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Soft substrate maintains stemness and pluripotent stem cell-like phenotype of human embryonic stem cells under defined culture conditions

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Abstract

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the pre-implantation blastocyst. Prior to embryo implantation, the ICM cells are surrounded by trophoblasts which have mechanical stiffness ranging from Pascal (Pa) to kilopascal (kPa). However, under in vitro conditions these cells are cultured on stiff tissue culture treated plastic plates (TCP) which have stiffness of approximately 1 gigapascal (GPa). This obvious dichotomy motivated us to investigate the fate of hESCs cultured on softer substrate, and to probe if the hESCs undergo differentiation or they retain pluripotency on soft substrates. We investigated the expression of pluripotency markers, and lineage-specific markers; we particularly looked at the expression of transcriptional coactivator YAP (Yes-associated protein), an important mediator of extracellular matrix (ECM) mechanical cues and a known downstream transducer of Hippo pathway. Downregulation of YAP has been correlated to the loss of multipotency of human mesenchymal stem cells (hMSCs) and pluripotency in mouse ESCs (mESCs); but we report that hESCs maintain their stemness on soft substrate of varying stiffness. Our findings revealed that on soft substrate hESCs express pluripotency markers and does not undergo substrate-mediated differentiation. Interestingly we show that hESCs maintained basal level of YAP expression for cell survival and proliferation, but YAP expression does not correlate directly with pluripotency in hESCs. To summarize, our results show that hESCs retain their stemness on soft substrate despite downregulation of YAP.

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Data availability

The data that support the findings of this study are available from the corresponding author upon a reasonable request.

References

  • Abbas Y, Carnicer-Lombarte A, Gardner L, Thomas J, Brosens JJ, Moffett A, Sharkey AM, Franze K, Burton GJ, Oyen ML (2019) Tissue stiffness at the human maternal–fetal interface. Hum Reprod 34:1999–2008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arora S, Saha S, Roy S, Das M, Jana SS, Ta M (2015) Role of nonmuscle myosin II in migration of Wharton’s jelly-derived mesenchymal stem cells. Stem Cells Dev 24:2065–2077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brusatin G, Panciera T, Gandin A, Citron A, Piccolo S (2018) Biomaterials and engineered microenvironments to control YAP/TAZ-dependent cell behaviour. Nat Mater 17:1063–1075

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chan EH, Nousiainen M, Chalamalasetty RB, Schäfer A, Nigg EA, Sillje HH (2005) The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24:2076–2086

    Article  CAS  PubMed  Google Scholar 

  • Czechanski A, Byers C, Greenstein I, Schrode N, Donahue LR, Hadjantonakis AK, Reinholdt LG (2014) Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat Protoc 9:559–574

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, Gayyed MF, Anders RA, Maitra A, Pan D (2007) Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130:1120–1133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Driscoll TP, Cosgrove BD, Heo SJ, Shurden ZE, Mauck RL (2015) Cytoskeletal to nuclear strain transfer regulates YAP signaling in mesenchymal stem cells. Biophys J 108:2783–2793

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dumasia NP, Khanna AP, Pethe PS (2021) Sonic hedgehog signals hinder the transcriptional network necessary for pancreatic endoderm formation from human embryonic stem cells. Genes Cells 26:282–297

    Article  CAS  PubMed  Google Scholar 

  • Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore NS (2011) Role of YAP/TAZ in mechanotransduction. Nature 474:179–183

    Article  CAS  PubMed  Google Scholar 

  • Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689

    Article  CAS  PubMed  Google Scholar 

  • Evans ND, Minelli C, Gentleman E, LaPointe V, Patankar SN, Kallivretaki M, Chen X, Roberts CJ, Stevens MM (2009) Substrate stiffness affects early differentiation events in embryonic stem cells. J Eur Cells Mater 18:1–14

    Article  CAS  Google Scholar 

  • Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS (2004) Differences between human and mouse embryonic stem cells. Dev Biol 269:360–380

    Article  CAS  PubMed  Google Scholar 

  • Hadden WJ, Young JL, Holle AW, McFetridge ML, Kim DY, Wijesinghe P, Taylor-Weiner H, Wen JH, Lee AR, Bieback K, Vo BN (2017) Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels. Proc Natl Acad Sci USA 114:5647–5652

