Implantation-Competent Blastocyst-Like Structures from Mouse Pluripotent Stem Cells

Implantation-Competent Blastocyst-Like Structures from Mouse Pluripotent Stem Cells | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Implantation-Competent Blastocyst-Like Structures from Mouse Pluripotent Stem Cells Cody Kime , Hiroshi Kiyonari , Satoshi Ohtsuka , Eiko Kohbayashi , Michio Asahi , Shinya Yamanaka , Masayo Takahashi , Kiichiro Tomoda doi: https://doi.org/10.1101/309542 Cody Kime 1 Gladstone Institute of Cardiovascular Disease , San Francisco, CA 94158, USA 2 Lab of Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research , Kobe 650-0047, Japan 8 Lead Contact Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hiroshi Kiyonari 3 Animal Resource Development Unit and Genetic Engineering Team, RIKEN Center for Life Science Technologies , Kobe 650-0047, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Satoshi Ohtsuka 4 Department of Life Science, Medical Research Institute, Kanazawa Medical University , Ishikawa 9200293, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eiko Kohbayashi 5 Second Department of Internal Medicine, Osaka Medical College , Osaka 569-8686, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michio Asahi 6 Department of Pharmacology, Faculty of Medicine, Osaka Medical College , Osaka 569-8686, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shinya Yamanaka 1 Gladstone Institute of Cardiovascular Disease , San Francisco, CA 94158, USA 7 Center for iPS Cell Research and Application (CiRA), Kyoto University , Kyoto 606-8507, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Masayo Takahashi 2 Lab of Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research , Kobe 650-0047, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kiichiro Tomoda 1 Gladstone Institute of Cardiovascular Disease , San Francisco, CA 94158, USA 6 Department of Pharmacology, Faculty of Medicine, Osaka Medical College , Osaka 569-8686, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF SUMMARY Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous types of cells, including germ cells and extended pluripotency stem cells, have been generated from pluripotent stem cells (PSCs) in-vitro , generating functional BCs only from PSCs has not yet been reported. Here we describe induced self-organizing 3D BC-like structures (iBCs) generated from mouse PSC culture in-vitro . Resembling natural BCs, iBCs have a blastocoel-like cavity and were formed with outer cells that are positive for trophectoderm lineage markers and with inner cells that are positive for pluripotency markers. iBCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBCs are implantation-competent. iBC production required the transcription factor Prdm14 and iBC precursor intermediates concomitantly activate the MERVL totipotency related cleavage stage reporter. Thus, our system may contribute to understanding molecular mechanisms underpinning totipotency, embryogenesis, and implantation. HIGHLIGHTS -Pluripotent cells self-organize blastocyst-like structures in defined conditions. -Structures have several extraembryonic and embryonic characteristics of blastocysts. -Structures can implant in the uterus and grow before resorption. -Totipotency is implicated concomitantly at loci that originate induced blastocysts. INTRODUCTION During early mammalian development, a fertilized egg (zygote) completely intersects the animal life cycle upon zygotic genome activation (ZGA): the event where gamete totipotent genomes of the pronucleus are epigenetically activated and rapidly enter cleavage ( Seydoux and Braun, 2006 ; Wu et al., 2017 ). The zygote cleaves and symmetry later bifurcates to form the blastocyst (BC) in preparation for implantation and differentiation. The BC is a 3D ball-like structure of three characteristic parts: the extraembryonic (ExEm) outer layer of trophectoderm (TE) lineage cells, the pluripotent cells of the inner cell mass (ICM), and a fluid filled cavity called the blastocoel. Upon implantation, trophoblasts contribute to the ExEm tissue of the placenta, and the ICM gives rise to embryo proper and some ExEm tissues. Emerging trophoblasts and pluripotent cells result from the first differentiation event in mammalian development, initiated in cleaving totipotent cells that begin to polarize just before the BC forms ( Hirate et al., 2015 ; Nishioka et al., 2009 ; Yu et al., 2016 ; Stephenson et al., 2010 ). Implantation is crucial to natural development and establishes the physical connection between the mother and early embryo that supports embryonic (Em) development through the rest of the pregnancy. Implantation is tightly regulated at several molecular and cellular levels: apposition, adhesion and invasion of the TE lineage cells, and subsequent decidualization of the endometrial wall of the uterus. ExEm tissues thereafter increase growth and differentiation while the ICM differentiates to form the embryo proper and additional ExEm tissues. Implantation-competent BCs require TE cells expressing Cdx2 ( Meissner and Jaenisch, 2006 ), and molecular mechanisms involving Lpar3, Lif, Bmp, and others signal the interface between the TE and the receptive uterus ( Cha et al., 2012 ; Wang and Dey, 2006 ). These events must occur in a short developmental window: failed implantation is a major cause of early pregnancy loss in humans ( Norwitz et al., 2001 ; Cha et al., 2012 ). Defective embryos also fail later and begin the resorption process in which maternal immune cells degrade the embryo ( Cossée et al., 2000 ; Flores et al., 2014 ). The zygote and cleavage stages exhibit true totipotency, isogenically preceding all ExEm (vegetal) and Em (animal) cell bi-directional development toward entire organisms. From plant tissue cultures, specific cytokine, vitamin, and plant hormone (auxins) ratios are adjusted to induce totipotent transient cells for propagating isogenic embryos ( Steward et al., 1958 ). In mammals, isogenic 3D BCs from differentiated cells are both attractive and elusive. Recent progress in PSC research challenges this barrier by generating functional germ cells that give rise to offspring by in-vitro fertilization ( Hikabe et al., 2016 ), and some reports show stem cells with extended/bi-directional pluripotency in chimeric mice ( Macfarlan et al., 2012 ; Yang et al., 2017 ). However, experiments inducing implantation-competent isogenic BCs entirely from PSCs are unprecedented. In reprogramming and conversion experiments with specific cytokines, nutrient, and lipid ( Kime et al., 2016 ), we frequently observed tissues and hemispheres that resemble BCs. Inspired by these observations, we developed a stepwise regime to readily induce BC-like structures from PSCs in-vitro , which we term iBCs. iBCs demonstrate implantation-competence since transplant into pseudopregnant mice induced focal decidualization in the uterus, recruited a maternal blood supply, and expanded the embryonic cavity. Some implanted iBCs produced many cell types similar to implanted embryos but failed to develop further due to embryonic resorption. A live pluripotency reporter suggests pluripotency is partially recovered in the putative ICM of iBCs, and some iBC express Zscan4 . Utilizing the murine endogenous retrovirus ( MERVL ) live totipotency-related reporter, we found iBC precursors and cells where iBCs originate may indicate ZGA mechanisms ( Macfarlan et al., 2012 ; Wu et al., 2017 ). Further analysis of Yap protein distribution in iBC precursors and early iBCs was