Academic literature on the topic 'Trophoectoderm differentiation'

Abstract Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells (HSCs) acquiring mutations in the PIG-A gene. Its product (PIG-A protein) is required for biosynthesis of dozens of cell surface proteins to be linked to glycosyl-phosphatidyl-inositol (GPI) molecule and anchored on plasma membrane. The underlying mechanisms of PIG-A mutant clonal dominance in PNH patients and the close relationship of PNH to other bone marrow failure diseases (aplastic anemia and myelodysplasia syndrome) and leukemia remain unknown. Establishing a mutation of PIG-A gene in human HSCs from healthy donors remains unfeasible due to the current inability to expand and select clonal HSCs in culture. Although mouse models have been generated by disrupting conditionally the mouse Pig-a gene, the existing Pig-a null mice indeed lacking GPI-APs in blood cells did not replicate faithfully PNH pathological symptoms. To create a human cell-based, prospective experimental system to investigate the effects of PIG-A/GPI-AP deficiency and pathophysiology of PNH, we made mutated hES cells lacking GPI-APs. FACS analysis revealed that two independent hES clones lack cell-surface expression of CD55 and CD59, as well as CD90/Thy and Cripto that are preferentially expressed in undifferentiated hES cells. However, the cell-surface expression of these GPI-APs can be restored by a lentiviral vector inducibly expressing the PIG-A cDNA. Like mouse ES cells, lack of PIG-A/GPI-APs did not affect the growth of undifferentiated hES cells in culture. Unlike mouse ES cells, however, PIG-A/GPI-AP deficient hES cells formed embryoid bodies normally in culture. RT-PCR analysis of marker gene expression indicated that commitment to the 3 (somatic) germ layers appeared normal within embryonic bodies from either the mutated or wildtype hES cells. However, formation of extra-embryonic cells such as trophoblasts from the PIG-A/GPI-AP deficient hES cells is defective in both embryoid body formation and BMP4-induced assays. Expression of trophoectoderm-specific genes such as hCGalpha could not be induced in PIG-A/GPI-AP deficient hES cells upon BMP4 induced trophoectodermal differentiation. The induction of other trophoectoderm markers such as hCGbeta and CDX2 was also significantly reduced after the BMP4 treatment. The lack of tropho-ectoderm was further confirmed by lacking of hCG hormone production. The defect in trophoectoderm differentiation from the PIG-A/GPI-AP deficient hES cells was restored by the expression of the PIG-A cDNA in the mutated hES cells. For somatic cell differentiation, we are currently examining the effects of PIG-A/GPI-AP deficiency beyond the initial differentiation commitment during embryonic body formation stage. Along mesodermal and hematopoietic differentiation, we found that the PIG-A/GPI-AP deficiency in hES cells had little effect on the formation of CD34+ cells, a precursor cell population for human hematopoietic and endothelial lineages. We are currently examining the effects of PIG-A/GPI-AP deficiency on properties of hematopoietic cells derived from the mutated and normal hES cells. This study represents one of first cases that hES cells may provide a prospective research model to investigate genetic and developmental basis of human diseases.

