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Self-Organized Amniogenesis by Human Pluripotent Stem Cells in a Biomimetic Implantation-Like Niche

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Self-Organized Amniogenesis by Human Pluripotent Stem Cells in a Biomimetic Implantation-Like Niche LETTERS PUBLISHED ONLINE: 12 DECEMBER 2016 | DOI: 10.1038/NMAT4829 Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche Yue Shao1†, Kenichiro Taniguchi2†, Katherine Gurdziel3, Ryan F. Townshend2, Xufeng Xue1, Koh Meng Aw Yong1, Jianming Sang1, Jason R. Spence2, Deborah L. Gumucio2* and Jianping Fu1,2,4* Amniogenesis—the development of amnion—is a critical factors seen in the in vivo amniogenic niche: a three-dimensional developmental milestone for early human embryogenesis (3D) extracellular matrix (ECM) that is provided by the basement and successful pregnancy1,2. However, human amniogenesis membrane surrounding the epiblast during implantation11; and a is poorly understood due to limited accessibility to peri- soft tissue bed provided by the uterine wall and trophoblast to implantation embryos and a lack of in vitro models. Here support the developing amnion (Fig. 1a,b). Since amniogenesis ini- we report an ecient biomaterial system to generate human tiates from the expanding pluripotent epiblast, we utilized mTeSR1 amnion-like tissue in vitro through self-organized development medium and basement membrane matrix (Geltrex) to render the of human pluripotent stem cells (hPSCs) in a bioengineered culture permissive for pluripotency maintenance. niche mimicking the in vivo implantation environment. We In this culture system, H9 human embryonic stem cells (hESCs) show that biophysical niche factors act as a switch to toggle were plated as single cells at 30,000 cells cm−2 onto a thick, hPSC self-renewal versus amniogenesis under self-renewal- soft gel bed of Geltrex (with thickness ≥100 µm, bulk Young's permissive biochemical conditions. We identify a unique modulus ∼900 Pa, coated on a glass coverslip), in mTeSR1 medium molecular signature of hPSC-derived amnion-like cells and supplemented with the ROCK inhibitor Y27632 (Fig. 1b). At 24 h show that endogenously activated BMP–SMAD signalling is (day 1), medium containing Y27632 was replaced by fresh mTeSR1 required for the amnion-like tissue development by hPSCs. This supplemented with 4% (v/v) Geltrex to establish a 3D implantation- study unveils the self-organizing and mechanosensitive nature like niche (referred to henceforth as the `Gel-3D' condition). of human amniogenesis and establishes the first hPSC-based To assess the effect of ECM dimensionality and matrix rigidity, model for investigating peri-implantation human amnion respectively, several modifications of this Gel-3D condition were development, thereby helping advance human embryology and tested (Fig. 1b). First, the Geltrex supplement was excluded from reproductive medicine. the medium, with the gel bed retained (referred to henceforth as During implantation of a human embryo, amnion cells the `Gel-2D' condition). Second, the soft gel bed was replaced by (amnioblasts) are the first differentiated cell group emerging from a 1% Geltrex-coated glass coverslip (referred to henceforth as the an expanding pluripotent epiblast population and will give rise to a `Glass-3D' condition). Finally, a standard 2D culture, using a 1% polarized squamous amniotic epithelium that encloses the amniotic Geltrex-coated glass coverslip (referred to henceforth as the `Glass- cavity1,2 (Fig. 1a). Despite its basic and clinical significance, 2D' condition), was examined as a control that maintains hPSC amnion development in humans is poorly understood due to self-renewal. Culture medium was replenished daily. Analyses were limited studies on peri-implantation human embryos2 and drastic performed at day 5 unless otherwise noted (Fig. 1b). differences in amniogenesis between human and other commonly In the Glass-2D condition, apico-basally polarized hESC colonies used amniote models1,3. Even with recent progress in developing were observed at day 5. Strikingly, in the Gel-2D, Glass-3D in vitro systems4,5, including in vitro-cultured human embryos6,7, for and Gel-3D conditions, hESCs formed 3D cysts with EZRINC studying early human embryogenesis, the development of human apical surfaces facing inward, reflecting the intrinsic lumenogenic amnion remains mysterious. property of hESCs5,7. In both Gel-2D and Glass-3D, >90% Human pluripotent stem cells (hPSCs), which reside in a de- of lumenal cysts are made of tall, columnar E-CADHERINC velopmental state similar to pluripotent epiblasts8,9, have been suc- (ECADC) epithelial cells with apico-basally elongated nuclei and cessfully utilized for modelling post-gastrulation human embry- thick epithelium (Fig. 1c–e and Supplementary Fig. 1). In