similar with cleavage stage cells polarizing through compaction toward emerging blastocysts ( Nishioka et al., 2009 ; Stephenson et al., 2010 ; Bedzhov et al., 2014 ). We anticipate this approach may lead to simplified isogenic embryo production for research, medicine, and uncovering the intricacies of totipotency and implantation. RESULTS Efficient Conversion to Naive PSCs Produces Blastocyst-like Hemispheres In-vitro pluripotency is characterized in two distinct states: a post-implantation epiblast state (primed) and a pre-implantation blastocyst ICM state (naive). Primed female PSCs have one active and one inactive X chromosome (Xa/Xi), but naive female PSCs have two Xas (Xa/Xa) ( Payer et al., 2011 ). We previously presented defined conditions that enhanced iPS cell reprogramming, and in primed to naive PSC conversion experiments, those conditions formed structures resembling early embryonic material. In the naive conversion experiments, we used a primed female mouse epiblast stem cell (mEpiSC) line that harbors a silent green fluorescent protein (GFP) transgene on the Xi chromosome (Xi-GFP, XGFP-). GFP is expressed upon reactivation of Xi to Xa (Xa-GFP, XGFP+), a hallmark of the ICM and often of cleavage stage cells ( Kime et al., 2016 ; Monk and Harper, 1979 ; Okamoto et al., 2004 ; Bao et al., 2009 ). Robust naive conversion frequently produced hemispheres with morphological characteristics we suspected to resemble BCs ( Figure 1 ). XGFP+ cell clusters, which we found to be naive cells ( Kime et al., 2016 ), were polar and internal to fluid-filled hemispherical domes of distinct large flat cells with ectodermal morphology resembling trophoblasts. Using immunocytochemistry, we found that these hemispheres initiated the fluid-filled cavity and had NANOG+XGFP+ inner cells with no bright DNA-stain punctae, which may indicate loss of heterochromatin usually found in scarce transient Zscan4+ 2C-like state cells in mouse naive PSCs ( Akiyama et al., 2015 ; Wu et al., 2016 ). The inner cells were surrounded by spheroid DNA-stained punctae-enriched NANOG+XGFP− outer cells. These cell distributions and expression characteristics were similar to early differentiating cells of the morula. Also, the most-outward cells were flattened and NANOG-XGFP−, characteristic of lineage committed TE. ( Figure 1A , Figure S1A ). Download figure Open in new tab FIGURE 1: Blastocyst-Like Hemispheres Suggest Bi-Directional Potential For All: Green = XGFP+ (Xa/Xa-GFP), Red = NANOG, Light Blue = DNA; Hoechst 33342. All scale bars = 100 μm . A ) Blastocoel-like fluid filled oversized hemisphere with NANOG+XGFP+ inner cells, NANOG+XGFP-spheroid cells, and NANOG-XGFP− in flattened TE-like cells. XGFP+ cells exclusively indicate euchromatin characteristics ( Figure S1A ). B ) Late BC-like hemisphere with NANOG+ cells restricted to XGFP+ cells and NANOG-XGFP− TE-like cells surrounding the fluid filled cyst. C ) TE lineage marker positive cells (white; TROMA-I) surrounding the fluid filled hemisphere with oversized NANOG+XGFP+ polar mass. D ) X chromosome reactivation indicated by XGFP+ cells as polar masses among fluid filled hemispheres in naive conversion experiments. In a short time, the outer XGFP− cells completely flattened, took on morphology similar to TE, and surrounded the expanding fluid-filled cyst, wherein only the XGFP+ polar mass of the hemisphere maintained XGFP+ and NANOG+ expression ( Figure 1B,C , Figure S1B , Video S1). We regularly observed tens to hundreds of such characteristic hemispheres during our previous study ( Figure 1D , Figure S1A , Kime et al., 2016 ). Time-course reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments of naive conversion experiments revealed the induction of Prdml(Blimpl), Prdm14, Id1, Id2, Id3, and Id4 ( Figure S1C ). These powerful genes broadly regulate the genome and are curiously related to the cleavage stage, early embryo, and germ line preparation ( Yang et al., 2017 ; Hiller et al., 2010 ; Yamaji et al., 2008 ; Luna-Zurita and Bruneau, 2013 ; Burton et al., 2013 ). The composition and organization of hemispheres drew our attention since data continued to implicate a BC: the GFP+NANOG+ polar mass of cells corresponds to the ICM where pluripotent Xa/Xa naive cells exist, and the cavity to a blastocoel. XGFP-NANOG- flattened cells had the morphology and organization of TE cells. Therefore, we checked hemispheres with an antibody specific to TROMA-I (KRT8), a well-characterized TE lineage marker. Indeed, the flattened TE morphology cells, but not XGFP+NANOG+ cells, expressed TROMA-I, and thus, the hemispheres are surrounded by XGFP-NANOG- TROMA-I+ cells ( Figure 1C ). Taken together, our efficient conversion of the primed state mEpiSCs to naive PSCs is concurrent with the generation of highly self-organized BC-like hemispheres. SMAD2/3 Signaling Inhibition and Stepwise Treatment Produces Self-Organizing Floating BlastocystLike 3D Structures We tested several culture conditions to enhance the conversion efficiencies from primed to naïve PSCs. For example, adding the SMAD2/3 signaling pathway ALK5 inhibitor SB431542 had marginal effects on conversion efficiencies, yet we observed some small cell aggregates and BC-like spheres floating in the medium. We speculated that the floating spheres had BC-like properties as the hemispheres and the cell aggregates were precursors of the BC-like spheres. To yield floating BC-like structures more stably and efficiently, we optimized Phase 1 and 2 treatments with our defined supplements ( Figure 2A ), harvesting plate supernatants to low attachment plates on Day 6 and obtaining 5–30 floating BC-like structures by Day 7 ( Figure S2A , Table S1 ). On Day 6 of purification, the floating structures did not stick together. Like late-hatched BCs, on Day 7 or 8, as the BC-like structures expanded, they drastically slowed growth and readily stuck together. Thus, we routinely isolated and pooled these structures at Day 7 for most downstream experiments ( Figure 2B ). We stained the DNA to clarify cell nuclei and found a compact ICM-like region and large flat TE-like cells surrounding a possible blastocoel similar to the hemispheres ( Figure S2B ). From these observations and more hereafter, we termed these structures induced blastocysts (iBCs). Download figure Open in new tab FIGURE 2: Defined Conditions Release Early Embryo-Like iBC-PCs and Polarizing iBCs to Suspension A ) Two-phase iBC induction media timing to induce mEpiSC to iBCs. **Supernatant iBC-PC are collected to ultra-low attachment(ULA) wells on Day 6, and high-quality BC-like iBCs are selected on Day 7 by embryo pipette . B ) iBCs are pooled in ULA plate for downstream experiments. 