Elongation of the trophectoderm and gastrulation of the embryonic disc, observed during gestational Days 11 (D11) through 12 (D12), denote a critical period of porcine conceptus development. Serial analysis of gene expression identified genes involved in cellular differentiation/structure (cytokeratin-8 and -18) and growth/cell migration/mesoderm-epithelial interaction (stratifin and midkine), which could potentially be regulated by steroids such as estrogen. Characterization of these factors is lacking in porcine conceptuses, therefore, the current study investigated mRNA expression of these factors in elongating conceptuses and primordial tissues as well as protein expression and cellular localization to better define their biological significance. Conceptuses examined were of ovoid (D11; 6-10 mm), tubular (D11; 11-50 mm), or filamentous (D12; >100 mm) morphology. Cells of the conceptus were highly proliferative at all stages and the embryonic disc of the ovoid conceptus was already polarized as indicated by the protein expression of Ki67 and brachyury. Real-time PCR was utilized to determine the transcript expression profiles. Differential expression of cytokeratin-18 and midkine were not apparent; however, cytokeratin-8 was clearly down-regulated in filamentous compared to ovoid conceptuses. In contrast, stratifin mRNA levels were greatest in tubular conceptuses of 42-50 mm size. Transcripts for cytokeratin-8 and -18, stratifin, and midkine were detected in both cell types (endoderm and trophoblast) of the trophoectoderm. Western blotting and/or immunohistochemistry were utilized to examine protein expression and cellular localization. The embryonic disc of ovoid conceptuses was almost devoid of cytokeratin-18 protein, however, its distribution was uniform throughout the trophectoderm at all stages of elongation. Stratifin and midkine proteins demonstrated more unique expression patterns within the conceptus. Distinct cell populations of the embryonic disc and the trophoectoderm contained stratifin; cellular localization was predominantly cytoplasmic but occasional nuclear translocation was evident. Furthermore, total protein levels of stratifin were not different among ovoid, tubular, and filamentous conceptuses, but proteolysis of the protein was apparent at the filamentous stage. Midkine protein expression was prominent in the embryonic disc of ovoid conceptuses. In tubular conceptuses, midkine was associated with cells that appeared to be migrating away from embryonic disc as well as some concentrating in the tips of the trophectoderm. Our findings suggested that cytokeratin-8 and -18 are associated primarily with the trophectoderm, as seen in other species. Furthermore, the distribution and localization of stratifin and midkine proteins could reflect attributed functions of these factors, minimal anti-proliferative activity in the rapidly growing conceptuses and cell migration important for gastrulation/trophectoderm elongation, respectively.

Abstract Trophoblast stem cells (TSCs), which can be derived from the trophoectoderm of a blastocyst, have the ability to sustain self-renewal and differentiate into various placental trophoblast cell types. Meanwhile, essential insights into the molecular mechanisms controlling the placental development can be gained by using TSCs as the cell model. Esrrb is a transcription factor that has been shown to play pivotal roles in both embryonic stem cell (ESC) and TSC, but the precise mechanism whereby Esrrb regulates TSC-specific transcriptome during differentiation and reprogramming is still largely unknown. In the present study, we elucidate the function of Esrrb in self-renewal and differentiation of TSCs, as well as during the induced TSC (iTSC) reprogramming. We demonstrate that the precise level of Esrrb is critical for stem state maintenance and further trophoblast differentiation of TSCs, as ectopically expressed Esrrb can partially block the rapid differentiation of TSCs in the absence of fibroblast growth factor 4. However, Esrrb depletion results in downregulation of certain key TSC-specific transcription factors, consequently causing a rapid differentiation of TSCs and these Esrrb-deficient TSCs lose the ability of hemorrhagic lesion formation in vivo. This function of Esrrb is exerted by directly binding and activating a core set of TSC-specific target genes including Cdx2, Eomes, Sox2, Fgfr4, and Bmp4. Furthermore, we show that Esrrb overexpression can facilitate the MEF-to-iTSC conversion. Moreover, Esrrb can substitute for Eomes to generate GEsTM-iTSCs. Thus, our findings provide a better understanding of the molecular mechanism of Esrrb in maintaining TSC self-renewal and during iTSC reprogramming.

Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4mgmL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2μMmL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3mgmL−1 PVA, fixed for 15min at RT in 4% paraformaldehyde containing 10μgmL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30min at 37°C in a humid chamber. The embryos were then treated with RNase A (50UmL−1) for 30min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3UmL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than in zona-enclosed blastocysts (n=35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74±22) , and ICM/TE ratio (0.28±0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.

Embryonic stem cells (ESC) are a useful tool for studying embryonic development, cell differentiation, and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral, and religious considerations by scientists and laymen alike. Therefore, an animal model such as the pig (Sus scrofa) is valuable in overcoming such hurdles because this species holds physiologic parameters similar to humans. In spite of the great biomedical potential of ESC, many difficulties have been faced in maintaining these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro- and in vivo-produced swine blastocysts; (2) to compare 2 in vitro culture conditions to maintain isolated inner cell masses (ICM), murine embryonic fibroblasts (MEF), or Matrigel; and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2, and FoxD3 at ESC and in vitro- and in vivo-produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vivo-produced blastocysts were obtained by superovulation followed by AI of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing 5 days after ovulation. In vitro- and in vivo-produced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution, and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. Resultant ICM (14 and 66 ICM from in vitro- and in vivo-produced blastocysts, respectively) were cultured in stem cells media (GMEM added by 15% FCS, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng mL-1 of basic fibroblast growth factor) over monolayer of irradiated mouse embryonic fibroblasts (MEF) or Matrigel for 3 weeks. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro- and in vivo-produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis was performed only with whole in vitro- and in vivo-produced blastocysts. Results showed that Nanog and Sox2 were less expressed in in vitro-produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Because no ESC lineage was obtained in swine until now, we believe this species has different requirements compared with murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC. Acknowledgments are given to FAPESP (processes 06/58507-0 and 07/51732-0).

