Assessment of apoptosis and appearance of hepatocyte growth factor in placenta at different gestational ages: A cross-sectional study

Background: Fetal growth is determined by the interaction between mother and fetus using the placental interface throughout the pregnancy. Objective: To research apoptosis and appearance of hepatocyte growth factor (HGF) in placentas of different gestational ages and to describe the anthropometrical and clinical indices of mothers and newborns. Materials and Methods: The study material was obtained from 53 human immunodeficiency virus negative pregnant women of legal age without systemic diseases. The staining of placental apoptotic cells was processed by a standard in situ cell death detection kit. The detection of HGF was provided by the ImmunoCruz goat ABC Staining System protocol sc-2023. Relative distribution of positive structures was evaluated using the semiquantitative counting method. Results: The mean rank value of the amount of HGF-containing cells (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, Höfbauer cells, and cells of extraembryonic mesoderm) was 1.61 ± 0.94. Apoptotic cells (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and cells of extraembryonic mesoderm) were found in all placental samples of various gestational ages (term 13.00 ± 13.05 and preterm 27.00 ± 18.25); in general, their amount decreased with advancing gestational age of the placenta (p < 0.01). Conclusion: Weight of a placenta directly depends on the gestational age and correlates with the main fetal anthropometrical parameters (weight, length, and head and chest circumferences). The decrease in HGF-containing and apoptotic cells with advancing gestation depends on the adaptation potential of the placenta, proving the other ways of cellular disposition. Key words: Pregnancy, Placenta, Gestational age, Apoptosis, Immunohistochemistry.

P–381 Deciphering the genetic cause of recurrent and sporadic pregnancy loss

Study question To investigate the prevalence and effect of (mosaic) de novo genomic aberrations in recurrent pregnancy loss (RPL) and sporadic abortion (SA). Summary answer Prevalence of maternal uniparental disomies (UPDs) was high in both cohorts. While chromosomal UPDs were found in both cohorts, genome wide UPDs were RPL specific. What is known already Spontaneous abortion occurs in 10–15% of clinically recognized pregnancies and recurrent pregnancy loss in 1–3%. SA and RPL are associated with reduced quality of life. Multiple factors contribute to SA and RPL, such as uterine malformations and parental/fetal chromosomal abnormalities. However, in ∼60% of SA and RPL the cause remains unknown. UPD is defined as the presence of two homologues chromosomes originating from a single parent. This phenomenon can lead to imprinting disorders that are characterised by clinical features affecting growth, development and metabolism in liveborn offspring. However, it could also be responsible for pregnancy loss. Study design, size, duration We recruited 32 families with pregnancy loss (n = 16 RPL cohort, n = 16 SA cohort) with no known genetic predispositions and normal karyotyping results in both parents and the fetus. Average maternal age was 28.68 years (SD = 5.43), paternal age 30.3 years (SD = 5.53), and the gestational age at pregnancy loss was 8.65 weeks (SD = 2.47). The average number of miscarriages in the RPL group was 3.57 (SD = 0.84). We profiled the genomic landscape of both cohorts using SNP typing. Participants/materials, setting, methods We isolated DNA from blood of both parents and the placental tissues from the miscarried products of conception. The placenta tissues were sampled from two distinct extraembryonic and embryonic germ layers, the extraembryonic mesoderm and the chorionic villi cytotrophoblast. Subsequently, we performed SNP-genotyping using Illumina’s Global-Screening Array–24 v2.0 BeadChips and applied haplarithmisis to delineate allelic architecture of fetal tissues of both cohorts. This allowed us to detect large de novo copy-number and -neutral (&gt;10kb) changes. Main results and the role of chance In this pilot study, we have analyzed 132 DNA samples (n = 32 families), of which 16 families were in the RPL cohort and 16 in the SA cohort. Within the RPL cohort, we found: one family with mosaic genome wide hexaploidy both in the extraembryonic mesoderm and chorionic villi, one family with a non-mosaic genome wide hetero UPD of the chorionic villi tissue, one family with a mosaic UPD of chromosome 14 in both tissues and tetraploidy exclusively in the chorionic villi, one family with a mosaic UPD of chromosome 16 in both tissues, one family with a mosaic UPD of chromosome 6 in both tissues, and another family with a mosaic UPD of chromosome 5 in the extraembryonic mesoderm. Within the SA group, one family showed a UPD of chromosome 7 and another family showed a segmental UPD of chromosome 5 in both tissues. Strikingly, all the UPDs found in this study were maternal in origin. Limitations, reasons for caution The main limitation of this study is the resolution of detecting copy-neutral and copy-number variations, which is an inherent limiting factor of SNP-array technology. In addition, in the sample in which we observed non-mosaic genome wide UPD, maternal contamination is likely that can be investigated by other technologies. Wider implications of the findings: Multiple genome wide UPDs are found in the RPL group but none in the SA group, indicating an association between genome wide mosaic UPD and RPL. These findings could lead to a better understanding of causative factors for SA and RPL and the need for a SNP-based non-invasive prenatal testing. Trial registration number Not applicable