distinct onic development4,10. However, the applicability of hPSCs for mod- contrast, >90% of cysts formed in Gel-3D show a squamous elling peri-implantation, pre-gastrulation developmental events, epithelial morphology featuring flattened, laterally elongated cell such as amniogenesis, remains undetermined. Here we adapted a nuclei and reduced epithelium thickness, as well as unique biomimicry approach to engineer a biomaterial-based in vitro hPSC ECADC protrusions extending from basal surfaces (Fig. 1c–e and culture system for efficient generation of early human amniotic Supplementary Fig. 1). tissue. Specifically, we constructed a biomimetic implantation-like Notably, all 3D columnar epithelial cysts that formed in the niche for cultured hPSCs by implementing two major biophysical Gel-2D and Glass-3D conditions express the pluripotency markers 1Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA. 2Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. 3Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan 48202, USA. 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA. †These authors contributed equally to this work. *e-mail: [email protected]; [email protected] NATURE MATERIALS | VOL 16 | APRIL 2017 | www.nature.com/naturematerials 419 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. LETTERS NATURE MATERIALS DOI: 10.1038/NMAT4829 a b Plate hPSC on Glass or Gel bed Trophoblast +Y27632 Replenish culture medium Trophoblast Blastocoele Blastocoele Hypoblast Day 0 1 2 3 4 5 Primitive endoderm Epiblast Inner cell mass ± Amniotic cavity 3D ECM Analysis Amniogenesis Amnion/amnioblast −Y27632 (3D ECM, soft tissue bed) Maternal tissue Maternal tissue Glass-2D Glass-3D Gel-2D Gel-3D Glass Gel bed 3D ECM overlay c Glass-2D Gel-2D Glass-3D Gel-3D d Glass-2D Gel-2D Glass-3D Gel-3D Day 5 ECAD Hoechst section Y − X WGA Hoechst section Hoechst Z − EZRIN X e f Glass-2D Gel-2D Glass-3D Gel-3D api-bas lat api-bas lat g Epithelium thickness ( WGA ∗∗∗ NANOG ∗∗∗ ∗∗∗ 35 Glass-2DGlass-3DGel-2DGel-3D Columnar 2.5 ∗∗∗ 30 NANOG 25 2.0 OCT4 1.5 20 WGA 15 OCT4 SOX2 1.0 10 0.5 ECAD 5 μ m) (apico-basal/lateral) Squamous 0.0 0 GAPDH Normalized nucleus dimension Normalized WGA Gel-2D Gel-3D Gel-2D Gel-3D SOX2 Glass-3D Glass-3D Figure 1 | hPSCs form squamous cysts with amnion-like morphology in an implantation-like niche. a, Development of amnion/amnioblasts from epiblasts in a peri-implantation human embryo1. Amniogenesis in vivo occurs in a biophysical niche featuring a soft tissue bed (maternal tissue and invading trophoblasts) and a 3D extracellular matrix (ECM) provided by the overlying primitive endoderm/hypoblasts. b, hPSC amniogenesis assay. c, Schematic diagrams showing hPSC morphogenesis under dierent culture conditions (top). Confocal micrographs showing the X–Y (middle) and X–Z (bottom) sections of the hPSC monolayer and cysts formed in the indicated conditions at day 5. EZRIN (green) demarcates apical surfaces. Hoechst (blue) counterstains nuclei. d, Confocal micrographs showing staining of E-CADHERIN (ECAD, green; top), wheat germ agglutinin (WGA, red; bottom), and Hoechst (blue, nucleus) in hPSCs cultured under the indicated conditions. Squamous cells exhibit basal ECADC protrusions, highlighted by white arrowheads. nD16 independent experiments. e, Box charts showing normalized nucleus dimension (left) and epithelium thickness (right) for hPSC cysts in the indicated conditions (box: 25–75%, bar-in-box: median, and whiskers: 1% and 99%). The schematic diagrams show columnar versus flat, squamous cyst morphologies and the apico-basal (api-bas) and lateral (lat) directions. ncell D179, 254 and 243, and ncyst D11, 11 and 23, for Gel-2D, Glass-3D and ∗∗∗ Gel-3D, respectively. nD4 independent experiments. P values were calculated using unpaired, two-sided Student’s t-test. P<0.001. f, Confocal micrographs showing NANOG (top), OCT4 (middle), and SOX2 (bottom) immunostaining in hPSCs cultured under the indicated conditions. WGA co-staining shows cell morphology. nD9 independent experiments. g, Western blot showing protein levels of NANOG, OCT4, SOX2, ECAD and GAPDH in hPSCs cultured under the indicated conditions. nD3 independent experiments. Scale bars in c,d,f, 50 µm. NANOG, OCT4 and SOX2, consistent with the well-known markers, the squamous cyst maintains an epithelial phenotype, association between columnar epithelial morphology and retaining expression of ECAD/CDH1 and CLDN6 (ref. 12; Fig. 1d,g pluripotent epiblast in vivo2,6,9 (Fig. 1f,g). However, in squamous and Supplementary Fig. 2b). This spontaneously differentiated cysts that formed in Gel-3D, expression of NANOG, OCT4 and squamous epithelial cystic tissue is morphologically reminiscent of SOX2 protein is lost, suggesting that they are composed of a the developing amnion
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