5cale bar = 200 μm . C ) Isolated predicted iBC-PC developing into iBC over time. Scale bars = 100 μm . D ) iBC-PC stained for YAP (magenta) and DNA (light blue, Hoechst 33342). Scale bars = 50 μm. Nuclear-excluded YAP region is outlined with dotted white line . E ) Early iBCs and early BCs stained for YAP (magenta) and DNA (light blue, Hoechst 33342). Scale bars = 50 μm. Nuclear-excluded YAP region is outlined with dotted white line . Next, to ask if iBCs originate from a single source, we isolated the floating aggregate spheres that visibly lacked any particular polarity on Day 5.5 of induction ( Figure 2C , left panel). The isolated spheres were individually cultured and regularly observed. Most grew and changed morphology: some developed into iBCs ( Figure 2C ). In some occasions, the isolated sphere appeared to change in the first 17 hours, resulting in mostly large round cells among a few cell types. In the next 28 hours, the cells divided and grew to further change morphology, forming an apparent cavity and became an iBC ( Figure 2C ). Prolonged culture thereafter resulted in paused growth and slightly reduced size of the iBC structure. These observations led us to speculate that stepwise treatment of PSC culture in-vitro induces floating aggregates as iBC precursors (iBC-PC) that morphologically develop and expand as iBCs. To examine early polarity and inner/outer cell likeness, we collected iBC-PCs for immunofluorescent staining of YAP, a transcription factor involved in the first positional information and differentiation of the outer and inner cells of the early mouse embryo ( Nishioka et al., 2009 , Bedzhov et al., 2014 ). Similar to late cleavage stage non-polarized and early embryos ( Hirate et al., 2015 ; Nishioka et al., 2009 ; Yu et al., 2016 ), YAP distribution was homogenously cytosolic and nuclear among all cells in some iBC-PCs with stochastic nuclear positioning ( Figure 2D , left). However, other iBC-PCs more closely reflected 8C/16C compacting embryos undergoing outer/inner cell polarization; YAP was excluded only from the nucleus of the inner cells ( Figure 2D , middle and right). We then examined emergent early iBCs and found that YAP was excluded from the nucleus of iBC inner cells and enriched in the nucleus of iBC outer cells, similar to natural early embryos ( Figure 2E , Figure 3B ), although iBC putative ICMs appeared smaller. These strikingly similar distributions of YAP suggest the same molecular mechanisms and signaling pathways of early embryos are installed in iBC-PCs and early iBCs. Download figure Open in new tab FIGURE 3: iBCs Share Many Molecular Characteristics with BCs A ) RT-qPCR of single BC, iBC, and mEpiSC cDNA samples, with Euclidean distance and clustering by average linkage, represented as a heat map of global ΔCT to Gapdh . B ) Early iBCs and early BCs stained for YAP (magenta), CDX2 (green) and DNA (light blue, Hoechst 33342). Scale bars = 50 μm. Nuclear-excluded YAP region is outlined with dotted white line . C ) Early iBC and early BC stained for for TROMA-I (white), OCT4 (yellow), and DNA (light blue, Hoechst 33342). Comparable microscopy setting detector gains have target matched colors (see methods; Table S2 ). Scale bars = 50μum . D ) mEpiSC express live pluripotency reporter EOS:: RFP, and late iBC above culture with EOS::RFP expression largely in the putative ICM. White stars label out of focus EOS:: RFP+ cells on the plate. Scale bars = 100 μm . BCs have an outer layer of trophoblast cells and an ICM of pluripotent cells. Thus, BCs express genes important for inducing and maintaining both lineages. To analyze BC gene expression, we extracted RNA from individually isolated BCs and iBCs, along with isolated mEpiSC colonies for a similar sized control ( Figure 3A ). We also sampled RNA from earlier emerging individually isolated BCs and iBCs and mEpiSC colonies accordingly ( Figure S2C ). RT-qPCR experiments revealed that each iBC exhibits slightly different gene expression patterns as do BCs, suggesting there is a difference in the quality, developmental timing, or both, in iBC preparations. However, overall, the key genes analyzed are expressed in iBCs at closer levels to BCs than mEpiSCs except for a few genes ( Figure 3A ). Many of the genes examined that are first activated or maintained in totipotent cleavage stage cells were found strongly upregulated in iBCs to match BCs when compared with mEpiSCs ( Figure 3A , Figure S2C ). Interestingly, one early BC and one early iBC had comparable detectable levels of Zscan4 ( Figure S2C ). In addition, the cleavage stage and naive pluripotency-related gene Zfp42 (Rex1) was activated in early iBCs sampled (four of six), despite being undetectable in mEpiSCs ( Figure S2C ). Among those genes, Atp1b1 expression was particularly striking since Atp1b1 encodes a subunit of Na+/K+ ATPase pump, and its expression was comparable to BCs where it is essential for blastocoel formation and proper tight junctions of trophoblast cells ( Hamatani et al., 2004 ; Madan et al., 2007 ). Thus, Atp1b1 expression may be consistent with formation of a blastocoel-like fluid-filled cavity and outer layer cell tight junctions in iBCs. Genes that are involved in the outer cell lineage induction and/or function (e.g., Cdx2, Gata3 , and Krt8 (Troma-I)) were also strongly induced in iBCs ( Figure 3A ). Cdx2 was not equally expressed to BCs, but Gata3 and Krt8 were comparable. These data, with the morphology of iBC outer layer cells, suggest that a functional TE lineage may be established. In contrast, master pluripotent transcription factors Nanog and Pou5f1 (Oct4) were lower in iBCs than in BCs and mEpiSCs, but still detected in many iBCs ( Figure 3A , Figure S2C ). Sox2 was only detected in the Zscan4+ iBC ( Figure S2C ). Furthermore, we rarely observed XGFP+ cells in iBCs (data not shown). These expressions of the pluripotency genes and XGFP are different from those in naive conversion hemispheres on the plate where XGFP+ cells expressed pluripotency genes similar to other PSCs. However, even during the naive conversion, Nanog is once downregulated but quickly recovers to the same level as in PSCs, dependent on LIF signaling ( Kime et al 2016 ). Thus, early pluripotency may be induced but is not activated strongly during iBC induction. Low-level expression of Cdx2 and Oct4 mRNA raised the question of whether iBCs correctly possess the TE lineage outer and ICM inner cell populations and are organized like BCs. To address this, we examined detection and localization of CDX2 and OCT4, along with YAP and the TE marker TROMA-I (KRT8), in early iBCs and early BCs by staining with well-characterized antibodies. The iBC inner cells downregulated CDX2 and YAP similar to BCs, but CDX2 in iBC outer cells was evenly localized and was not enriched in many nuclei ( Figure 3B ). These results suggest that expression and phosphorylation of CDX2 in iBCs are poorly regulated ( Rings et al., 2001 ). Immunostaining iBCs with TROMA-I antibody revealed that outer layer TE-like flat cells are strongly positive for TROMA-I, similar to BCs ( Figure 3C ). Additionally, the inner cell region of the iBCs did not exhibit strong TROMA-I signals, similar to BCs, which suggested that the inner cells are not in the TE lineage. Conversely, immunostaining iBCs with OCT4 antibody clearly showed nuclear OCT4 in the inner cell region ( Figure 3C ; Bulut-Karslioglu et al., 2016 ; Ralston and Rossant, 2008 ). Like BCs, The nuclear signals of iBC inner cells were stronger than those in the outer layer flat cells, although iBCs have a weaker overall OCT4 signal than BCs. To investigate these differences further, we used two mainstream confocal microscopes to prepare four early iBC images as control settings to compare with early BCs. The detector gains required for a comparable image capture suggested that the strong TROMA-I signals in the outer layer cells are consistent with higher expression levels of Troma-I (Krt8) mRNA in iBCs. However, between iBCs and BCs, the detector gain difference for OCT4 signal was less than that of Pou5f1 (Oct4) mRNA, suggesting post-transcriptional regulation of Oct4 ( Figure 3A,C , Figure S2C , Table S2 ). At last, to examine Oct4 function as a transcription factor, we used the EOS-S(4+) synthetic live pluripotency reporter from the Sox2 genetic element Srr2 to drive a red fluorescent protein (RFP, EOS:: RFP). Srr2 elements require a heterodimer of pluripotency transcription factors OCT4 and SOX2 to activate ( Hotta et al., 2009 ; Tomioka et al., 2002 ). We introduced the reporter construct into mEpiSCs and established recombinant EOS:: RFP mEpiSCs ( Figure 3D ). As expected, mEpiSCs were strongly EOS:: RFP positive, and the RFP was not detected in differentiated cells, as reported ( Figure 3D , Figure S2E ; Hotta et al 2009 ; Tomioka et al 2002 ). We generated iBCs from EOS:: RFP mEpiSCs, and the resulting iBCs often exhibited RFP signals ( Figure 3D ). Importantly, the RFP signals were stronger in the putative ICM than outer cells, suggesting OCT4 and SOX2 in the inner cell regions of iBCs better activate or maintain EOS:: RFP expression. Yields from three iBC production wells were 14, 20, and 27 iBCs, and among those iBCs, 12, 13, and 20 had detectable EOS:: RFP+ ( Figure S2F ). These data suggest iBCs have an ICM-like region where some pluripotency transcription network is operative. Thus, iBC-PCs and iBCs exhibit several unique features that are common with preimplantation embryos at morphological and molecular levels. We concluded that the stepwise manipulation of signaling pathways triggered, to some extent, a dynamic self-organizing event reminiscent of the first polarization, differentiation, and morphogenesis of early embryonic development. Reproducibility of iBC Generation Our various XGFP mEpiSC reporter sub-lines performed similarly throughout iBC induction. Two published mEpiSC lines reacted similarly throughout and produced iBCs with lower yields ( Figure S2G ; Tesar et al., 2007 ; Ohtsuka et al., 2012 ). Another mEpiSC line with apparent cell culture characteristic differences failed completely ( Parchem et al., 2014 ). Therefore, iBC generation should be possible with many but not all mEpiSC lines. Purified iBCs Implant, Induce Decidualization, and Grow in Pseudopregnant Mice Implantation/Decidualization Rates of iBCs The characteristic similarities between iBCs and BCs led us to examine implantation-competence and developmental potency of iBCs in-utero . We collected iBC-PCs at Day 6 or 7 of induction and allowed them about 18 hours to expand toward iBCs that were purified with an embryo pipette by visual assessment and loosely pooled. Purified iBCs were transferred to the uterus horn of sterile-male bred pseudopregnant mice, and positive control BCs were transferred separately; we also transferred large mEpiSC colony clusters and embryoid bodies (EBs) prepared from mEpiSCs as controls. We dissected the transplanted mice between E5.5 and E7.5 and found that transferring iBCs induced deciduae in the pseudopregnant mice while mEpiSC clusters and EBs failed ( Figure 4A,B ). Deciduae induced by iBC transfer were similar in focal morphology to BC-induced deciduae although they were, on average, smaller in size than those from BC transfers. Importantly, many iBC-induced deciduae recruited large maternal blood vessels seen in the uterus, and sectioning showed red brown color in the decidua basalis region, similar to those from BCs, indicating significant blood supply from the mother ( Figure 4B ). These observations clearly show that iBCs induce decidualization of the uterus and recruit the maternal blood supply, which result from iBC implantation. Still, the smaller sized deciduae from iBCs suggested postimplantation developmental delay or resorption. Download figure Open in new tab FIGURE 4: iBC Uterus Transfer Decidualization in Pseudopregnant Mice Embryo pipettes indicate when a new pipette was used . A ) Single source uterus transfer experiment diagram. Observed deciduae in uterus horns with respect to EB, mEpiSC clusters, iBC, or control BC single source uterus transfers. B ) Uterus horn of mouse with iBC implanted deciduae (left, yellow arrows), prepared for cryosection (right). C ) EB or mEpiSC clusters or iBC co-transfer with BCs uterus transfer experiment diagram. Observed deciduae from EB co-transfer, mEpiSC Cluster co-transfer, and iBC co-transfer. * = control BC decidualization rate (69.2%, Figure 4A ); Red box indicates decidualization gained from iBCs.) D ) LCM genomic DNA PCR test for mouse genomic DNA universal and iBC-specific hygromycin resistance. LCM samples from figure H&E slides are shown in Figure S4A . By counting transferred iBCs or BCs and the resulting deciduae obtained, we calculated frequencies of decidualization ( Table S3 ). iBCs and BCs induced deciduae at 6.7% (10/149) and 69.2% (36/52), respectively ( Figure 4A ). Thus, iBCs alone induce decidualization but less frequently than BCs. The lower frequency by iBCs may be due to different iBC developmental size and timing among a heterogeneity in quality ( Figure 2 , Figure 3 , Figure S2 ). Co-transferring control embryos that easily implant improves implantation rates of difficult embryos in assisted reproductive settings ( Mochida et al., 2014 ). We therefore transferred iBCs in co-transfer with control BCs ( Figure 4C ). In total, we transferred 186 iBCs along with 109 BCs to numerous uteri, and upon dissection, we frequently observed more deciduae than the number of control BCs, implicating iBCs as the source of increased implantation. Among all co-transfer experiments, the total number of 116 observed deciduae divided by the total number of 109 control BCs would mean an impossible implantation rate of 106.4% (116/109) if control BCs were the only source ( Figure 4C , Table S3 ). We note that, since control BCs transferred alone implanted at 69.2% ( Figure 4A ) and others reported control BC implantation rates of 43-44% ( Bulut-Karslioglu et al., 2016 ), iBCs should be the source of the 37.2% difference, or increase the implantation of control BCs, or a combination therein ( Figure 4C ). Yet even if we assumed 100% success from all BCs, we performed 26 total iBC+BC co-transfer experiments, and 15 of those experiments (57.7%) induced more focal deciduae than the number of BCs ( Table S3 ). Given that co-transfer is more robust, we also examined co-transfer of mEpiSC clusters or EBs from mEpiSCs along with BCs. We found no deciduae from the EB+BC co-transfers, meaning the size or cells of EBs might impair the implantation process ( Figure 4C ). mEpiSC clusters with BCs proved to be a better control co-transfer experiment, allowing for the likely 68.8% implantation of control BCs similar to control BCs alone ( Figure 4A,C ). iBC implantation and decidualization in co-transfer with BC are higher than as a single source. To confirm the origin of the deciduae from both types of experiments, we obtained genomic DNA from some deciduae cryosections by laser capture microdissection (LCM) and amplified a transgenic DNA region specific to cells that generate iBCs ( Figure 4D , Figure S2D , Figure S4A ). This analysis confirmed that iBCs from both the single source and co-transfer experiments induced deciduae with detectable tissue at the proper location for natural embryos ( Figure 4B ). We therefore recognize that cotransferring BCs with iBCs enhances the ability of iBCs to induce decidualization and may prove more useful in later studies. Cryosection and Analysis of Implanted iBC-Derived Tissues We performed hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) on cryosections of dissected deciduae from E7.5 iBC single source transfer experiments and compared them to control embryos at E7.5 and E6.5 ( Figure 5 , Figure S4B ). Similar to control deciduae, iBC-induced deciduae were surrounded by uterine tissue and had distinct