Establishment of Human Formative Pluripotent Stem Cell-Like Cells Exhibiting Amniotic Differentiation Potentials

Abstract

Human embryos undergo pivotal morphogenetic remodelling shortly after implantation. The understanding of this crucial stage is severely impeded by the scarcity of embryonic samples and ethical constraints. Pluripotent stem cells with the competence for somatic and germline differentiation serve as in vitro models of epiblast. In this study, we established human formative pluripotent stem cell-like cells (hfPSC-LCs) from naïve human embryonic stem cells (hESCs), conventional hESCs, human induced pluripotent stem cells (hiPSCs), as well as human blastocysts using the three-dimensional (3D) Matrigel culture system. Similar to pre-gastrula stage epiblast, hfPSC-LCs self-organise into self-renewing colonies with an apical lumen and exhibit several hallmarks of formative pluripotency, consistent with the properties observed in mouse fPSCs. Notably, single cells of hfPSC-LCs could differentiate into amnion-like precursor cells (hALPCs) which are transcriptionally and morphologically similar to the bona fide amnion. Meanwhile, hfPSC-LCs directly respond to primordial germ cell (PGC) induction signals, generating PGC-like cells (PGCLCs) either as single-cell aggregates or intact colonies, with an efficiency of approximately 50%. Chromatin accessibility analysis revealed that the differentiation capacity of hfPSC-LCs for gametes and amnion lineages might correlate with the accessible chromatin architecture of PGC and amnion associated genes. Loss of 3D-Matrigel niche disrupts formative pluripotency in both mouse and human, manifesting as downregulated formative markers and compromised differentiation capacity. Collectively, our findings establish hfPSC-LCs as a 3D model for investigating formative pluripotency of humans, thereby probably addressing a critical gap in the understanding of human pluripotency transitions.

Keywords

3D culture / epiblast / human amnion-like precursor cells (hALPCs) / human early embryogenesis / human formative pluripotent stem cell-like cells (hfPSC-LCs) / pluripotent stem cells (PSCs)

References

[1] A. G. Smith, “Embryo-Derived Stem Cells: Of Mice and Men,” Annual Review of Cell and Developmental Biology 17, no. 1 (2001): 435–462.

[2] M. F. Pera, “Human Embryo Research and the 14-Day Rule,” Development 144, no. 11 (2017): 1923–1925.

[3] T. E. Ludwig, M. E. Levenstein, J. M. Jones, et al., “Derivation of Human Embryonic Stem Cells in Defined Conditions,” Nature Biotechnology 24, no. 2 (2006): 185–187.

[4] K. Takahashi, K. Tanabe, M. Ohnuki, et al., “Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors,” Cell 131, no. 5 (2007): 861–872.

[5] J. I.-E. J. F.-S. Thomson, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall, and J. M. Jones, “Embryonic Stem Cell Lines Derived From Human Blastocysts,” Science 282, no. 5391 (1998): 1145–1147.

[6] J. Yu, M. A. Vodyanik, K. Smuga-Otto, et al., “Induced Pluripotent Stem Cell Lines Derived From Human Somatic Cells,” Science 318, no. 5858 (2007): 1917–1920.

[7] Z. Ai, B. Niu, Y. Yin, et al., “Dissecting Peri-Implantation Development Using Cultured Human Embryos and Embryo-Like Assembloids,” Cell Research 33, no. 9 (2023): 661–678.

[8] Y. Gong, B. Bai, N. Sun, et al., “Ex Utero Monkey Embryogenesis From Blastocyst to Early Organogenesis,” Cell 186, no. 10 (2023): 2092–2110 e2023.

[9] H. Ma, J. Zhai, H. Wan, et al., “In Vitro Culture of Cynomolgus Monkey Embryos Beyond Early Gastrulation,” Science 366, no. 6467 (2019): eaax7890.

[10] R. C. V. Tyser, E. Mahammadov, S. Nakanoh, L. Vallier, A. Scialdone, and S. Srinivas, “Single-Cell Transcriptomic Characterization of a Gastrulating Human Embryo,” Nature 600, no. 7888 (2021): 285–289.

[11] L. Xiang, Y. Yin, Y. Zheng, et al., “A Developmental Landscape of 3D-Cultured Human Pre-Gastrulation Embryos,” Nature 577, no. 7791 (2020): 537–542.