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Heng BC, Zhang X, Aubel D, Bai Y, Li X, Wei Y, Fussenegger M, Deng X (2020) Role of YAP/TAZ in cell lineage fate determination and related signaling pathways. Front Cell Dev Biol 8:735

    Article  PubMed  PubMed Central  Google Scholar 

  • Hsiao C, Lampe M, Nillasithanukroh S, Han W, Lian X, Palecek SP (2016) Human pluripotent stem cell culture density modulates YAP signaling. Biotechnol J 11:662–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang J, Wu S, Barrera J, Matthews K, Pan D (2005) The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell 122:421–434

    Article  CAS  PubMed  Google Scholar 

  • Ireland RG, Simmons CA (2015) Human pluripotent stem cell mechanobiology: manipulating the biophysical microenvironment for regenerative medicine and tissue engineering applications. Stem Cells 33:3187–3196

    Article  PubMed  Google Scholar 

  • Keung AJ, Asuri P, Kumar S, Schaffer DV (2012) Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells. Integr Biol 4:1049–1058

    Article  CAS  Google Scholar 

  • Kim MK, Jang JW, Bae SC (2018) DNA binding partners of YAP/TAZ. BMB Rep 51:126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar N, Hinduja I, Nagvenkar P, Pillai L, Zaveri K, Mukadam L, Telang J, Desai S, Mangoli V, Mangoli R, Padgaonkar S (2009) Derivation and characterization of two genetically unique human embryonic stem cell lines on in-house-derived human feeders. Stem Cells Dev 18:435–446

    Article  CAS  PubMed  Google Scholar 

  • Lee S, Stanton AE, Tong X, Yang F (2019) Hydrogels with enhanced protein conjugation efficiency reveal stiffness-induced YAP localization in stem cells depends on biochemical cues. Biomaterials 202:26–34

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lian I, Kim J, Okazawa H, Zhao J, Zhao B, Yu J, Chinnaiyan A, Israel MA, Goldstein LS, Abujarour R, Ding S (2010) The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev 24:1106–1118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ma X, Jana SS, Anne Conti M, Kawamoto S, Claycomb WC, Adelstein RS (2010) Ablation of nonmuscle myosin II-B and II-C reveals a role for nonmuscle myosin II in cardiac myocyte karyokinesis. Mol Biol Cell 21:3952–3962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McClatchey AI, Yap AS (2012) Contact inhibition (of proliferation) redux. Curr Opin Cell Biol 24:685–694

    Article  CAS  PubMed  Google Scholar 

  • Musah S, Wrighton PJ, Zaltsman Y, Zhong X, Zorn S, Parlato MB, Hsiao C, Palecek SP, Chang Q, Murphy WL, Kiessling LL (2014) Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification. Proc Natl Acad Sci USA 111:13805–13810

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ohgushi M, Minaguchi M, Sasai Y (2015) Rho-signaling-directed YAP/TAZ activity underlies the long-term survival and expansion of human embryonic stem cells. Cell Stem Cell 17:448–461

    Article  CAS  PubMed  Google Scholar 

  • Oka T, Mazack V, Sudol M (2008) Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP). J Biol Chem 283:27534–27546

    Article  CAS  PubMed  Google Scholar 

  • Oliver-De La Cruz J, Nardone G, Vrbsky J, Pompeiano A, Perestrelo AR, Capradossi F, Melajová K, Filipensky P, Forte G (2019) Substrate mechanics controls adipogenesis through YAP phosphorylation by dictating cell spreading. Biomaterials 205:64–80

    Article  CAS  PubMed  Google Scholar 

  • Pathak MM, Nourse JL, Tran T, Hwe J, Arulmoli J, Dai Trang TL, Bernardis E, Flanagan LA, Tombola F (2014) Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells. Proc Natl Acad Sci USA 111:16148–16153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pelham RJ, Wang YL (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA 94:13661–13665

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pethe P, Pursani V, Bhartiya D (2015) Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro. Cell Biol Int 39:600–610

    Article  CAS  PubMed  Google Scholar 

  • Pijuan-Galitó S, Tamm C, Annerén C (2014) Serum inter-α-inhibitor activates the Yes tyrosine kinase and YAP/TEAD transcriptional complex in mouse embryonic stem cells. J Biol Chem 289:33492–33502