sub-regions; among those, the decidua basalis showed vascular sinus foldings and red blood cells, indicating maternal blood supply ( Figure 5 , Figure S3 , Figure S4A,B ). Sections often showed distinct disfigured tissues in the presumptive embryonic region with surrounding ExEm-like cells and internal small dark stained cells resembling the embryonic portion ( Figure 5A,C , Figure S3 , Figure S4A ). We tested proximal cryosections of the same deciduae for ExEm lineage TROMA-I and found that the surrounding iBC-derived ExEm-like tissues were TROMA-I+ and had invaded the deciduae, growing to a total size similar to or in excess of a control E6.5 embryo ( Figure 5B ). The cryosections showed tissues resembling a retracting parietal yolk sac cavity, a degrading putative Reichert’s membrane, and TROMA-I+ cells surrounding internal TROMA-I- cells that we speculated to be Em portion cells based on location and H&E stain characteristics ( Figure 5 , Figure S3 , Figure S4A ). While iBC-derived E7.5 tissues were larger than E6.5 control embryo tissues, many cells appeared pycnotic and lacked a healthy appearance, and were collectively smaller than a E7.5 control embryo ( Figure 4B ; Gardner and Johnson, 1972 ). Download figure Open in new tab FIGURE 5: iBCs Implant and Partially Develop Before Resorption EC, Embryonic Cavity A) H&E-stained proximal cryosections of deciduae from iBC single source transfer. Higher magnification is indicated and shows iBC-derived tissue resembling large cell masses of resorbing tissues. Scale Bars: Upper panels = 500 μm, Lower Left = 100 μm, and Lower Right = 200 μm . B) Cryosection IHC for ExEm TROMA-I (white), and DNA (light blue; Hoechst 33342). E6.5 and E7.5 control embryos show healthy size and structure. E7.5 iBC-derived tissues from cryosections proximal to Figure 5A and Figure 5C are labeled. EP, ectoplacental cone; EX, extraembryonic portion; EM, embryonic portion; YC, yolk sac cavity; RM & dotted line, Reichert’s Membrane labeled on one side for clarity. Scale bars = 100 μm . C) H&E stained decidua from iBC single source transfer section shows high presence of immune cells resorbing a mass of cells with ExEm-like and Em-like stain and morphology. Scale bar = 100 μm . To further examine the development of iBC-derived ExEm tissues in implanted deciduae, we performed IHC for ExEm markers trophoblast-specific protein alpha (TPBPA) and placental lactogen 1 (PL-I). TPBPA is expressed in ectoplacental cone, spongiotrophoblasts and precursors of trophoblast giant cells (TGCs) that originate from TE ( Simmons and Cross, 2005 ; Simmons et al., 2007 ). TPBPA was detected in the larger cells surrounding the embryonic cavity in both iBC and BC-implanted deciduae at E6.5 ( Figure S4C ). At the same time, PL-I expressing cells characteristic of parietal TGCs lined the embryonic cavity in iBC deciduae similar to the control embryo where it was also detected on the visceral endoderm ( Figure S4C ; Chen et al., 2016 ; Peng et al., 2015 ; Simmons and Cross, 2005 ; Simmons et al., 2007 ; Screen et al., 2008 ). Some TPBPA+ or PL-I+ cells had larger more brightly stained nuclei and scattered far from the cavity, suggesting they are polyploid scattering TGCs ( Figure S4C ). Implanted iBCs May Develop Briefly Before Resorption We noticed that iBC-implanted deciduae were often variably smaller than control deciduae of the same timing. Histology frequently showed evidence of a retracting post-implantation embryonic cavity ( Figure 5A,C , Figure S4 ). We observed blood cells within the blood sinuses and around the iBC-derived implanted tissues ( Figure S3 ), but the high presence of lymphoid and myeloid cells around the disfigured pycnotic tissues indicated the embryo resorption process ( Figure 5C , Figure S3 ; Cossée et al., 2000 ; Flores et al., 2014 ). Still, upon closer observation, iBC-derived non-decidual tissues were markedly diverse and had morphology and localization similar to invading trophoblasts, ectoplacental cone, ExEm portion, Em portion, and yolk sac cavity, when compared to a previous report of natural resorbing embryos ( Figure 4 , Figure S3 ; Cossée et al., 2000 ). These data show that some iBCs are functionally competent to implant, induce decidualization, and grow to greater cell numbers while developing over several days. Implanted iBC tissues displayed varied natural analogous characteristics while providing distinct evidence of ExEm tissue differentiation surrounding internal cells resembling the Em lineage, and in total, apparently following a natural progression of embryonic resorption. Further Characterization of the iBC Generation Process Establishing Pluripotency Is Insufficient in iBCs, Yet Possible in Outgrowths Mouse embryonic stem (ES) cells are a naive PSC-derived from the ICM of BCs. In addition, trophoblast stem (TS) cells can be established from BCs using different conditions. To determine if ES-like or TS-like cells could be derived from iBCs, we isolated iBCs/iBC-PCs with XGFP and EO5/:D2nRFP dual reporters and plated them on feeder cells. In ES cell derivation conditions ( Czechanski et al., 2014 ), outgrowths proliferated, and some cells expressed XGFP and EOS:: D2nRFP ( Figure S5A ). These cells could be passaged and enriched like naive ES cells and were comparable to naive ES cells when stained for OCT4, NANOG, and YAP ( Figure 6A ). We also examined expression of several important pluripotency and early embryonic genes with RT-qPCR and found that iBC/iBC-PC derived cells were generally comparable to ES cells ( Figure 6B ). Interestingly, iBC/iBC-PC-derived cells expressed notably higher levels of Zscan4 and Zfp42 (Rex1). Download figure Open in new tab FIGURE 6: iBC/iBC-PC-Derived Outgrowths Suggest In-Vitro Bi-Directional Potential. A ) Mouse ES cells and iBC/iBC-PC-derived ES-like cells stained for OCT4, NANOG, or YAP (magenta), and DNA (light blue, Hoechst 33342). iBC/iBC-PC-derived ES-like cells demonstrate X chromosome reactivation (XGFP+) and express EO5:: D2nRFP. 5cale bars = 50 μm . B ) RT-qPCR of mouse ES cells, iBC/iBC-PC-derived ES-like cells, and mEpiSC cDNA samples, shown as mean ΔCT to Gapdh . Data represents two biological samples per type and all probes tested in technical triplicate. Error bars represent standard deviation between the two biological samples. * = no detectable Zscan4 signal in mEpiSC samples. ** = one mEpiSC biological sample did not have detectable Zfp42 (Rex1), and therefore no standard deviation. C ) Left: Live imaging of iBC derived TE-like cells. Right: TE-like cells were stained for CDX2 (Green) and DNA (light blue, Hoechst 33342). Channels shown separately and merged. 