Self-patterning of human stem cells into post-implantation lineages.

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

The Presence or Absence of Alkaline Phosphatase Activity to Discriminate Pluripotency Characteristics in Porcine Epiblast Stem Cell-Like Cells.

Porcine embryonic stem cells (pESCs) would provide potentials for agricultural- and biotechnological-related applications. However, authentic pESCs have not been established yet because standards for porcine stem cell-specific markers and culture conditions are not clear. Therefore, the present study reports attempts to derive pluripotent epiblast stem cells either from in vitro or in vivo derived porcine embryos. Nine epiblast cell lines (seven lines from Berkshire and two lines from Duroc) could only be isolated from day 9- to 9.5-old in vivo derived early conceptuses. Pluripotency features were analyzed in relation to the presence or absence of alkaline phosphatase (AP) activity. Interestingly, the mRNA expression of several marker genes for pluripotency or epiblast was different between putative epiblast stem cells of the two groups [AP-positive (+) pEpiSC-like cell 2 line and AP-negative (-) pEpiSC-like cell 8 line]. For example, expressions of OCT-3/4, NANOG, SOX2, c-MYC, FGF2, and NODAL in AP-negative (-) porcine epiblast stem cell (pEpiSC)-like cells were higher than those in AP-positive (+) pEpiSC-like cells. Expression of surface markers differed between the two groups to some extent. SSEA-1 was strongly expressed only in AP-negative (-) pEpiSC-like cells, whereas AP-positive (+) pEpiSC-like cells did not express. In addition, we report to have some differences in the in vitro differentiation capacity between AP-positive (+) and AP-negative (-) epiblast cell lines. Primary embryonic germ layer markers (cardiac actin, nestin, and GATA 6) and primordial germ cell markers (Dazl and Vasa) were strongly expressed in embryoid bodies (EBs) aggregated from AP-negative (-) pEpiSC-like cells, whereas EBs aggregated from AP-positive (+) pEpiSCs did not show expression of primary embryonic germ layers and primordial germ cell markers except GATA 6. These results indicate that pEpiSC-like cells display different pluripotency characteristics in relation to AP activity.

Embryonic stem cell identity grounded in the embryo.

Pluripotent embryonic stem cells (ESCs) can be derived from blastocyst-stage mouse embryos. However, the exact in vivo counterpart of ESCs has remained elusive. A combination of expression profiling and stem cell derivation identifies epiblast cells from late-stage blastocysts as the source, and functional equivalent, of ESCs.

The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification.

The precise relationship of embryonic stem cells (ESCs) to cells in the mouse embryo remains controversial. We present transcriptional and functional data to identify the embryonic counterpart of ESCs. Marker profiling shows that ESCs are distinct from early inner cell mass (ICM) and closely resemble pre-implantation epiblast. A characteristic feature of mouse ESCs is propagation without ERK signalling. Single-cell culture reveals that cell-autonomous capacity to thrive when the ERK pathway is inhibited arises late during blastocyst development and is lost after implantation. The frequency of deriving clonal ESC lines suggests that all E4.5 epiblast cells can become ESCs. We further show that ICM cells from early blastocysts can progress to ERK independence if provided with a specific laminin substrate. These findings suggest that formation of the epiblast coincides with competence for ERK-independent self-renewal in vitro and consequent propagation as ESC lines.

Dynamics of Akt activation during mouse embryo development: distinct subcellular patterns distinguish proliferating versus differentiating cells.

