h and stained at E4.5 equivalent time. (b) Immunostaining for CDX2 and KRT18 as markers for the polar and mural axis in E4.5 blastocysts. Parts of the polar and mural trophectoderm (white boxes) of the blastocyst are enlarged and shown on the right. (c) Transcription analysis of MTSCs and TSCs. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. Number of up-regulated genes, 1822; Number of down-regulated genes, 894. (d) Venn diagram analysis of differentially expressed genes among 32-TE, MTSCs, and TSCs. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. (e) Heatmap analysis of TE-specific and core TSC gene expression between MTSCs and TSCs. (f) The expression of polar and mural trophectoderm (TE) enriched genes in MTSCs and TSCs shown by a heatmap. (g) The expression of morula trophectoderm and blastocyst trophectoderm enriched genes in MTSCs and TSCs shown by a heatmap. (h) Correlation heatmap analysis of 32-TE, MTSCs, and TSCs. (i) Differentially expressed genes between 32-TE, MTSCs, and TSCs are shown by cluster analysis in a heatmap. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcription levels. Experiments were repeated three times (a, b) with similar results. Scale bars: 50 μm in a, and b.

Extended Data Fig. 5 Comparison of molecular properties between MTSCs, TSCs, and TESCs.

(a) Clone formation and cell proliferation from 32-cell stage embryo cultured in the TESC induction medium for 6 days. The red circles indicate TESC clones; green arrows indicate trophoblast giant cells (TGCs). (b) Immunostaining for CDX2 of MTSCs, TESCs, and TSCs. BF, Bright field. (c) Analysis of fluorescence intensity of CDX2 positive cells in TSCs, TESCs, and MTSCs. (d) mRNA expressions of Tead4, Klf5, Cdx2, Eomes, Ly6a, Gost1, and Sox2 in TSCs, TESCs, MTSCs, and Diff-TSCs detected by qRT-PCR. diff-TSC, TSCs after removal of FGF4 for 2 days. (f) mRNA expressions of Gcm1, Ctsq, Tpbpa, Prl3d1, Krt8, and Krt18 in TSCs, TESCs, MTSCs, and Diff-TSCs were detected by qRT-PCR. diff-TSC, TSCs after removal of FGF4 for 2 days. (g) Transcription analysis of differentially expressed genes between MTSCs, TESCs, and TSCs. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. blue bar, up-regulated genes; orange bar, down-regulated genes; green, total differentially expressed genes. (h) Immunostaining for CDX2, YAP, and E-CAD of TESCs and MTSC cultured in the TESC medium for 48 h. (i) Heatmap analysis of differentially expressed genes between TESCs, MTSCs, TSCs, and differentiated TSCs (Diff-TSC). “Diff-TSC” refers to TSCs after FGF4 removal for 2 days. Differentially expressed genes were those with Padj < 0.05 and log2FoldChange ≥ 1 in transcriptome sequencing analysis. Experiments were repeated three times (a, b, g, h) with similar results. Data are shown as mean ± SEM from three independent experiments (d, e). Numerical data are available as source data. Scale bars: 50 μm in a; 20 μm in b, g, and h.

Extended Data Fig. 6 Single cell sequencing analysis of gene expression and epithelial protein marker analysis in TSCs, TESCs, and MTSCs.

(a) UMAP embeddings of TSCs, TESCs, MTSCs, and MTSCs cultured in TESCM for 48 h (MITE). These were integrated with published datasets of developmental stages from zygote to E6.5 mouse embryos (related to Fig. 4a). (b) UMAP TSC cells after integration, each sample was randomly sampled to 5000. Data were generated by 10× Genomics scRNA-seq. Colors indicate cluster membership. (c) UMAP TESC cells after integration, each sample was randomly sampled to 5000. Data were generated by 10× Genomics scRNA-seq. Colors indicate cluster membership. (d) Expression of Elf5 and Esrrb RNAs in TSCs, TESCs, and MTSCs. (e) Expression of Dmkn, Plac1, Muc1, and Prl3d1 RNAs in TSCs, TESCs, and MTSCs. (f) Expression of Cdh1 and Tjp1 RNAs in TSCs, TESCs, and MTSCs. (g) Immunostaining of E-CAD and ZO-1 in TSCs, TESCs, and MTSCs (related to Figure S6f). Experiments were performed with cell numbers indicated. Scale bar, 20 μm.