5cale bars = 100um . We asked if iBCs/iBC-PCs give rise to TE lineage progeny on feeder cells and first saw colonies of slow growing TE-like cells expressing CDX2 ( Figure 6C ). We also tried a defined TS cell medium to derive TS cells or their progeny ( Latos and Hemberger, 2016 ; Ohinata and Tsukiyama, 2014 ), but we could not obtain stable TS cells. However, we could expand TE-like cells for a few passages in the defined condition ( Figure S5B ), and those cells passed through a binucleate phase that is characteristic of TE-derivative cell cultures that produce trophoblast giant cells ( Figure S5B , mid panel; Ilgren, 1981 ). The culture of cells slowed almost to a halt of large single and binucleate cells that stained strongly but variably for the TE-lineage markers PL-I and TPBPA ( Figure S5B ; Awonuga et al., 2011 ). These outgrowth results suggest that iBCs/iBC-PCs hold increased potential whereby additional culture time might fully establish naive pluripotency. In addition, our TS cell culture outgrowth did not establish stabilized TS cells, but yielded cells with important post-implantation ExEm markers and characteristics in-vitro to strengthen our cryosection IHC detection of iBC-derived implanted tissues ( Figure S4C ). iBC Induction Requires Prdm14 and May Concomitantly Activate a Totipotency-Related Cell Program Because iBCs coordinately differentiated evidence of a bi-directional 3D cyst expressing cleavage stage-initiated genes, such as Atplbl , and possibly had interim loss of pluripotency, we hypothesized that iBCs emerge from totipotent-like cells. The totipotent genome is prepared in the germ line and activated by ZGA as the zygote enters 2C and cleavage. Phase 1 of iBC induction has defined molecules involved in germ cell differentiation ( Chen et al., 2012 ; Hikabe et al., 2016 ; Yamaji et al., 2008 ; Yang et al., 2017 ), and Phase 2 has defined molecules that induce naive pluripotency and TE lineage transdifferentiation ( Figure 2A ; Bao et al., 2009 ; Hayashi et al., 2010 ; Kime et al., 2016 ). In this sense, we looked to Prdm14, a major gene regulatory factor shared in the germ line and early embryo ( Nakaki and Saitou, 2014 ; Hackett et al., 2017 ). RT-qPCR of mEpiSCs showed very low detection of Prdm14 in large samples, and Prdm14 was undetectable in small isolated colonies; however, in experiments for isolated BC and iBC samples, some iBCs expressed Prdm14 at significant or comparable levels to BCs ( Figure 7A ), suggesting that this important transcription factor is induced in the iBC process. Download figure Open in new tab FIGURE 7: iBC Generation Requires Prdm14 and Activates MERVL Reporter in iBC-PCs A ) Single isolated BC and iBC RT-qPCR for Prdm14 . B ) Control and Prdm14 KD mEpiSC are plated for iBC induction. Loci that originate iBC-PC are initiated in both experiments by Day 3 (yellow arrows). Control cells maintain iBC-PC induction through Day 6 (yellow arrows) and Prdm14 KD cells abort iBC-PCs among cell debris. Scale bars = 200um . C ) RT-qPCR of control and Prdm14 KD cell plate cDNA samples for Prdm14 in iBC generation, shown as mean ΔCT to Gapdh . Error bars represent standard deviation from technical triplicate . D ) iBC induction Day 6 colocalized expression of MERVL:: RFP and XGFP+ reporters. Scale bar = 100 μm . E ) After iBC are collected, iBC generation plate on Day 7 retained some larger MERVL:: RFP+ cells with cleavage stage cell-like morphology. Scale bar = 100um . F ) Live fluorescent image of iBC-PCs expressing MERVL:: RFP in ULA plate on Day 6. Scale bar = 100 μm . G ) Live fluorescent image of MERVL:: RFP expressed strongly in iBC-PC yet poorly detected in emergent iBC, seen in culture on Day 8. Scale bar = 100 μm . We tested our constitutive shRNA Prdm14 knockdown(KD) mEpiSCs and found with daily microscopy that the experiment began similar to control cells and initiated the compacted iBC-PC/iBC originating loci ( Figure 7B ). However, by Day 6, the iBC-PCs that may become iBCs were nearly completely aborted, and the peripheral cells appear to degrade ( Figure 7B ). Supernatants collected from all iBC experiments with Prdm14 KD cells failed to yield favorable iBCs and had increased cell debris. For reference, when control cell iBC-PC were not harvested by agitation to supernatant on Day 6, they still differentiated as gently attached expanded iBC above such concentrated cell loci ( Figure 3D , Figure 7G ). To examine Prdm14 expression in control and Prdm14 KD cells, we collected mRNA from cell populations at the end of Phase 1 on Day 4, when the cultures appear to perform similarly, and on Day 6, immediately before iBC-PCs are usually harvested or lost in the Prdm14 KD cell population ( Figure 7B ). Compared to the detection of Gapdh , control cell iBC induction populations had notable Prdm14 expression by Day 4, and further increased by Day 6. Prdm14 KD cells showed significantly reduced Prdm14 expression on Day 4 and a much lower proportional expression by Day 6 ( Figure 7C ). The lower detection of Prdm14 in Prdm14 KD cells on Day 6 suggests that the lost iBC-PCs required Prdm14 and that iBC-PC might represent most of the detectable Prdm14 at that time ( Figure 7B,C ). To further elucidate a possible relationship between totipotency and iBC generation, we cloned the well-studied 2C MERVL live totipotency-related reporter to drive an RFP( MERVL:: RFP) in XGFP mEpiSCs to operate dual reporters ( Figure S2D ). These reporters were undetectable in mEpiSCs in agreement with previous reports ( Bao et al., 2009 ; Macfarlan et al., 2012 ; Wu et al., 2017 ). On Days 5-6 of iBC induction, we observed some of the characteristic loci where iBC-PC originate had MERVL:: RFP+ cells, and many cells expressed XGFP, suggesting X chromosome reactivation to Xa/Xa ( Figure 7D ). We speculated that cells with dual reporter activation implicates ZGA mechanisms since both are reported characteristics of 2C cleavage stage cells ( Figure 7D ; Monk and Harper, 1979 ; Okamoto et al., 2004 , Wu et al., 2017 ). Interestingly, when iBC-PC were harvested, some remaining attached MERVL:: RFP+ cells variably lost XGFP expression and often became larger and more rounded with cleavage stage-like cell morphology ( Figure 7E ). Furthermore, despite the low overall frequency of MERVL + reporter cell loci on the plate at Day 6, many of the iBC-PCs harvested by agitation were composed of MERVL:: RFP+ cells ( Figure 7F ). MERVL:: RFP expression in iBC-PC was usually weaker than the MERVL:: RFP+ cells seen on the plate ( Figure 7D,E,F,G ). Also, XGFP was generally not observed in MERVL:: RFP+ collected iBC-PCs, suggesting that both reporters were down regulated at that critical stage similar to compacting 8C/16C embryos that precede BCs. Strengthening this observation, emergent iBCs had far reduced detectable MERVL:: RFP ( Figure 7G ). Of further interest, MERVL:: RFP+ and MERVL:: RFP+/XGFP+ cells were variably maintained on the plate for several days in Phase 2 media after iBC-PC harvest. Collectively, our results indicated that the iBC production process required Prdm14 . MERVL:: RFP+/XGFP+ subpopulations preceded and reported from the characteristic loci where Prdm14 dependent iBC-PC emerged, and many harvested iBC-PCs expressed the MERVL:: RFP reporter. These data show an unknown intermediate role for Prdm14-dependent iBC formation among plate loci where totipotent-cell characteristics may be differentially but strongly induced. Since Prdm14 KD compromised the entire iBC formation via abortive loss of iBC-PCs, and both the TE-like and putative ICM-like cells were lost, Prdm14 may be key to improved induction of totipotent 2C-like cells or the specification of lineages in iBCs. DISCUSSION This study showed that sequential treatment of mouse PSC culture with defined molecules reproducibly induces BC-like 3D structures with several distinct features of BCs. Remarkably, the structures emerged from floating small cell clumps resembling denuded 8C/16C-compact embryos wherein cells lack morphological differences or polarity. As such, differentiation to the iBC structure is emergent. iBCs exhibited signs of implantation when transplanted into pseudopregnant surrogates, indicating implantation-competence. In previous work of mouse PSC-derived oogenesis, rare BC-like structures from 40+ day long-term differentiation experiments were partially described ( Hübner et al., 2003 ). However, whether those