Akt is a highly conserved serine-threonine protein kinase which has been implicated in a wide variety of cellular functions, from the regulation of growth and metabolism, to activation of pro-survival pathways and cell proliferation, and promotion of differentiation in specific cell types. However, very little is known about the spatial and temporal pattern of Akt activity within cells and whether this pattern changes as cells enter and proceed in their differentiation programs. To address this issue we profiled Akt activation in E8.5-E13.5 mouse embryos and in C2C12 cells. We used a commercial antibody against Akt, phosphorylated on one of its activating residues, Thr-308, and performed high resolution confocal imaging of the immunofluorescence in labeled embryos. We observe strong Akt activity during mitosis in the dermomyotome, the neuroepithelium and some mesenchymal cells. This burst of activity fills the whole cell except for heterochromatin-positive areas in the nucleus. A surge in activity during mitosis is also observed in subconfluent C2C12 cells. Later on in the differentiation programs of skeletal muscle and neural cells, derivatives of the dermomyotome and neuroepithelium, respectively, we find robust, sustained Akt activity in the cytoplasm, but not in the nucleus. Concomitantly with skeletal muscle differentiation, Akt activity becomes concentrated in the sarcomeric Z-disks whereas developing neurons maintain a uniform cytoplasmic pattern of activated Akt. Our findings reveal unprecedented cellular and subcellular details of Akt activity during mouse embryo development, which is spatially and temporally consistent with proposed functions for Akt in mitosis and myogenic and neural differentiation and/or survival. Our results thus demonstrate a subcellular change in the pattern of Akt activation when skeletal muscle and neural progenitor cells cease dividing and progress in their differentiation programs.

JAK/STAT3 signalling is sufficient and dominant over antagonistic cues for the establishment of naive pluripotency.

Induced pluripotency depends on cooperativity between expression of defined factors and the culture environment. The latter also determines the pluripotent cell state, that is, naïve or primed. LIF-JAK/STAT3 signalling was recently shown to be a limiting factor for reprogramming to naïve pluripotency. Here we show that sufficient activation of JAK/STAT3 overcomes the reprogramming block of cell intermediates and enables somatic cell reprogramming in absence of otherwise essential pluripotency medium requisites. Activation of FGF-ERK signalling, which promotes exit of naïve pluripotent cells from self-renewal, does not prevent JAK/STAT3 induced post-implantation epiblast-derived stem cell conversion into naïve pluripotency. Moreover, even in the presence of FGF plus Activin, which instructs and maintains the primed state, JAK/STAT3 enforces naïve pluripotency in epiblast stem cells. We conclude that JAK/STAT3 signalling can be sufficient and dominant over antagonistic cues to enable the induction of a naïve pluripotent state.

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos.

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

Human hypoblast formation is not dependent on FGF signalling.

Mouse embryos segregate three different lineages during preimplantation development: trophoblast, epiblast and hypoblast. These differentiation processes are associated with restricted expression of key transcription factors (Cdx2, Oct4, Nanog and Gata6). The mechanisms of segregation have been extensively studied in the mouse, but are not as well characterised in other species. In the human embryo, hypoblast differentiation has not previously been characterised. Here we demonstrate co-exclusive immunolocalisation of Nanog and Gata4 in human blastocysts, implying segregation of epiblast and hypoblast, as in rodent embryos. However, the formation of hypoblast in the human is apparently not dependent upon FGF signalling, in contrast to rodent embryos. Nonetheless, the persistence of Nanog-positive cells in embryos following treatment with FGF inhibitors is suggestive of a transient naïve pluripotent population in the human blastocyst, which may be similar to rodent epiblast and ES cells but is not sustained during conventional human ES cell derivation protocols.

The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.

Early steps in neural development.

We studied early neurulation events in vitro by transplanting quail Hensen's node, central prenodal regions (before the nodus as such develops), or upper layer parts of it on the not yet definitively committed upper layer of chicken anti-sickle regions (of unincubated blastoderms), eventually associated with central blastoderm fragments. We could demonstrate by this quail-chicken chimera technique that after the appearance of a pronounced thickening of the chicken upper layer by the early inductive effect of neighboring endophyll, a floor plate forms by insertion of Hensen's node-derived quail cells into the median part of the groove. This favors, at an early stage, the floor plate "allocation" model that postulates a common origin for notochord and median floor plate cells from the vertebrate's secondary major organizer (Hensen's node in this case). A comparison is made with results obtained after transplantation of similar Hensen's nodes in isolated chicken endophyll walls or with previously obtained results after the use of the grafting procedure in the endophyll walls of whole chicken blastoderms.

