ISRIB facilitates the co-culture of human trophoblast stem cells and embryonic stem cells

Abstract

The embryo-like structures (embryoids) constructed by aggregating embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have provided revolutionary tools for studying the intricate interaction between embryonic and extra-embryonic tissues during early embryonic development, which has been achieved in mice. However, due to the opposite dependence on some signalling pathways for in vitro culture of human ESCs (hESCs) and TSCs (hTSCs), particularly WNT and TGFβ signalling pathways, which limits the construction of human post-implantation embryoids by aggregating hESCs and hTSCs. To overcome this challenge, here, by screening 1639 chemicals, we found that an inhibitor of integrated stress response, ISRIB, can replace WNT agonists and TGFβ inhibitors to maintain the stemness and differentiation capacity of hTSCs. Thus, we developed an ISRIB-dependent in vitro culture medium for hTSCs, namely nTSM. Furthermore, we demonstrated that ISRIB could also maintain the hESC stemness. Using a 3D co-culture system (hESCs and hTSCs aggregate, ETA), we demonstrated that a 1:1 mixture of hESC culture medium (ESM) and nTSM improved the cell proliferation and organisation of both hESC- and hTSC-compartments and the lumenogenesis of hESC-compartment in ETAs. Overall, our study provided an ISRIB-dependent system for co-culturing hESCs and hTSCs, which facilitated the construction of human embryoids by aggregating hESCs and hTSCs.

References

[1] Rossant J, Tam PPL. Early human embryonic development: blastocyst formation to gastrulation. Dev Cell. 2022;57(2):152-165.

[2] Maltepe E, Fisher SJ. Placenta: the forgotten organ. Annu Rev Cell Dev Biol. 2015;31:523-552.

[3] Xiao Z, Yan L, Liang X, Wang H. Progress in deciphering trophoblast cell differentiation during human placentation. Curr Opin Cell Biol. 2020;67:86-91.

[4] Li Q, Wu H, Wang Y, Wang H. Current understanding in deciphering trophoblast cell differentiation during human placentation. Biol Reprod. 2022;107(1):317-326.

[5] Sozen B, Amadei G, Cox A, et al. Publisher correction: self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures. Nat Cell Biol. 2018;20(10):1229.

[6] Bernardo AS, Faial T, Gardner L, et al. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell. 2011;9(2):144-155.

[7] Lee CQ, Gardner L, Turco M, et al. What is trophoblast? A combination of criteria define human first-trimester trophoblast. Stem Cell Rep. 2016;6(2):257-272.

[8] Bredenkamp N, Stirparo GG, Nichols J, Smith A, Guo G. The cell-surface marker sushi containing domain 2 facilitates establishment of human naive pluripotent stem cells. Stem Cell Rep. 2019;12(6):1212-1222.

[9] Osnato A, Brown S, Krueger C, et al. TGFβ signalling is required to maintain pluripotency of human naïve pluripotent stem cells. Elife. 2021;10:e67259.

[10] Okae H, Toh H, Sato T, et al. Derivation of human trophoblast stem cells. Cell Stem Cell. 2018;22(1):50-63.e6.

[11] Pollheimer J, Loregger T, Sonderegger S, et al. Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast. Am J Pathol. 2006;168(4):1134-1147.

[12] Pham TXA, Panda A, Kagawa H, et al. Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells. Cell Stem Cell. 2022;29(9):1346-1365.e10.

[13] Vallier L, Mendjan S, Brown S, et al. Activin/nodal signalling maintains pluripotency by controlling Nanog expression. Development. 2009;136(8):1339-1349.

[14] Io S, Kabata M, Iemura Y, et al. Capturing human trophoblast development with naive pluripotent stem cells in vitro. Cell Stem Cell. 2021;28(6):1023-1039.e13.

[15] Wang Y, Wu H, Jiang X, et al. LMNA determines nuclear morphology during Syncytialization of human trophoblast stem cells. Front Cell Dev Biol. 2022;10:836390.

[16] Wu H, Wang Y, Wang H. Generation of human trophoblast stem cell-dependent placental in vitro models. Methods Mol Biol. 2022.

[17] Wu H, Wang Y, Wang H. Workflow for performing genetic manipulation in human trophoblast stem cells using CRISPR/Cas9 technology. Methods Mol Biol. 2022.