structures were developmentally competent in-utero is unknown. Furthermore, using TS cells, PSCs and PSC-derived bi-directionally contributing cells has never been demonstrated to contribute to animal development in transplanted pseudopregnant mothers without donor cells or chimerism for support ( Macfarlan et al., 2012 ; Yang et al., 2017 ). Therefore, generation of fully functional iBCs, which give rise to newborn animals in an isogenic setting, may uncover a maximum differentiation potential of PSCs. To our knowledge, this is the first demonstration that PSC culture can generate the 3D architecture with cellular materials and implantation-competence resembling BCs. Until now, only implantation-competent BCs or their trophoblasts ( Gardner and Johnson, 1972 ), chimeras thereof, or specific melanoma cells were reported to induce deciduae in sterile-male bred pseudopregnant mice ( Wilson, 1963 ). A related field of uterine environment study involves deliberate uterus disruption often combined with injected progesterone and estrogen hormone treatments to induce deciduomas ( Herington and Bany, 2007 ; Lee et al., 2007 ). Deciduomas are composed of homogenous decidual cells, and focal deciduomas that better resemble individual natural deciduae requires concanavalin A-coated Sepharose beads ( Herington et al., 2009 ). Unlike deciduomas, our iBC-induced deciduae rely exclusively on sterile-male bred pseudopregnant surrogates and have correct positional implanted tissues in focal deciduae that resemble natural deciduae or deciduae from BC-derived trophoblast vesicles ( Gardner and Johnson, 1972 ). To exclude the possibility of deciduomas, we molecularly characterized iBC-derived implanted tissues and emphasize that we do not use the materials or methods required to produce deciduoma. In our experiments, co-transfer with control embryos may greatly improve the implantation of the iBC, as with difficult mouse strains ( Mochida et al., 2014 ), yet iBC alone demonstrate implantation-competence. Molecular Considerations in the iBC Process We showed that YAP localization implicates non-polarized and polarized iBC-PC, and polarized early iBCs, that are critically similar to early embryos. Intermediate MERVL reporter and comparable Zscan4 activation furthers that prospect. Signaling inputs that we provided during iBC production may mimic developmental cues of embryogenesis. Using a synthetic LPA (OMPT) in our cocktail may be striking because LPA treated BCs exhibit enhanced embryogenesis by activating YAP in-vitro and in-utero ( Yu et al., 2016 ). iBCs expressed Cdx2 at a lower level than BCs and had some nuclear CDX2 localization while most iBC outer cells retained cytosolic CDX2. During early development, Cdx2 is preferentially upregulated around the 8C stage to specify committed outer cells ( Strumpf et al., 2005 ; Ralston and Rossant, 2008 ). Cdx2 is crucial for development since Cdx2-deficient BCs cannot implant in the uterus despite having functional pluripotent cells in the ICM ( Meissner and Jaenisch, 2006 ). We found iBCs exhibit a TROMA-I+ and nuclear-enriched YAP outer layer and a blastocoel-like cavity and are implantation-competent. Additionally, TROMA-I+ cells from transplanted iBCs grew well, invaded the uterus to decidua reaction, and developed different morphologies and detectable markers (PL-I, TPBPA), depending on their positions in the embryonic cavity. These results further indicate the proliferation and differentiation capacity of the iBC outer cells within the TE lineage after implantation despite weak iBC Cdx2 characteristics. Additionally, post-implantation proliferation of trophoblast progeny depends on ICM-derived tissues in normal development, which suggests that some of the larger more developed iBC-derived implanted tissue may have been helped by cells from the iBC ICM-like region ( Gardner and Johnson, 1972 , 1975 ; Rossant and Ofer, 1977 ; Simmons and Cross, 2005 ). iBCs plated on feeder cells produced TE-like colonies uniformly expressing CDX2 protein, and when grown in defined TE cell culture conditions, the iBC/iBC-PC-derived cells were characteristically similar to dissociated cultured TE derivatives. Thus, Cdx2 expression in iBCs may be sufficient to induce functional TE lineage cells that enable iBCs to implant and develop for several days. However, we observed that iBCs had uneven implantation, implicating molecular pathways that interface between the TE-like cells and receptive uterus are incorrect. Optimizing our regimen based on known conditions that induce trophoblasts from PSCs may improve Cdx2 expression to correct abnormalities ( Hayashi et al., 2010 ). Our results show that iBCs have a putative ICM with OCT4 and nuclear-excluded YAP and downregulated both CDX2 and TROMA-I protein. Notably, the exclusion of nuclear YAP is a characteristic of pluripotent cells in the ICM of BCs that is critically different from in-vitro cultured mouse pluripotent ES cells that have nuclear-enriched YAP ( Tamm et al., 2011 ; Figure 6A ). We speculate that the putative ICM in iBCs became the central TROMA-I- cells we observed in iBC-derived cryosections. However, the key pluripotent transcription factor Oct4 mRNA was expressed at lower levels in iBCs than in BCs. Since precise expression of Oct4 is crucial for establishing and/or maintaining pluripotency, lower expression suggests suboptimal re-establishment of pluripotency in iBCs ( Niwa et al., 2000 ). This may explain why iBC-derived post-implantation proliferation and development eventually delayed or ceased ( Gardner and Johnson, 1972 ). Additionally, we rarely observed Xi-GFP reactivation in iBCs, consistent with the lower expression of Nanog ( Silva et al., 2009 ). However, we could establish ES cell-like cells from iBCs/iBC-PCs in naive PSC derivation conditions, suggesting that authentic pluripotency could be reestablished in altered conditions. We therefore speculate that minor critical adjustments to iBC generation conditions may improve intermediate iBC-PC and iBC cell states. The findings of the insufficient pluripotency in iBCs are in stark contrast to what we observed during the hemisphere formation experiments where naive pluripotency was robustly established ( Kime et al., 2016 ). In that study, we showed that BMP4 signaling, with LIF and ascorbic acid, greatly increased Prdm14 expression during conversion of mEpiSCs to the naive state ( Kime et al., 2016 ); but we also measured the induction of Prdm1(Blimp1) and Id gene family mRNAs, which we reported here. SMAD2/3 signaling inhibition stimulates BMP induction, and BMP4 can replace serum in naive PSC culture by inducing Id genes toward self-renewal ( Ying et al., 2003 ). BMP signaling and these critical Prdm and Id family genes are shared among germ cell development and cleavage through preimplantation embryonic development ( Yang et al., 2017 ; Hiller et al., 2010 ; Yamaji et al., 2008 ). Interestingly, Id2 is comparably expressed in outer and inner lineage early embryonic cells ( Ying et al., 2003 ; Tang et al., 2010 ; Wu et al., 2016 ). One considerable difference between our hemisphere/naive conversion and iBC generation is in the induction regimes. SMAD2/3 signaling inhibition may be necessary to generate iBCs, but SMAD2 specific inhibition induces TE and germ cell differentiation while suppressing pluripotency expression via Nanog inhibition ( Chen et al., 2012 ; Sakaki-Yumoto et al., 2013 ). We anticipate that SMAD2/3 signaling inhibition may require further adjustment to achieve sufficient