Genotypically unbalanced diploid<==>diploid foetal mouse chimaeras: possible relevance to human confined mosaicism.

Two series of mouse chimaeras were produced by aggregating pairs of eight-cell embryos that differed at the Gpi-1s locus, encoding glucose phosphate isomerase (GPI-1); the paired embryos were respectively homozygous Gpi-1sa/Gpi-1sa and Gpi-1sb/Gpi-1sb. Chimaeric blastocysts were transferred to pseudopregnant females, that were homozygous Gpi-1sc/Gpi-1sc and produced only GPI-1C enzyme. Quantitative electrophoresis of GPI-1 was used to estimate the contribution of each embryo (GPI-1A and GPI-1B enzyme activity) to the foetus, placenta and other extraembryonic tissues of 12 1/2 day chimaeric conceptuses. For both series of chimaeras, the distributions of %GPI-1A in different tissues were classified as (1) balanced and typical, (2) balanced but atypical or (3) unbalanced. One series of chimaeras was clearly unbalanced, so that the cells derived from the (C57BL x CBA/Ca)F2 embryo (Gpi-1sb/Gpi-1sb) predominated over those derived from the BALB/c inbred strain (Gpi-1sa/Gpi-1sa) in most foetuses. Two significant observations were made concerning this unbalanced series. Firstly, the mean composition of the placenta and other extraembryonic tissues was similar to that in the foetus, i.e. also unbalanced with (C57BL x CBA/Ca)F2 (abbreviated to BF2) cells predominating. Secondly, despite this generalized deficiency of BALB/c cells, there were differences in the frequency of non-chimaeric tissues between different developmental lineages. In 20/38 [corrected] chimaeric conceptuses in the unbalanced series only BF2 cells were detected in the foetus, whereas both BF2 and BALB/c cells were present in at least one of the extraembryonic tissues. This group of chimaeras, therefore, shows some similarities to human confined mosaicism. Although chimaerism occurred more often in the primitive endoderm (hypoblast) lineage (yolk sac endoderm and parietal endoderm) than in the placenta, this may also be the case in human mosaics. The mosaic status of the human yolk sac endoderm is usually unknown so it is possible that mosaicism often occurs in the yolk sac endoderm as well as the trophectoderm in human 'confined placental mosaicism'. The uniformly unbalanced phenotype seen in the mouse chimaeras may be a result of generalized cell selection against BALB/c cells in all tissues. As an alternative explanation, we propose that most of the BALB/c cells in the blastocyst are allocated to the mural trophectoderm, which has a limited mitotic potential and so contributes little to the mid-gestation conceptus. Further work is required to test these possibilities.

Isoenzyme changes during rat facial development.

A method is presented by which rat facial processes from different stages were obtained in pure fraction. The morphology, and protein and DNA contents in free dissected facial processes were determined. Facial processes of embryonic rats aged 9-15 days were analyzed by isoelectric focusing for their isoenzymic distribution of four enzymes: lactate dehydrogenase, creatine phosphokinase, fructose diphosphate aldolase and phosphoglycerate mutase. A dominance of LDH-5, LDH-4 and LDH-3 isoenzymes was observed. As a comparison, LDH isoenzymes from mandibular and maxillary processes of rat embryos aged 9-11 days only revealed LDH-5 and to a smaller extent LDH-4. The results support the presence of a prominent anaerobic metabolism in these tissues during early facial development. The change to LDH-3 development correlates well with the formation of new blood vessels. From the ninth embryonic day, isoenzyme BB of creatine phosphokinase was present and during days 10-15 MB and MM developed. Isoenzyme A4 of fructose diphosphate aldolase was present from the ninth embryonic day and isoenzymes A3C and A2C2 developed during days 10-15. From the tenth embryonic day, isoenzyme BB of phosphoglycerate mutase was present and during days 10-15 isoenzyme MB and MM developed. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal and medial nasal processes, and it preceded morphologic evidence of skeletal muscle formation.