[12] H. Xie, C. An, B. Bai, et al., “Modeling Early Gastrulation in Human Blastoids With DNA Methylation Patterns of Natural Blastocysts,” Cell Stem Cell 32, no. 3 (2025): 409–425 e408.

[13] J. Zhai, Y. Xu, H. Wan, et al., “Neurulation of the Cynomolgus Monkey Embryo Achieved From 3D Blastocyst Culture,” Cell 186, no. 10 (2023): 2078–2091 e2018.

[14] T. Boroviak, R. Loos, P. Bertone, A. Smith, and J. Nichols, “The Ability of Inner-Cell-Mass Cells to Self-Renew as Embryonic Stem Cells Is Acquired Following Epiblast Specification,” Nature Cell Biology 16, no. 6 (2014): 516–528.

[15] Y. Kojima, K. Kaufman-Francis, J. B. Studdert, et al., “The Transcriptional and Functional Properties of Mouse Epiblast Stem Cells Resemble the Anterior Primitive Streak,” Cell Stem Cell 14, no. 1 (2014): 107–120.

[16] J. A. Hackett and M. A. Surani, “Regulatory Principles of Pluripotency: From the Ground State Up,” Cell Stem Cell 15, no. 4 (2014): 416–430.

[17] J. Nichols and A. Smith, “Naive and Primed Pluripotent States,” Cell Stem Cell 4, no. 6 (2009): 487–492.

[18] P. J. Tesar, J. G. Chenoweth, F. A. Brook, et al., “New Cell Lines From Mouse Epiblast Share Defining Features With Human Embryonic Stem Cells,” Nature 448, no. 7150 (2007): 196–199.

[19] Q. L. Ying, J. Wray, J. Nichols, et al., “The Ground State of Embryonic Stem Cell Self-Renewal,” Nature 453, no. 7194 (2008): 519–523.

[20] K. C. Davidson, E. A. Mason, and M. F. Pera, “The Pluripotent State in Mouse and Human,” Development (Cambridge) 142, no. 18 (2015): 3090–3099.

[21] A. De Los Angeles, F. Ferrari, R. Xi, et al., “Hallmarks of Pluripotency,” Nature (London) 525, no. 7570 (2015): 469–478.

[22] A. De Los Angeles, Y. H. Loh, P. J. Tesar, and G. Q. Daley, “Accessing Naive Human Pluripotency,” Current Opinion in Genetics & Development 22, no. 3 (2012): 272–282.

[23] J. Rossant, “Mouse and Human Blastocyst-Derived Stem Cells: Vive Les Differences,” Development 142, no. 1 (2015): 9–12.

[24] Y. Takashima, G. Guo, R. Loos, et al., “Resetting Transcription Factor Control Circuitry Toward Ground-State Pluripotency in Human,” Cell 158, no. 6 (2014): 1254–1269.

[25] T. W. Theunissen, B. E. Powell, H. Wang, et al., “Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency,” Cell Stem Cell 15, no. 4 (2014): 471–487.

[26] G. Guo, F. Von Meyenn, F. Santos, et al., “Naive Pluripotent Stem Cells Derived Directly From Isolated Cells of the Human Inner Cell Mass,” Stem Cell Reports 6, no. 4 (2016): 437–446.

[27] S. Bao, W. W. Tang, B. Wu, et al., “Derivation of Hypermethylated Pluripotent Embryonic Stem Cells With High Potency,” Cell Research 28, no. 1 (2018): 22–34.

[28] P. Du, M. Pirouz, J. Choi, et al., “An Intermediate Pluripotent State Controlled by MicroRNAs Is Required for the Naive-To-Primed Stem Cell Transition,” Cell Stem Cell 22, no. 6 (2018): 851–864 e855.

[29] M. Kinoshita, M. Barber, W. Mansfield, et al., “Capture of Mouse and Human Stem Cells With Features of Formative Pluripotency,” Cell Stem Cell 28, no. 3 (2021): 453–471 e458.

[30] M. Kinoshita, T. Kobayashi, B. Planells, et al., “Pluripotent Stem Cells Related to Embryonic Disc Exhibit Common Self-Renewal Requirements in Diverse Livestock Species,” Development 148, no. 23 (2021): dev199901.

