Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment.

Activin, a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Steroids Regulate SLC2A1 and SLC2A3 to Deliver Glucose Into Trophectoderm for Metabolism via Glycolysis.

The conceptuses (embryo/fetus and placental membranes) of pigs require energy to support elongation and implantation, and amounts of glucose and fructose increase in the uterine lumen during the peri-implantation period. Conceptuses from day 16 of pregnancy were incubated with either 14C-glucose or 14C-fructose and amounts of radiolabeled CO2 released from the conceptuses measured to determine rates of oxidation of glucose and fructose. Glucose and fructose both transport into conceptuses, and glucose is preferentially metabolized in the presence of fructose, whereas fructose is actively metabolized in the absence of glucose and to a lesser extent in the presence of glucose. Endometrial and placental expression of glucose transporters SLC2A1, SLC2A2, SCL2A3, and SLC2A4 were determined. SLC2A1 messenger RNA (mRNA) and protein, and SLC2A4 mRNA were abundant in the uterine luminal epithelium of pregnant compared to cycling gilts, and increased in response to progesterone and conceptus-secreted estrogen. SLC2A2 mRNA was expressed weakly by conceptus trophectoderm on day 15 of pregnancy, whereas SLC2A3 mRNA was abundant in trophectoderm/chorion throughout pregnancy. Therefore, glucose can be transported into the uterine lumen by SLC2A1, and then into conceptuses by SLC2A3. On day 60 of gestation, the cell-specific expression of these transporters was more complex, suggesting that glucose and fructose transporters are precisely regulated in a spatial-temporal pattern along the uterine-placental interface of pigs to maximize hexose sugar transport to the pig conceptus/placenta.

Non-cytotoxic silver nanoparticle levels perturb human embryonic stem cell-dependent specification of the cranial placode in part via FGF signaling.

Silver nanoparticles (AgNPs) are compounds used in numerous consumer products because of their desirable optical, conductive and antibacterial properties. However, several in vivo and in vitro studies have raised concerns about their potential developmental toxicity. Here, we employed a human embryonic stem cell model to evaluate the potential ectodermal toxicity of AgNPs, at human relevant concentrations. Among the four major ectodermal lineages tested, only cranial placode specification was significantly affected by AgNPs and AgNO3, morphology-wise and in the expression of specific markers, such as SIX3 and PAX6. Mechanistically, we found that the effects of AgNPs on the cranial placode differentiation were probably due to Ag ion leakage and mediated by the FGF signaling. Thus, AgNPs may have the ability to alter the early stages of embryonic development.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

Type I interferon response impairs differentiation potential of pluripotent stem cells.

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.

ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation.

Significant embryo loss remains a serious problem in pig production. Reactive oxygen species (ROS) play a critical role in embryonic implantation and placentation. However, the potential mechanism of ROS on porcine trophectoderm (pTr) cell fate during the peri-implantation period has not been investigated. This study aimed to elucidate the effects of ROS on pTr cell phenotypes and the regulatory role in cell attachment and differentiation. Herein, results showed that exogenous H2O2 inhibited pTr cell viability, arrested the cell cycle at S and G2/M phases, and increased cell apoptosis and autophagy protein light chain 3B and Beclin-1, whereas these effects were reversed by different concentrations of N-acetyl-l-cysteine (NAC) posttreatment. In addition, NAC abolished H2O2-induced autophagic flux, inhibited intracellular and mitochondrial ROS, and restored expression of genes important for mitochondrial DNA and biogenesis, cell attachment, and differentiation. NAC reversed H2O2-activated MAPK and Akt/mammalian target of rapamycin pathways in dose-dependent manners. Furthermore, analyses with pharmacological and RNA interference approaches suggested that autophagy regulated cell apoptosis and gene expression of caudal-related homeobox 2 and IL-1ß. Collectively, these results provide new insights into the role of the ROS-induced autophagy in pTr cell apoptosis, attachment, and differentiation, indicating a promising target for decreasing porcine conceptus loss during the peri-implantation period.