pluripotency in iBCs and note that a necessary adjustment of SB431542 concentration was mEpiSC line specific. Prdm14 KD experiments suggest that Prdm14 has a pivotal role during iBC induction although the exact mechanisms are unknown. In the iBC system, Prdm14 was greatly enriched by Day 4, prior to LIF in Phase 2, which contrasts with conventional roles of LIF in ES cell pluripotency ( Ying et al., 2003 ). Taken together with SMAD2/3 signaling inhibition, early increases of Prdm14 observed in iBC generation may be partial to germ cell induction mechanisms. Prdm14 is a transcription factor whose role is both powerful and unclear: reported to be dispensable in BCs yet a major epigenetic regulator expressed in the 2C cleavage stage that may direct lineage commitment in the BC ( Yamaji et al., 2008 ; Luna-Zurita and Bruneau, 2013 ; Burton et al., 2013 ). Prdm14 is involved in dynamic biological events that accompany epigenetic reprogramming, such as PGC specification, X chromosome reactivation, and conversion from primed to naive state pluripotency ( Yamaji et al., 2008 ; Gillich et al., 2012 ; Payer et al., 2013 ; Kime et al., 2016 ). Intriguingly, iBC induction with Prdm14 KD cells proceeded typically for several days but caused iBC-PC cell death at the time when iBC-PCs should begin to polarize and differentiate to iBCs. How Prdm14 is involved in iBC production warrants further investigation. Notably, our defined conditions resemble germ cell induction medium, suggesting a PGC/germ cell specification process may be involved; the germ line prepares a totipotent genome that is not yet activated epigenetically. Since BCs naturally differentiate from homogenous totipotent cells, the 2C stage ZGA mechanism suggested by MERVL may also play a role ( Wu et al., 2017 ). Supporting this notion, we observed the compacting iBC-PC/iBC originating loci with concomitant MERVL:: RFP+ cells curiously demonstrating large cleavage-stage cell size and morphology. Some previous studies of MERVL-enriched PSCs touched upon the implication of totipotent hallmarks, ZGA, and 2C-like expression, yet these reports did not demonstrate similar apparent morphological changes ( Macfarlan et al., 2012 ; Blaschke et al., 2013 ; Ishiuchi et al., 2015 ). Several reports found ES cell colonies have rare transient MERVL+ cells that return to the ES state. In iBC experiments, we see several cells at iBC-PC loci, or iBC-PC, activating MERVL:: RFP simultaneously and with relatively consistent sustained expression. We also note that X reactivation suggested by XGFP+ cells among our MERVL:: RFP+ cells presents a ratio quite different from distinct features of naive PSCs, which should be completely Xa/Xa and have rare MERVL reporter activation ( Macfarlan et al., 2012 ; Payer et al., 2011 ). Our observation of MERVL::RFP+ cells with less X reactivation may reflect the varied Xa/Xa state of cleavage stage cells rather than naive PSCs. The enrichment of Atp1b1 in isolated iBCs was also remarkable since natural embryos activate Atp1b1 in cleavage stage cells in preparation to act in cell junctions of compaction and as a Na+/K+ ATPase pump subunit to fill the blastocoel ( Hamatani et al., 2004 ; Madan et al., 2007 ; Stephenson et al., 2010 ). Isolated iBCs could also show Zfp42 (Rex1) and Zscan4 , which are also cleavage stage-induced genes with different roles in pluripotent cells. Moreover, Zfp42 (Rex1) may negatively regulate 2C-related gene expression ( Schoorlemmer et al., 2014 ), yet iBC/iBC-PC-derived ES-like cells have notably increased expression of both Zfp42 (Rex1) and Zscan4 RNAs when compared to ES cells. With these observations, a thorough molecular elucidation of iBC generation would likely improve their quality to obtain full functionality. Generation of iBCs requires stringent PSC preparation and rounds of iBC purification, yet most of the experiment involves 1 week of simplified defined-media changes. Thus, we envision that iBC technology readily opens avenues in several fields, such as embryology and implantation biology among its promise in early embryogenesis. For instance, even though iBCs cannot develop completely, transplanting iBCs from gene knockdown or knockout PSCs may make it easier to elucidate the molecular mechanisms governing implantation. In any case, our findings may offer a new step in expanding knowledge in pluripotency, totipotency, and embryogenesis, which may be necessary to significantly advancing PSC technologies and related fields. AUTHOR CONTRIBUTIONS Conceptualization, C.K.; Methodology, C.K. and K.T.; Validation, S.O.; Formal Analysis, C.K. and K.T.; Investigation, C.K., K.T., and H.K.; Resources, C.K., K.T., S.Y., and M.T.; Writing - Original Draft, C.K. and K.T.; Writing - Review & Editing, C.K. and K.T.; Visualization, C.K.; Supervision, C.K. and K.T.; Project Administration, C.K.; Funding Acquisition, S.Y., M.A., and M.T. Conflict of Interest C. K and K.T. have applied for patents related to this technology and extended works. S.Y. is a scientific advisor of iPS Academia Japan without salary. ACKNOWLEDGEMENTS We honor the help of Dr. Hitoshi Niwa for critical input and for providing the Rabbit anti-Mouse CDX2 antibody. We are grateful to Drs. Siqin Bao and Azim Surani for their female XGFP mEpiSC. We also thank Drs. Robert Blelloch and Paul Tesar for providing their mEpiSC for research. MERVL 2C and EOS-S(4+) reporter DNA was provided by Addgene ( http://www.addgene.org ) under MTA and subcloned into our systems. TROMA-I (Krt8) monoclonal antibody developed by Institut Pasteur was obtained from the Developmental Studies Hybridoma Bank(DSHB), created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. We greatly thank the Yamanaka and Takahashi labs for the support and research environment that made this work possible. REFERENCES ↵ Abe , T. , Kiyonari , H. , Shioi , G. , Inoue , K.-I. , Nakao , K. , Aizawa , S. , and Fujimori , T. ( 2011 ). 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Share Implantation-Competent Blastocyst-Like Structures from Mouse Pluripotent Stem Cells Cody Kime , Hiroshi Kiyonari , Satoshi Ohtsuka , Eiko Kohbayashi , Michio Asahi , Shinya Yamanaka , Masayo Takahashi , Kiichiro Tomoda bioRxiv 309542; doi: https://doi.org/10.1101/309542 Share This Article: Copy Citation Tools Implantation-Competent Blastocyst-Like Structures from Mouse Pluripotent Stem Cells Cody Kime , Hiroshi Kiyonari , Satoshi Ohtsuka , Eiko Kohbayashi , Michio Asahi , Shinya Yamanaka , Masayo Takahashi , Kiichiro Tomoda bioRxiv 309542; doi: https://doi.org/10.1101/309542 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7969) Biochemistry (18623) Bioengineering (14765) Bioinformatics (44155) Biophysics (22457) Cancer Biology (19585) Cell Biology (26737) Clinical Trials (138) Developmental Biology (13900) Ecology (20880) Epidemiology (2067) Evolutionary Biology (25315) Genetics (16100) Genomics (23400) Immunology (18607) Microbiology (42209) Molecular Biology (17939) Neuroscience (92895) Paleontology (693) Pathology (2969) Pharmacology and Toxicology (5063) Physiology (8064) Plant Biology (15901) Scientific Communication and Education (2091) Synthetic Biology (4538) Systems Biology (10184) Zoology (2376) window.__CF$cv$params={r:'a376d3194f6aada1',t:'MTc4ODc5NTc1OQ==',u:'01a07c89434a7933aa17a26a4b9348b6',ut:'iwUy7stPfMZp4g1Op8rIUu988m3.UbVjvZqwI11QOdU-1788795765-1.2.1.1-brXZwoyAlV5F6m.Kml0gqQGGy52QLqx9l56LiSc5cRgaJeVhcsGhGMz1fY4Yq7ht.xrt.FgZNfuFqcHDgws_o_piGuGW6SPCis0TFaBwees',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();