Investigation of variability among mouse aggregation chimaeras and X-chromosome inactivation mosaics.

Mouse aggregation chimaeras were produced by aggregating C3H/HeH and C3H/HeHa-Pgk-1a/Ws embryos. At mid-term the proportions of the two cell populations in these conceptuses and the X-inactivation mosaic female progeny of C3H/HeH female X C3H/HeHa-Pgk-1a/Ws male matings were estimated using quantitative electrophoresis of phosphoglycerate kinase (PGK-1) allozymes. The percentage of PGK-1B was more variable in the foetus, amnion and yolk sac mesoderm of the chimaeras than in the corresponding tissues of the mosaic conceptuses. Positive correlations were found for the percentage of PGK-1B between these three primitive ectoderm tissues in both chimaeras and mosaics and between the two primitive endoderm tissues (yolk sac endoderm and parietal endoderm) of the chimaeras. There was no significant correlation between the primitive ectoderm and primitive endoderm tissues of the chimaeras. The results suggest that unequal allocation of cell populations to the primitive ectoderm and primitive endoderm considerably increases the variability among chimaeras but variation probably exists before this segregation occurs. The variation that arises before and at this allocation event is present before X-chromosome inactivation occurs in the primitive ectoderm lineage and explains why the proportions of the two cell populations are more variable among chimaeras than mosaics. Additional variation arises within the primitive ectoderm lineage, after X-inactivation. This variation may be greater in chimaeras than mosaics but the evidence is inconclusive. The results also have some bearing on the nature of the allocation of cells to the primitive ectoderm and primitive endoderm lineages and the timing of X-chromosome inactivation in the primitive ectoderm lineage.

X-chromosome inactivation mosaicism in the three germ layers and the germ line of the mouse embryo.

Electrophoretic variant forms of the X-linked enzyme phosphoglycerate kinase (PGK-1, E.C.2,7,2,3) have been used to examine X-chromosome mosaicism in tissues from 12 1/2-day post coitum heterozygous female mouse embryos. Samples of yolk-sac endoderm, neural ectoderm, heart (mesoderm), liver (endoderm) and germ cells were analysed from each embryo. In all tissues except yolk-sac endoderm, both PGK-1 isozymes were expressed. The extent of covariance among tissues with respect to the PGK-1 isozyme contribution is consistent with all tissues being derived from the same pool of cells after X-inactivation. The covariance among tissues gives an estimate of the size of this pool (47 cells) and places the earliest time of X-inactivation in epiblast cells between 4 1/2 and 5 1/2 days post coitum. From the independent variance among tissues within an individual, the average primordial precursor pool size for the three germ layers and the germ line itself was estimated as 193 cells.

Cyclic nucleotides phosphodiesterase activity changes in early chick development.

Differences in the activity of cyclic nucleotides phosphodiesterase develop in different germ layers during the gastrulation of the chick embryo.

[Development of the adenylate cyclase activity of the embryonic chorda-mesoderm and endoderm during the migration of primordial germ cells in Xenopus laevis (anuran amphibian)]. / Evolution de l'activité adénylate cyclasique du cordomésoderme et de l'endoderme embryonnaires pendant la migration des cellules germinales primordiales chez Xenopus laevis (amphibien anoure).

In the embryo of Xenopus laevis, adenylate cyclase activity is higher in the chorda-mesoderm than in the endoderm. The peak of activity in the chorda-mesoderm is observed at the beginning of the migration of the primordial germ cells (PGC). There could be a correlation between the adenylate cyclase activity of the chorda-mesoderm and the intraendodermic migration of the PGC.