Murine exposure to gold nanoparticles during early pregnancy promotes abortion by inhibiting ectodermal differentiation.

BACKGROUND: Gold nanoparticles (AuNPs) have been widely studied for biomedical applications, although their safety and potential toxicity in pregnancy remains unknown. The aim of this study is to explore the effect of AuNPs maternal exposure at different gestational ages on fetal survival and development, as well as the potential mechanism of AuNPs affecting embryos and fetuses. METHODS: Thirty nm polyethylene glycol (PEG)-coated AuNPs (A30) were administered to pregnant mice via intravenous injection (5 µg Au/g body weight) over three days at either early or late pregnancy. Fetal abortion rate and morphological development in E16.5 were then detected in detail. The pregnant mice physiological states with A30 exposure were examined by biochemical, histological or imaging methods; and materno-fetal distribution of gold elements was assayed by electron microscopy and mass spectrometry. Murine embryonic stem cells derived embryoid-bodies or neuroectodermal cells were treated with A30 (0.0025 to 0.25 µg Au/mL) to examine A30 effects on expression levels of the germ differentiation marker genes. Tukey's method was used for statistical analysis. RESULTS: Exposure to A30 during early (A30E) but not late (A30L) pregnancy caused a high abortion rate (53.5%), lower fetal survival rate and abnormal decidualization compared with non-exposed counterparts. The developmental damage caused by A30 followed an "all-or-nothing" pattern, as the non-aborted fetuses developed normally and pregnancies maintained normal endocrine values. A30 caused minor impairment of liver and kidney function of A30E but not A30L mice. TEM imaging of fetal tissue sections confirmed the transfer of A30 into fetal brain and live as aggregates. qPCR assays showed A30 suppressed the expression of ectodermal, but not mesodermal and endodermal differentiation markers. CONCLUSIONS: These results illustrate that maternal A30 exposure in early pregnant results in A30 transfer into embryonic tissues, inhibiting ectodermal differentiation of embryonic stem cells, leading to abnormal embryonic development and abortion. While exposure to A30 during late pregnancy had little or no impact on dams and fetuses. These findings suggest the safety of biomedical applications employing AuNPs during pregnancy is strongly influenced by fetal maturity and gestational age at exposure and provide the clues for AuNPs safe application period in pregnancy.

Quantitative Analysis of the Area of the Apical Ectodermal Ridge in Chick Appendages Using Image-J.

OBJECTIVE: To determine the effect of sodium phenytoin on the apical ectodermal ridges (AER) of chick wing buds by using the software program Image-J. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Anatomy, Regional Center, College of Physicians and Surgeons Pakistan (CPSP), Islamabad, from January 2014 to January 2015. METHODOLOGY: Sixty fertilised chicken eggs of 'Egyptian fayoumi' breed were selected and separated into experimental (B) and control (A) groups, each having 30 eggs. A single dose of 3.5 mg sodium phenytoin was injected into each egg of the experimental group. The controls were injected with the same volume of normal saline. Developing embryos were extracted 96 hours (day 4) after incubation and histological sections were cut at 5 µm thickness. These sections were stained with Feulgen Nuclear and Light Green. The area of apical ectodermal ridges of chick wing buds was calculated by employing Image-J and subjected to statistical analysis. RESULTS: The difference between the mean values of the area of apical ectodermal ridges of experimental and control groups, as calculated by Image-J, was found to be statistically insignificant. CONCLUSION: Change in the area of the apical ectodermal ridges in experimental chicks, following phenytoin exposure, was insignificant as proven on the basis of quantification by Image-J.

Retinoic acid signaling and neurogenic niche regulation in the developing peripheral nervous system of the cephalochordate amphioxus.