The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein inin vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout the blastocysts. In contrast,in vivo-produced day-7 to -10 blastocysts showed greatly reduced trophoectodermal POU5F1 protein expression. To determine whether the uterine environment modulates POU5F1 expression, IVP blastocysts were transferred to the uteri of mares, then recovered 2–3 days later (IVP-ET embryos). These embryos showed similar POU5F1 expression as thein vivo-produced embryos. Levels ofPOU5F1,SOX2, andNANOGmRNA in IVP-ET blastocysts were significantly higher in the inner cell mass than in trophectoderm (TE) cells. These data suggest that the differentiation of equine TE, as indicated by loss of POU5F1 expression, is impaired duringin vitroculture, but proceeds normally when the embryos are exposed to the uterine environment. Previously reported differences in trophectodermal expression of POU5F1 among species may thus be in part artifactual, i.e. related toin vitroculture. Failure for correction of such changes by the uterine environment is a potential factor in the placental abnormalities seen after transfer of cultured embryos in some species.

The use of human induced pluripotent stem cells (hiPSCs) has tremendous potential for regenerative medicine by providing an unlimited source of personalized cells. A number of protocols have been established for efficient differentiation of hiPSCs to the desired lineage in vitro, such as cardiomyocytes and blood. However, the field lacks an in vivo system to evaluate the differentiation potential and quality of hiPSCs. Developmental potential of stem cells derived from experimental animals can be readily assessed by generating blastocyst chimeras and examination of the contribution to the embryos, or by the potential of teratoma formation. However, this is not possible in the case of humans. As a potential solution for this issue, we examined whether porcine parthenotes could be used as an experimental model to test the developmental potential of the hiPSCs. Parthenotes are generated by electrical activation of the oocytes collected at the abattoir and will develop up to gestational day 53 if transferred to a pseudo-pregnant sow. The embryonic culture conditions have also been established and the zygotes can develop normally to the expanded blastocyst stage (day 7 post fertilization/activation), in vitro. We took advantage of this in vitro system and examined the ability of hiPSCs to proliferate and integrate into the parthenogenetic embryos. Parthenogenetic embryos were injected with ten undifferentiated hiPSCs at day 4 (8 cell ~ morula stage) and cultured up to 72 hours. During this period, parthenotes underwent blastocoel cavity formation and hatching. Cell tracing experiments demonstrated that hiPSCs proliferated and integrated into the parthenotes. They retained pluripotency marker expression during this period. hiPSCs and their derivatives were found both in trophoectoderm and embryo proper. We further observed that the hiPSCs underwent cellular proliferation and promoted developmental progression of the parthenote in vitro. In summary, the porcine parthenote model system is an efficient high throughput system to examine the developmental capacity of human stem cell populations.

The presence of rare transient cells within pluripotent stem cell population that resemble functional and transcriptional features of totipotent 2-cell-stage embryos has emerged several questions regarding their mechanism of reactivation. Although few recent works have aimed to characterize them both at transcriptional and functional level, the mechanism of transition to 2C-like state is poorly investigated. Here we identified a new pathway, whose activation through several compounds could induce transition to 2C-like state. Interestingly, inhibition of this pathway by specific inhibitors could collectively restrain the majority of the 2C-like state transcriptional alterations including MuERV-L and Zscan4 gene family. Finally, we found that developmental potential of induced 2C-like cells could be extended to embryonic plus extra-embryonic tissues in contrast to embryonic stem cells in culture. Our finding provides a better understanding of cellular plasticity at early embryonic state, which could be important for the potential therapeutic avenues.