[31] A. Neagu, E. Van Genderen, I. Escudero, et al., “In Vitro Capture and Characterization of Embryonic Rosette-Stage Pluripotency Between Naive and Primed States,” Nature Cell Biology 22, no. 5 (2020): 534–545.

[32] A. Smith, “Formative Pluripotency: The Executive Phase in a Developmental Continuum,” Development 144, no. 3 (2017): 365–373.

[33] X. Wang, Y. Xiang, Y. Yu, et al., “Formative Pluripotent Stem Cells Show Features of Epiblast Cells Poised for Gastrulation,” Cell Research 31, no. 5 (2021): 526–541.

[34] L. Yu, Y. Wei, H. X. Sun, et al., “Derivation of Intermediate Pluripotent Stem Cells Amenable to Primordial Germ Cell Specification,” Cell Stem Cell 28, no. 3 (2021): 550–567 e512.

[35] T. Kalkan and A. Smith, “Mapping the Route From Naive Pluripotency to Lineage Specification,” Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 369, no. 1657 (2014): 20130540.

[36] M. Kinoshita and A. Smith, “Pluripotency Deconstructed,” Development, Growth & Differentiation 60, no. 1 (2018): 44–52.

[37] T. Nakamura, I. Okamoto, K. Sasaki, et al., “A Developmental Coordinate of Pluripotency Among Mice, Monkeys and Humans,” Nature 537, no. 7618 (2016): 57–62.

[38] M. Rostovskaya, S. Andrews, W. Reik, and P. J. Rugg-Gunn, “Amniogenesis Occurs in Two Independent Waves in Primates,” Cell Stem Cell 29, no. 5 (2022): 744–759 e746.

[39] M. N. Shahbazi, A. Scialdone, N. Skorupska, et al., “Pluripotent State Transitions Coordinate Morphogenesis in Mouse and Human Embryos,” Nature 552, no. 7684 (2017): 239–243.

[40] Y. Shao, K. Taniguchi, R. F. Townshend, T. Miki, D. L. Gumucio, and J. Fu, “A Pluripotent Stem Cell-Based Model for Post-Implantation Human Amniotic Sac Development,” Nature Communications 8, no. 1 (2017): 208.

[41] Y. Zheng, R. Z. Yan, S. Sun, et al., “Single-Cell Analysis of Embryoids Reveals Lineage Diversification Roadmaps of Early Human Development,” Cell Stem Cell 29, no. 9 (2022): 1402–1419 e1408.

[42] G. Guo, G. G. Stirparo, S. E. Strawbridge, et al., “Human Naive Epiblast Cells Possess Unrestricted Lineage Potential,” Cell Stem Cell 28, no. 6 (2021): 1040–1056 e1046.

[43] S. Io, M. Kabata, Y. Iemura, et al., “Capturing Human Trophoblast Development With Naive Pluripotent Stem Cells in Vitro,” Cell Stem Cell 28, no. 6 (2021): 1023–1039 e1013.

[44] Y. Shao, K. Taniguchi, K. Gurdziel, et al., “Self-Organized Amniogenesis by Human Pluripotent Stem Cells in a Biomimetic Implantation-Like Niche,” Nature Materials 16, no. 4 (2017): 419–425.

[45] W. P. Luckett, “The Development of Primordial and Definitive Amniotic Cavities in Early Rhesus Monkey and Human Embryos,” American Journal of Anatomy 144, no. 2 (1975): 149–167.

[46] N. Bredenkamp, J. Yang, J. Clarke, et al., “Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency,” Stem Cell Reports 13, no. 6 (2019): 1083–1098.

[47] P. W. Burridge, E. Matsa, P. Shukla, et al., “Chemically Defined Generation of Human Cardiomyocytes,” Nature Methods 11, no. 8 (2014): 855–860.

[48] S. M. Chambers, C. A. Fasano, E. P. Papapetrou, M. Tomishima, M. Sadelain, and L. Studer, “Highly Efficient Neural Conversion of Human ES and iPS Cells by Dual Inhibition of SMAD Signaling,” Nature Biotechnology 27, no. 3 (2009): 275–280.

