Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species

ABSTRACT Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated ‘The people behind the papers’ interview.

45 Embryonic disc development invitro in ovine embryos

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo and the epiblast forms a flat embryonic disc (ED) that initiates gastrulation. The aim of this study was to develop an invitro culture system to support sheep embryo development after the blastocyst stage. Day 6/7 invitro-produced blastocysts were cultured over agarose gels to prevent attachment and allocated to different media: synthetic oviductal fluid with 10% fetal bovine serum (SOF-FBS, n=52), an invitro culture medium (hIVC, n=35) supporting ED formation in human embryos (Deglincerti et al. 2016 Nature 533, 251-254; https://doi.org/10.1038/nature17948), and chemically defined N2B27 medium (n=38) supporting ED formation in bovine embryos (Ramos-Ibeas et al. 2020 Reproduction 160, 579-589, https://doi.org/10.1530/REP-20-0243). At Day 14, survival and embryo area were recorded, the abundance of transcripts encoding interferon Tau (TP1) and metabolic enzymes was analysed by RT-qPCR, and the development of epiblast and hypoblast was assessed by immunostaining for SOX2 and SOX17. Embryo survival and size and the percentage of embryos achieving complete hypoblast migration were significantly reduced in SOF-FBS (Chi-squared test and one-way ANOVA; P<0.05). Only N2B27 medium supported epiblast survival in 11/28 embryos. TP1 expression increased at Day 14 in all culture conditions and metabolism-related genes revealed a shift from anaerobic glycolysis to oxidative phosphorylation after culture in hIVC and N2B27. Next, to promote epiblast development, we allocated blastocysts to N2B27 medium supplemented with activin A (n=45), rho-associated protein kinase (ROCK) inhibitor (ROCKi, n=42), or insulin growth factor 1 (IGF1, n=29). IGF1 reduced significantly the percentage of embryos showing an ED-like structure, whereas activin A supplementation significantly increased epiblast survival (Chi-squared test; P<0.05) and SOX2+ cell number was higher in embryos cultured with ROCK inhibitor. When we combined activin A and ROCKi supplementation (N2B27+A+R, n=151), SOX2+ cell number and the percentage of embryos showing an ED-like structure increased significantly (165.1±53.1 and 31/35 embryos with SOX2+ epiblast cells; ∼89%) compared with N2B27 alone (49.4±12.7 and 5/11; ∼45%) (one-way ANOVA and Chi-squared test; P<0.05). Moreover, 18/31 (∼58%) ED-like structures developed in N2B27+A+R lost the Rauber’s layer (polar trophoblast), and BRACHYURY expression, denoting the onset of gastrulation, was observed in 3/14. In conclusion, we have developed a culture system that supports complete hypoblast migration and ED development invitro, which represents a valuable tool to explore early embryo mortality in livestock species without the need for experimental animals.

Embryonic disc formation following post-hatching bovine embryo development in vitro

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

Onfalocele gigante y acretismo placentario

El onfalocele se define como un defecto congénito de la pared abdominal por fallo en la fusión de los pliegues del disco embrionario y, como consecuencia, el contenido intraabdominal se encuentra herniado; esto debe estar cubierto por peritoneo, gelatina de Wharton, amnios, centrado en el orificio umbilical con el cordón, originándose del ápex del mismo; la incidencia es de 1 de cada 5000 recién nacidos vivos. Con la intención de describir un caso atípico, se presenta el de una paciente gestante de 35 años de edad, Gesta: IV, Cesáreas: II, Abortos: I y 13/08/2013 como fecha de última menstruación. La paciente fue atendida en la Unidad Ecográfica de Alto Riesgo del Hospital Central Pedro Emilio Carillo (Trujillo, Venezuela), por presentar onfalocele gigante; se sometió a cirugía con los diagnósticos preoperatorios siguientes: 1) embarazo simple de 35 semanas y 2 días por biometría fetal; 2) pródromo de trabajo de parto; 3) onfalocele gigante; y 4) útero cicatrizal. Se le practicó cesárea corporal, histerectomía total con conservación de anexos. El recién nacido vivo presentó onfalocele gigante. Se evidenció placenta previa oclusiva más acretismo placentario, por lo que en el presente trabajo se pretende explicar la importancia del diagnóstico temprano y manejo terapéutico, así como la posible relación entre el onfalocele y placenta acreta a propósito de este caso. Palabras clave: Bolsa de Bogotá, cordón umbilical, acretismo placentario, pentalogía de Cantrell, síndrome de Beckwith-Wiedemann. Abstract Omphalocele is defined as a congenital defect of the abdominal wall due to failure in the fusion of the folds of the embryonic disc and, as a consequence, the intra-abdominal content is herniated; this must be covered by peritoneum, Wharton's jelly, amnion, centered in the umbilical orifice with the cord, originating from the apex thereof; the incidence is 1 in 5 000 live newborns. With the intention of describing an atypical case, that of a 35-year-old pregnant woman, Gesta: IV, Caesarean section: II, Abortion: I, and 08/13/2013 as the date of the last menstruation, is presented. The patient was treated at the High Risk Ultrasound Unit of the Pedro Emilio Carillo Central Hospital (Trujillo, Venezuela), for presenting a giant omphalocele; she underwent surgery with the following preoperative diagnoses: 1) simple 35-week and 2-day pregnancy by fetal biometry; 2) labor prodrome; 3) giant omphalocele; and 4) scar uterus. Body caesarean section was performed, total hysterectomy with preservation of annexes. The live newborn had a giant omphalocele. The occlusive placenta prae plus placental accretion was evidenced, therefore, in this work we try to explain the importance of early diagnosis and therapeutic management, as well as the possible relationship between omphalocele and placenta accreta in this case. Keywords: Bogota bag, umbilical cord, placental accreta, pentalogy of Cantrell, Beckwith Wiedemann syndrome.

Broca�s Area and Language Disorders in the Child

Language, the second signaling system, complexly integrates the body into the environment, assures interference with fellows, and modulates the thoughts and feelings to be communicated to others. The embryonic development of the encephalus is performed early on days 16-20 and determines the appearance of the neuroenteric canal and the appearance of the tridermic embryonic disc. The sketch of the Broca area has been researched from anatomopathological point of view, following a study carried out on fetal brain samples taken postpartum, shows that the sketches of the future Broca area are observed in gestation at week 23. Neural migration and glial cell distribution reveal the existence of a detectable neuroblasts reservoir, beginning at week 18, with exuberant evolution to a organized histoarchitectonics at 20-23 week. The premises of a future impairment in the development of the Broca area and, implicitly, of language can be established early in fetal life.

A pluripotent stem cell-based model for post-implantation human amniotic sac development

Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.

Gene expression analysis of bovine embryonic disc, trophoblast and parietal hypoblast at the start of gastrulation

SummaryIn cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFβ, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.