[18] Wu X, Zhao W, Wu H, et al. An aggregation of human embryonic and trophoblast stem cells reveals the role of trophectoderm on epiblast differentiation. Cell Prolif. 2023;56(5):e13492.

[19] Ohgushi M, Taniyama N, Vandenbon A, Eiraku M. Delamination of trophoblast-like syncytia from the amniotic ectodermal analogue in human primed embryonic stem cell-based differentiation model. Cell Rep. 2022;39(12):110973.

[20] Lee CQE, Bailey A, Lopez-Tello J, et al. Inhibition of phosphoinositide-3-kinase signaling promotes the stem cell state of trophoblast. Stem Cells. 2019;37(10):1307-1318.

[21] Krivega M, Essahib W, Van de Velde H. WNT3 and membrane-associated β-catenin regulate trophectoderm lineage differentiation in human blastocysts. Mol Hum Reprod. 2015;21(9):711-722.

[22] Ullah Z, Kohn MJ, Yagi R, Vassilev LT, DePamphilis ML. Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity. Genes Dev. 2008;22(21):3024-3036.

[23] Li G, Lin L, Wang YL, Yang H. 1,25(OH)2D3 protects trophoblasts against insulin resistance and inflammation via suppressing mTOR signaling. Reprod Sci. 2019;26(2):223-232.

[24] Lackner A, Müller M, Gamperl M, et al. The Fgf/erf/NCoR1/2 repressive axis controls trophoblast cell fate. Nat Commun. 2023;14(1):2559.

[25] Das P, Ezashi T, Schulz LC, Westfall SD, Livingston KA, Roberts RM. Effects of fgf2 and oxygen in the bmp4-driven differentiation of trophoblast from human embryonic stem cells. Stem Cell Res. 2007;1(1):61-74.

[26] Fan X, Rai A, Kambham N, et al. Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications. J Clin Invest. 2014;124(11):4941-4952.

[27] Ma Y, Yu X, Li YX, Wang YL. HGF/c-met signaling regulates early differentiation of placental trophoblast cells. J Reprod Dev. 2021;67(2):89-97.

[28] Rabouw HH, Langereis MA, Anand AA, et al. Small molecule ISRIB suppresses the integrated stress response within a defined window of activation. Proc Natl Acad Sci U S A. 2019;116(6):2097-2102.

[29] Zyryanova AF, Weis F, Faille A, et al. Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B. Science. 2018;359(6383):1533-1536.

[30] Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO Rep. 2016;17(10):1374-1395.

[31] Sidrauski C, McGeachy AM, Ingolia NT, Walter P. The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly. Elife. 2015;4:e05033.

[32] Racca AC, Ridano ME, Camolotto S, Genti-Raimondi S, Panzetta-Dutari GM. A novel regulator of human villous trophoblast fusion: the Krüppel-like factor 6. Mol Hum Reprod. 2015;21(4):347-358.

[33] Ferreira LMR, Meissner TB, Tilburgs T, Strominger JL. HLA-G: at the Interface of maternal-fetal tolerance. Trends Immunol. 2017;38(4):272-286.

[34] Kaneko KJ, DePamphilis ML. TEAD4 establishes the energy homeostasis essential for blastocoel formation. Development. 2013;140(17):3680-3690.

[35] Paul S, Home P, Bhattacharya B, Ray S. GATA factors: master regulators of gene expression in trophoblast progenitors. Placenta. 2017;60(Suppl 1):S61-S66.

[36] Rumer KK, Post MD, Larivee RS, et al. Siglec-6 is expressed in gestational trophoblastic disease and affects proliferation, apoptosis and invasion. Endocr Relat Cancer. 2012;19(6):827-840.

[37] Moore KH, Murphy HA, Chapman H, George EM. Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts. Am J Physiol Cell Physiol. 2021;321(4):C694-C703.

[38] Meinhardt G, Haider S, Kunihs V, et al. Pivotal role of the transcriptional co-activator YAP in trophoblast stemness of the developing human placenta. Proc Natl Acad Sci U S A. 2020;117(24):13562-13570.

[39] Krishnamoorthy J, Rajesh K, Mirzajani F, Kesoglidou P, Papadakis A, Koromilas AE. Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications. Cell Cycle. 2014;13(5):801-806.

[40] Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004;11(4):381-389.

[41] Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis. 2002;7(4):335-345.

[42] Chen Y, Tristan CA, Chen L, et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat Methods. 2021;18(5):528-541.

RIGHTS & PERMISSIONS

2024 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.