[49] Y. Yu, X. Wang, X. Zhang, et al., “ERK Inhibition Promotes Neuroectodermal Precursor Commitment by Blocking Self-Renewal and Primitive Streak Formation of the Epiblast,” Stem Cell Research & Therapy 9, no. 1 (2018): 2.

[50] F. W. Pagliuca, J. R. Millman, M. Gurtler, et al., “Generation of Functional Human Pancreatic Beta Cells in Vitro,” Cell 159, no. 2 (2014): 428–439.

[51] K. Sasaki, S. Yokobayashi, T. Nakamura, et al., “Robust in Vitro Induction of Human Germ Cell Fate From Pluripotent Stem Cells,” Cell Stem Cell 17, no. 2 (2015): 178–194.

[52] K. Hayashi, H. Ohta, K. Kurimoto, S. Aramaki, and M. Saitou, “Reconstitution of the Mouse Germ Cell Specification Pathway in Culture by Pluripotent Stem Cells,” Cell 146, no. 4 (2011): 519–532.

[53] K. J. Livak and T. D. Schmittgen, “Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta Delta C(T)) Method,” Methods 25, no. 4 (2001): 402–408.

[54] D. Kim, J. M. Paggi, C. Park, C. Bennett, and S. L. Salzberg, “Graph-Based Genome Alignment and Genotyping With HISAT2 and HISAT-Genotype,” Nature Biotechnology 37, no. 8 (2019): 907–915.

[55] Y. L. Shi, G. K. Smyth, and W. Shi, “featureCounts an Efficient General Purpose Program for Assigning Sequence Reads to Genomic Features,” Bioinformatics 30, no. 7 (2014): 923–930.

[56] M. I. Love, W. Huber, and S. Anders, “Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data With DESeq2,” Genome Biology 15, no. 12 (2014): 550.

[57] B. Langmead and S. L. Salzberg, “Fast Gapped-Read Alignment With Bowtie 2,” Nature Methods 9, no. 4 (2012): 357–359.

[58] F. Krueger and S. R. Andrews, “Bismark: A Flexible Aligner and Methylation Caller for Bisulfite-Seq Applications,” Bioinformatics 27, no. 11 (2011): 1571–1572.

[59] K. Danielski, “Guidance on Processing the 10× Genomics Single Cell Gene Expression Assay,” in Single Cell Transcriptomics: Methods and Protocols, ed. R. A. Calogero and V. Benes (Springer US, 2023), 1–28.

[60] D. Cornacchia, C. Zhang, B. Zimmer, et al., “Lipid Deprivation Induces a Stable, Naive-To-Primed Intermediate State of Pluripotency in Human PSCs,” Cell Stem Cell 25, no. 1 (2019): 120–136 e110.

[61] K. Watanabe, M. Ueno, D. Kamiya, et al., “A ROCK Inhibitor Permits Survival of Dissociated Human Embryonic Stem Cells,” Nature Biotechnology 25, no. 6 (2007): 681–686.

[62] J. Bayerl, M. Ayyash, T. Shani, et al., “Principles of Signaling Pathway Modulation for Enhancing Human Naive Pluripotency Induction,” Cell Stem Cell 28, no. 9 (2021): 1549–1565 e1512.

[63] D. Chen, W. Liu, J. Zimmerman, et al., “The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation,” Cell Reports 25, no. 13 (2018): 3591–3602 e3595.

[64] M. Nakanishi, R. R. Mitchell, Y. D. Benoit, et al., “Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells,” Cell 177, no. 4 (2019): 910–924.e922.

[65] K. T. Minn, S. Dietmann, S. E. Waye, S. A. Morris, and L. Solnica-Krezel, “Gene Expression Dynamics Underlying Cell Fate Emergence in 2D Micropatterned Human Embryonic Stem Cell Gastruloids,” Stem Cell Reports 16, no. 5 (2021): 1210–1227.

[66] J. Wu, J. Xu, B. Liu, et al., “Chromatin Analysis in Human Early Development Reveals Epigenetic Transition During ZGA,” Nature 557, no. 7704 (2018): 256–260.

[67] H. Mohammed, I. Hernando-Herraez, A. Savino, et al., “Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions During Mouse Early Gastrulation,” Cell Reports 20, no. 5 (2017): 1215–1228.