The retinoic acid (RA) signaling pathway regulates axial patterning and neurogenesis in the developing central nervous system (CNS) of chordates, but little is known about its roles during peripheral nervous system (PNS) formation and about how these roles might have evolved. This study assesses the requirement of RA signaling for establishing a functional PNS in the cephalochordate amphioxus, the best available stand-in for the ancestral chordate condition. Pharmacological manipulation of RA signaling levels during embryogenesis reduces the ability of amphioxus larvae to respond to sensory stimulation and alters the number and distribution of ectodermal sensory neurons (ESNs) in a stage- and context-dependent manner. Using gene expression assays combined with immunohistochemistry, we show that this is because RA signaling specifically acts on a small population of soxb1c-expressing ESN progenitors, which form a neurogenic niche in the trunk ectoderm, to modulate ESN production during elongation of the larval body. Our findings reveal an important role for RA signaling in regulating neurogenic niche activity in the larval amphioxus PNS. Although only few studies have addressed this issue so far, comparable RA signaling functions have been reported for neurogenic niches in the CNS and in certain neurogenic placode derivatives of vertebrates. Accordingly, the here-described mechanism is likely a conserved feature of chordate embryonic and adult neural development.

Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca2+ signals play an essential role in the active cell migration during gastrulation.

DEP and DBP induce cytotoxicity in mouse embryonic stem cells and abnormally enhance neural ectoderm development.

Diethyl phthalate (DEP) and dibutyl phthalate (DBP) are two typical small phthalate esters, extensively used in personal care and consumer products. Although previous studies have linked phthalate esters to several health issues, it is still unclear whether they can affects the early stages of embryonic development. In this study, we evaluated the early developmental neurotoxicity as well as the cytotoxicity of DEP and DBP, using mouse embryonic stem cells (mESCs). Our results showed that both DEP and DBP could decrease mESC viability in a dose-dependent manner. Moreover, while DBP could activate the caspase-3/7 enzymes and cause cell membrane damage as well as intracellular ROS accumulation, interestingly DEP treatment only showed stimulation of ROS production. In addition, DEP and DBP treatment at non-cytotoxic concentrations, abnormally altered the expression levels of several vitally important regulators of embryo development. For instance, neural ectoderm markers, such as Pax6, Nestin, Sox1 and Sox3, were significantly up-regulated upon DEP and DBP exposure. In conclusion, our work suggests a potential developmental toxicity of DEP and DBP on mammals, especially for neural ectoderm specification. Our findings help better understand the association between health problems and DEP/DBP exposure and most significantly remind us of the importance of additional health risk tests for these two largely used chemicals.

Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells.

Paracrine signals maintain developmental states and create cell fate patterns in vivo and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.

Bisphenol A Represses Dopaminergic Neuron Differentiation from Human Embryonic Stem Cells through Downregulating the Expression of Insulin-like Growth Factor 1.

Bisphenol A (BPA) is a ubiquitous compound emerging as a possible toxicant during embryonic development. Human embryonic stem cell (hESC) promises a valuable model for evaluating the effects of environmental chemicals on human prenatal development. In our study, 1 µM BPA were applied to hESC-derived embryoid bodies (hEBs) and effects of BPA on neural cell differentiation were investigated. The expression level of insulin-like growth factor 1 (IGF-1) and marker genes for ectoderm, neuron progenitor cells, and dopaminergic (DA) neurons were all repressed upon BPA exposure. The population of hESC-derived neural precursor cells (NPCs) and DA neurons were decreased. Furthermore, yield of DA neuron-secreted tyrosine hydroxylase (TH) and dopamine were also reduced. When recombinant IGF-1 supplied, BPA-caused repressions were partially or completely relieved. Our further methylation microarray analysis indicated that there was a higher methylation level on the promoter of SRY-related HMG-box 5 (SOX5), a possible enhancer of IGF-1. Consistently, next quantitative polymerase chain reaction (qPCR) results confirmed that SOX5 expression was downregulated. Our investigation suggests that BPA represses DA neuron differentiation mainly through downregulating IGF-1 expression, which may attribute to the altered methylation level on the promoter of IGF-1 upstream genes. Our findings first elaborate the mechanism of IGF-1-mediated BPA effects on neuronal differentiation, which is helpful to illuminate the unique mechanism of BPA toxicity on prenatal neurodevelopment.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRß, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

In vitro cultures of ectodermal monolayers from the model sea anemone Nematostella vectensis.