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Price AJ, Huang EY, Sebastiano V, Dunn AR (2017) A semi-interpenetrating network of polyacrylamide and recombinant basement membrane allows pluripotent cell culture in a soft, ligand-rich microenvironment. Biomaterials 121:179–192

    Article  CAS  PubMed  Google Scholar 

  • Qin H, Hejna M, Liu Y, Percharde M, Wossidlo M, Blouin L, Durruthy-Durruthy J, Wong P, Qi Z, Yu J, Qi LS (2016) YAP induces human naive pluripotency. Cell Rep 14:2301–2312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rosado-Olivieri EA, Anderson K, Kenty JH, Melton DA (2019) YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells. Nat Commun 10:1–11

    Article  CAS  Google Scholar 

  • Sun Y, Yong KMA, Villa-Diaz LG, Zhang X, Chen W, Philson R, Weng S, Xu H, Krebsbach PH, Fu J (2014) Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nat Mater 13:599–604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676

    Article  CAS  PubMed  Google Scholar 

  • Tamm C, Böwer N, Annerén C (2011) Regulation of mouse embryonic stem cell self-renewal by a Yes-YAP-TEAD2 signaling pathway downstream of LIF. J Cell Sci 124:1136–1144

    Article  PubMed  Google Scholar 

  • Udan RS, Kango-Singh M, Nolo R, Tao C, Halder G (2003) Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway. Nat Cell Biol 5:914–920

    Article  CAS  PubMed  Google Scholar 

  • Varelas X, Sakuma R, Samavarchi-Tehrani P, Peerani R, Rao BM, Dembowy J, Yaffe MB, Zandstra PW, Wrana JL (2008) TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal. Nat Cell Biol 10:837–848

    Article  CAS  PubMed  Google Scholar 

  • Vining KH, Mooney DJ (2017) Mechanical forces direct stem cell behaviour in development and regeneration. Nat Rev Mol Cell Biol 18:728–742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walker A, Su H, Conti MA, Harb N, Adelstein RS, Sato N (2010) Non-muscle myosin II regulates survival threshold of pluripotent stem cells. Nat Commun 1:1–9

    Article  CAS  Google Scholar 

  • Wen JH, Vincent LG, Fuhrmann A, Choi YS, Hribar KC, Taylor-Weiner H, Chen S, Engler AJ (2014) Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nat Mater 13:979–987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu S, Huang J, Dong J, Pan D (2003) Hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with Salvador and Warts. Cell 114:445–456

    Article  CAS  PubMed  Google Scholar 

  • Zhao Y, Fei X, Guo J, Zou G, Pan W, Zhang J, Huang Y, Liu T, Cheng W (2017) Induction of reprogramming of human amniotic epithelial cells into iPS cells by overexpression of Yap, Oct4, and Sox2 through the activation of the Hippo-Yap pathway. Exp Ther Med 14:199–206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We gratefully acknowledge Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, New Delhi for financially supporting this project (Grant No. BT/PR28474/MED/31/393/2018); and Human Resource Development Group, Council of Scientific and Industrial Research (CSIR-HRDG), Government of India, New Delhi for awarding Jasmeet Kaur Virdi with Senior Research Fellowship. We would also like to acknowledge Sunandan Divatia School of Science, SVKM’s NMIMS (deemed to-be) University and Symbiosis Centre for Stem Cell Research (SCSCR), Symbiosis International University, for providing the necessary resources and infrastructure.

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  1. Department of Biological Sciences, Sunandan Divatia School of Science, SVKM’s NMIMS (deemed-to-be) University, Mumbai, Maharashtra, 400056, India

    Jasmeet Kaur Virdi

  2. Symbiosis Centre for Stem Cell Research (SCSCR), Symbiosis School of Biological Sciences (SSBS), Symbiosis International University, Pune, Maharashtra, 412115, India

    Prasad Pethe

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  1. Jasmeet Kaur Virdi
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PP proposed the concept of the study. JKV performed the experiments and wrote the manuscript. All authors contributed in design of the project, acquisition, data analysis and data interpretation. All authors were involved in revising the manuscript for important intellectual content and approval of the final version of the manuscript.

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Correspondence to Prasad Pethe.

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Virdi, J.K., Pethe, P. Soft substrate maintains stemness and pluripotent stem cell-like phenotype of human embryonic stem cells under defined culture conditions. Cytotechnology 74, 479–489 (2022). https://doi.org/10.1007/s10616-022-00537-z

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