[68] X. Lian, C. Hsiao, G. Wilson, et al., “Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells via Temporal Modulation of Canonical Wnt Signaling,” Proceedings of the National Academy of Sciences of the United States of America 109, no. 27 (2012): E1848–E1857.

[69] Y. Zhang, N. Cao, Y. Huang, et al., “Expandable Cardiovascular Progenitor Cells Reprogrammed From Fibroblasts,” Cell Stem Cell 18, no. 3 (2016): 368–381.

[70] J. Baulida, V. M. Diaz, and A. G. Herreros, “Snail1: A Transcriptional Factor Controlled at Multiple Levels,” Journal of Clinical Medicine 8, no. 6 (2019): 757.

[71] N. Irie, L. Weinberger, W. W. Tang, et al., “SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate,” Cell 160, no. 1–2 (2015): 253–268.

[72] B. Gharibi, O. C. K. Inge, I. Rodriguez-Hernandez, et al., “Post-Gastrulation Amnioids as a Stem Cell-Derived Model of Human Extra-Embryonic Development,” Cell 188, no. 14 (2025): 3757–3774 e3720.

[73] J. G. L. Neupane, T. Gross-Thebing, M. L. Ruiz Tejada Segura, et al., “The Emergence of Human Primordial Germ Cell Like Cells in Stem Cell Derived Gastrloids,” Science Advances 1350, no. 10 (2025): 1350.

[74] L. P. Liu, D. X. Zheng, Z. F. Xu, et al., “Transcriptomic and Functional Evidence Show Similarities Between Human Amniotic Epithelial Stem Cells and Keratinocytes,” Cells 11, no. 1 (2021): 70.

[75] J. K. Yoon, “Advanced 3D Cell Culture Platform for Tissue Engineering,” Tissue Engineering and Regenerative Medicine 20, no. 4 (2023): 519–521.

[76] H. K. Kleinman and G. R. Martin, “Matrigel: Basement Membrane Matrix With Biological Activity,” Seminars in Cancer Biology 15, no. 5 (2005): 378–386.

[77] H. K. Kleinman, M. L. McGarvey, L. A. Liotta, P. G. Robey, K. Tryggvason, and G. R. Martin, “Isolation and Characterization of Type IV Procollagen, Laminin, and Heparan Sulfate Proteoglycan From the EHS Sarcoma,” Biochemistry 21 (1982): 6188–6193.

[78] W. Roslyn, P. G. Orkin, B. Ermona, et al., “A Murine Tumor Producing a Matrix of Basement,” Journal of Experimental Medicine 145 (1977): 204–220.

[79] C. S. Hughes, L. M. Postovit, and G. A. Lajoie, “Matrigel: A Complex Protein Mixture Required for Optimal Growth of Cell Culture,” Proteomics 10, no. 9 (2010): 1886–1890.

[80] Z. Ai, B. Niu, K. Duan, et al., “Modulation of Wnt and Activin/Nodal Supports Efficient Derivation, Cloning and Suspension Expansion of Human Pluripotent Stem Cells,” Biomaterials 249 (2020): 120015.

[81] G. Chen, D. R. Gulbranson, Z. Hou, et al., “Chemically Defined Conditions for Human iPSC Derivation and Culture,” Nature Methods 8, no. 5 (2011): 424–429.

[82] P. Zhu, B. Zhang, R. Sun, et al., “Derivation of New Pluripotent Stem Cells From Human Extended Pluripotent Stem Cells With Formative Features and Trophectoderm Potential,” Cell Proliferation 56, no. 11 (2023): e13480.

[83] C. Kyprianou, N. Christodoulou, R. S. Hamilton, et al., “Basement membrane remodelling regulates mouse embryogenesis,” Nature 582, no. 7811 (2020): 253–258.

[84] I. Bedzhov and M. Zernicka-Goetz, “Self-Organizing Properties of Mouse Pluripotent Cells Initiate Morphogenesis Upon Implantation,” Cell 156, no. 5 (2014): 1032–1044.

[85] S. Dupont, L. Morsut, M. Aragona, et al., “Role of YAP/TAZ in Mechanotransduction,” Nature (London) 474, no. 7350 (2011): 179–183.

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