Unveiling Complexity and Multipotentiality of Early Heart Fields

Rationale: Extraembryonic tissues, including the yolk sac and placenta, and the heart within the embryo, work to provide crucial nutrients to the embryo. The association of congenital heart defects (CHDs) with extraembryonic tissue defects further supports the potential developmental relationship between the heart and extraembryonic tissues. Although the development of early cardiac lineages has been well-studied, the developmental relationship between cardiac lineages, including epicardium, and extraembryonic mesoderm remains to be defined. Objective: To explore the developmental relationships between cardiac and extraembryonic lineages. Methods and Results: Through high-resolution single cell and genetic lineage/clonal analyses, we show an unsuspected clonal relationship between extraembryonic mesoderm and cardiac lineages. Single-cell transcriptomics and trajectory analyses uncovered two mesodermal progenitor sources contributing to left ventricle cardiomyocytes, one embryonic and the other with an extraembryonic gene expression signature. Additional lineage-tracing studies revealed that the extraembryonic-related progenitors reside at the embryonic-extraembryonic interface in gastrulating embryos, and produce distinct cell types forming the pericardium, septum transversum, epicardium, dorsolateral regions of the left ventricle and atrioventricular canal myocardium, and extraembryonic mesoderm. Clonal analyses demonstrated that these progenitors are multipotent, giving rise to not only cardiomyocytes and serosal mesothelial cell types but also, remarkably, extraembryonic mesoderm. Conclusions: Overall, our results reveal the location of previously unknown multipotent cardiovascular progenitors at the embryonic-extraembryonic interface, and define the earliest embryonic origins of serosal mesothelial lineages, including the epicardium, which contributes fibroblasts and vascular support cells to the heart. The shared lineage relationship between embryonic cardiovascular lineages and extraembryonic mesoderm revealed by our studies underscores an underappreciated blurring of boundaries between embryonic and extraembryonic mesoderm. Our findings suggest unexpected underpinnings of the association between congenital heart disease and placental insufficiency anomalies, and the potential utility of extraembryonic cells for generating cardiovascular cell types for heart repair.

Identification of Distinct Pathways Involved in Regulation of Mesenchymoangioblasts and Hemangioblasts Development From Mesoderm