Extended Data Fig. 7 MTSC medium promotes TSC core gene expression, supports feeder-free culture, and can reprogram ESCs into MTSC-like cells.

(a) Immunostaining for CDX2 and YAP1 in TSCs cultured in the MTSC medium and MTSCs cultured in the TSC medium. (b) Immunostaining of CK7 in TSCs cultured with the MTSC medium and MTSCs cultured in the TSC medium. (c) Morphology of TSCs, MTSCs, TSCs-MTSCM, and MTSCs-TSCM. TSCs-MTSCM, TSCs cultured in the MTSC medium for 72 hrs; MTSCs-TSCM, MTSCs cultured in the TSC medium for 72 hrs. (d) Immunostaining of TFAP2C, KRT8, and E-CAD in feeder-free MTSC culture. (e) Immunostaining of CDX2, YAP1, and E-CAD in feeder-free MTSC culture. (f) Immunostaining of CDX2, YAP1, and E-CAD in feeder-free TSC cultured in the MTSC medium. (g) Morphology of ESCs cultured in the TSC and MTSC medium for 2, 6, and 10 days. (h) qRT-PCR analysis of mRNA expression of Oct4 and Nanog in ESCs cultured in the MTSC or TSC medium for 4, 6, 8, and 10 days. (i) qRT-PCR analysis of mRNA expression of Gata4, Gata6, and Pdgfra in ESCs cultured in the MTSC or TSC medium for 4, 6, 8, and 10 days. (j) Flow cytometry analysis of TFAP2C and CK7 double-positive ESCs cultured in the MTSC medium for 10 days. Experiments were repeated three times (a-g, j) with similar results. Data are shown as mean ± SEM from three independent experiments (h, i), analyzed by ANOVA-LSD post hoc test without adjustment. Numerical data are available as source data. Scale bars: 20 μm in a-g.

Extended Data Fig. 8 Differentiation of MTSCs in vitro and in vivo.

(a) Immunostaining of TFAP2C and CK7 in ESCs cultured in the MTSC medium for 10 or 20 days. (b) Immunostaining of CDX2 and GATA6 in ESCs cultured in the MTSC medium for 10 days. Stages 1, 2, and 3 represent three distinct phases during the ESC to MTSC transition. Emerging CDX2 positive cells (white arrowheads). (c) Chimeric blastocysts formed after injection of 4-6 MTSCs (GFP positive) into 32-cell embryos. Bottom: green fluorescent images; Top: merged light field and green fluorescent images. (d) Deciduas of E8.5 embryos from the MTSC chimeric blastocysts; progenies of MTSCs (GFP positive) contributed to the placenta formation of E14.5. (e) Flow cytometry analysis of percentage of GFP positive cells in E16.5 mouse conceptuses (related to Fig. 7k). (f) Immunostaining of CK7, TPBPA, and SYNA proteins after MTSCs were cultured in the differentiation medium on day zero (related to Fig. 7h). (g) Immunostaining for TPBPA, SYNA, and CK7 in TSCs after removal of FGF4 for 2 or 4 days. BF, bright field. (h) Immunostaining for TPBPA, SYNA, and CK7 of the E16.5 chimeric placenta (progenies of MTSCs, GFP labeled). (i) Proliferation of trophoblast organoids derived from MTSCs cultured in vitro for 6 days. Experiments were repeated three times (a-i) with similar results. Scale bars: 20 μm in b and f; 50 μm in a, c, and g-i; 1 mm in d.