We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24 h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2 h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72 h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R 2 = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2 h after isolation and then replaced by standard medium, showed, at 6 days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.

Transcriptome and proteome characterization of surface ectoderm cells differentiated from human iPSCs.

Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye, and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFß superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFß-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis, and development of regenerative medicine approaches.

MAPK signaling is necessary for neurogenesis in Nematostella vectensis.

BACKGROUND: The nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood. RESULTS: We show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages. CONCLUSIONS: Together, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.

Correlation between IL-6 and invasiveness of ectoderm cells of embryo in early pregnancy.

This study aimed to explore the correlation between Interleukin-6 (IL-6) and invasiveness of ectoderm cells of embryo in early pregnancy, in order to further discuss whether IL-6 can enhance invasiveness of ectoderm cells. The study lays the foundation for determination of pathogenesis of some gestation period-related diseases. Differences in mRNA and protein expression of trophoblastic cell line JEG-3 cells in IL-6, matrix metalloproteinase-2 (MMP-2) and MMP-9 were analyzed; the regulating effect of different concentrations of IL-6 on invasive ability of trophoblast cells was studied by Transwell assay; the effect of IL-6 on proliferation of ectodermal cell line JEG-3 of embryo was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The invasive number of JEG-3 cells incubated by IL-6 (10 ng/ml) was higher than that of the control group, and the difference had statistical significance (p < 0.05). Results of using MMT assay to detect the effect of IL-6 on proliferation of trophoblastic cell line JEG-3 showed that JEG-3 cells before and after processing had no significant difference from the control group (p >0.05). Therefore, IL-6 can enhance invasiveness of ectoderm cells of embryo through activation of MMP-2.

Single-cell analysis delineates a trajectory toward the human early otic lineage.

Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Single-cell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types.

Lysophosphatidic Acid (LPA) Receptor 3-Mediated LPA Signal Transduction Pathways: A Possible Relationship with Early Development of Peri-Implantation Porcine Conceptus.

Lysophosphatidic acid (LPA) is a phospholipid with a variety of fatty acyl groups that mediates diverse biological effects on various types of cells through specific G protein-coupled receptors. LPA appears to play a significant role in many reproductive processes, including luteolysis, implantation, and placentation. Our previous study in pigs demonstrated that LPA and the LPA receptor system are present at the maternal-conceptus interface and that LPA increases uterine endometrial expression of prostaglandin-endoperoxide synthase 2 (PTGS2) through LPA receptor 3 (LPAR3). However, the role of LPA in conceptuses during early pregnancy has not been determined. Therefore, this study examined the effects of LPA in cell proliferation, migration, and activation of the intracellular signaling pathway in porcine conceptuses by using an established porcine trophectoderm (pTr) cell line isolated from Day 12 conceptuses. All examined LPA species with various fatty acid lengths increased proliferation and migration of pTr cells as the dosage increased. Immunoblot analyses found that LPA activated intracellular signaling molecules, extracellular signal-regulated kinase 1/2 (ERK1/2), ribosomal protein S6 kinase 90 kDa (P90RSK), ribosomal protein S6 (RPS6), and P38 in pTr cells. Furthermore, LPA increased expression of PTGS2 and urokinase-type plasminogen activator (PLAU), and the LPA-induced increases in PTGS2 and PLAU expression were inhibited by LPAR3 siRNA. Collectively, these results showed that LPA promotes proliferation, migration, and differentiation of pTr cells by activating the ERK1/2-P90RSK-RPS6 and P38 pathways, indicating that the LPA-LPAR3 system may be involved in the development of trophoblast during early pregnancy in pigs.