Abstract 1326 Using hESC differentiation system we recently identified a mesenchymoangioblast (MAB) as a novel precursor for mesenchymal stem cells (MSCs) and endothelial cells and demonstrated that mesenchymal and hematopoietic cells develop sequentially from mesodermal precursors with primary angiogenic potential – MAB and hemangioblasts (HB), respectively. In addition, we found that angiogenic mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by surface expression of apelin receptor (APLNR) and lack of expression of typical endothelial (CD31, CD144), hematopoietic (CD43, CD45) and mesenchymal (CD73, CD195) markers i.e. as EHMlin-APLNR+ cells. (Vodyanik et al. Cell Stem Cell 2010;7:718). In response to FGF2 APLNR+ cells form morphologically distinct compact mesenchymal or MAB colonies and disperse hematopoietic or blast (HB) colonies in serum-free clonogenic semisolid medium. When transferred to the adherent cultures in serum-free medium with FGF2, individual colonies gave rise to multipotential mesenchymal cell lines with typical phenotype (CD146+CD105+CD73+CD31-CD43-45-), differentiation (chondro-, osteo-, and adipogenesis) and robust proliferation (>80 doublings) potentials. In contrast, HB colonies consisted almost entirely of CD235a and CD41a expressing cells with morphology resembling erythroblasts. Replating of HB colonies in hematopoietic serum-free and serum-containing clonogenic medium demonstrated that they gave rise to erythroid, megakaryocytic and mixed colonies composed of erythroid, megakaryocytic cells and macrophages indicating that BL-CFCs probably reflected the first wave of embryonic hematopoiesis initiated in the yolk sac. To define pathways involved in MAB and HB development we tested the effect of different growth factors and cytokines on mesenchymal and blast colony formation. We confirmed that the development of MAB and HB depends on FGF2, by demonstrating complete suppression of blast and mesenchymal colony formation by elimination of FGF2 from clonogenic culture or by abrogation of FGF2 signaling using PD 173074 inhibitor of FGF2 receptor autophosphorylation. The formation of MAB and HB colonies was also completely abrogated by adding TGFb or activin A, and increased in the presence of SB431542 TGFb signaling inhibitor. PDGF-BB alone lacked colony-forming activity, but its addition to FGF2 significantly increased the frequency and size of mesenchymal but not blast colonies. In contrast, the addition of VEGF essentially abrogated mesenchymal colony formation. Although VEGF had little effect on BL-CFCs, the addition KI8751 KDR inhibitor significantly decreased the number of blast colonies, confirming that their development depends on VEGF signaling. The addition of individual hematopoietic cytokines to FGF2 had a relatively mild effect on the number of blast colonies. However, they increased the size of colonies, which was especially obvious with the addition of EPO. When IL3, IL6, EPO, TPO, and SCF were added to clonogenic cultures together with FGF2, we observed a significant increase in the number and size of blast colonies, some of which had grown into very large grape-like structures. Because we found that emerging CD144+CD235a+ cells generated hematopoietic colonies morphologically resembling blast colonies in the presence of FGF2 and hematopoietic cytokines, we concluded that hematopoietic cytokine-free clonogenic cultures would be more appropriate for detection of BL-CFCs and enhancing the specificity of this assay. The finding that mesenchymal and blast CFCs arise from cells expressing APLNR prompted us to test whether this receptor is involved in the regulation of MAB and HB development. We found that APLNR agonist aplein-12 inhibits mesenchymal colonies, while it significantly increases the formation of blast colonies in synergy with VEGF. Together, these studies demonstrated that the activation of multiple but different signaling pathways regulates the formation of mesenchymal and blast colonies. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership.

Deficiency of Zonula Occludens-1 Causes Embryonic Lethal Phenotype Associated with Defected Yolk Sac Angiogenesis and Apoptosis of Embryonic Cells

Zonula occludens (ZO)-1/2/3 are the members of the TJ-MAGUK family of membrane-associated guanylate kinases associated with tight junctions. To investigate the role of ZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1−/−) mice were generated by gene targeting. Although heterozygous mice showed normal development and fertility, delayed growth and development were evident from E8.5 onward in Tjp1−/− embryos, and no viable Tjp1−/− embryos were observed beyond E11.5. Tjp1−/− embryos exhibited massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E)9.5. In the yolk sac, the ZO-1 deficiency induced defects in vascular development, with impaired formation of vascular trees, along with defective chorioallantoic fusion. Immunostaining of wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2 were expressed in almost all embryonic cells, showing tight junction-localizing patterns, with or without ZO-3, which was confined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expression without influence on ZO-2/3 expression. In Tjp1+/+ yolk sac extraembryonic mesoderm, ZO-1 was dominant without ZO-2/3 expression. Thus, ZO-1 deficiency resulted in mesoderms with no ZO-1/2/3, associated with mislocalization of endothelial junctional adhesion molecules. As a result, angiogenesis was defected in Tjp1−/− yolk sac, although differentiation of endothelial cells seemed to be normal. In conclusion, ZO-1 may be functionally important for cell remodeling and tissue organization in both the embryonic and extraembryonic regions, thus playing an essential role in embryonic development.

Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors

Flk1, the gene encoding the vascular endothelial growth factor receptor 2 (VEGFR-2), is a well-known marker for vascular and hematopoietic progenitors and is indispensable for normal hematopoiesis and vasculogenesis. Here we show that Flk1 expression in the early mouse embryo marks a broad spectrum of mesodermal progenitors exiting the primitive streak as well as later mesodermal cell types including some cardiomyocytes, portions of the somites, and all extraembryonic mesoderm cells. These findings made use of an Flk1-lacZ knock-in allele in which the neomycin selection cassette was removed, which resulted in full replication of the endogenous expression of Flk1. Targeted deletion of a region in intron 1 that has been proposed to direct endothelial expression produced no alteration in either endothelial or broader mesodermal expression of the Flk1-lacZ allele. Examination of lacZ expression in homozygotes for the Flk1lacZ neo-out allele revealed that lacZ-expressing mesodermal cells persisted in nonvascular regions. Thus, Flk1 expression marks progenitors with broad mesodermal potential but is not absolutely required for the development of all mesodermal lineages in which it is expressed.