Extended Data Fig. 9 Progenies of MTSCs generate trophoblast organoids and blastoids.

(a) Immunostaining of EPCAM and SYNA of trophoblast organoids derived from MTSCs cultured in vitro for 8 days. (b) Immunostaining of TPBPA and TFAP2C of trophoblast organoids derived from MTSCs cultured in vitro for 8 days. (c) Heatmaps showing the expression of marker genes in placental cells (snRNA-seq) and MTSCs organoids(scRNA-seq). (d) Expression levels of marker genes of different placenta-like cells in MTSCs organoids. (e) Generation of blastoids between MTSCs or TSCs aggregated with EPSCs in Aggrewell400 culture plates. EPSCs are GFP positive. (f) Morphology of blastoids from aggregation between ESCs with various trophectoderm stem cells. (g) Immunostaining of OCT4 and KRT18 of abnormal blastoids of mismatched TSCs/ESCs. (h) Immunostaining of OCT4 and KRT18 in a morula-like structure from MTSC and ESC aggregates. (i) Immunostaining of OCT4 and KRT18 in a blastoid from the MITE and ESC aggregates. MITE, MTSCs were cultured in the TESC medium for 48 h before the aggregation. Scale bars: 20 μm in a-b; 50 μm in e-i. Experiments were repeated three times (a, b, e-i) with similar results. Scale bars: 20 μm in a-b; 50 μm in e-i.

Extended Data Fig. 10 Blastoids derived from TSC, TESC, or MITE can establish axes.

(a) Immunostaining of OCT4 and KRT18 in TSC, TESC, and MITE blastoids. (b) Immunostaining for CDX2 and KRT18 as markers for axes in MITE blastoids. No polarity: a blastoid without a clear CDX2-KRT18 axis; Polarity: a blastoid with a CDX2-KRT18 axis. (c) Percentage of blastoids with asymmetric expression for CDX2 or KRT18, or for both markers (double). Data are shown as mean ± s.e.m. from three independent experiments (total 30 blastoids per group), analyzed by two-tailed t-test without adjustment. Experiments were repeated three times (a, b) with similar results. Numerical data are available as source data. Scale bars: 50 μm in a-b.

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Supplementary Fig. 1

The gating strategy for cell cycle analysis by flow cytometry. A total of 20,000–100,000 cells were used for FACS analysis. Live cells were identified with FSC-A/SSC-A gating and followed by FSC-A/FSC-H for single cells. Positive cells were gated on the basis of different channels (FITC for Alexa 488 or GFP and APC for Alexa 647). Representative flow plots are shown depicting the complete gating strategy for analyzing marker protein positive cells in different cell lines (related to Figs. 5d,k and 6a,b and Extended Data Fig. 7j) or GFP-positive cells in mouse chimera conceptuses (related to Fig. 6k and Extended Data Fig. 8e). The numbers in the plots represent the frequency of the population within the drawn gates. a, Sequential gating strategies for control cells. b, Sequential gating strategies for treatment cells.

Supplementary Table 1

Reagents and resources.

Supplementary Table 2

qRT–PCR primers.

Supplementary Table 3

Differentially expressed genes.

Supplementary Table 4

Efficiency of derivation.

Supplementary Table 5

Screening of inducers.

Supplementary Table 6

Efficiency of aggregation or implantation.

Supplementary Table 7

Percentage of blastoids with axis.

Source data

Source Data Figs. 1–3 and 5–8

Source data for required figures.

Source Data Extended Data Figs. 1–3, 5, 7 and 10

Source data for required extended figures.

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Gao, Y., Li, M., Guan, W. et al. Mouse trophectoderm stem cells generated with morula signalling inducers capture an early trophectoderm state. Nat Cell Biol 27, 1572–1586 (2025). https://doi.org/10.1038/s41556-025-01732-8

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