Sylvia Lui

Transplantation of 3D scaffolds seeded with human embryonic stem cells: biological features of surrogate tissue and teratoma-forming potential

AIM To generate complex surrogate tissue by transplanting 3D scaffolds seeded with human embryonic stem cells (hESCs) between the liver lobules of severe combined immunodeficient (SCID) mice and to assess the teratoma-forming potential. MATERIALS & METHODS 3D poly-(lactic-co-glycolic acid) (PLGA) scaffolds coated with laminin were seeded with hESCs and then transplanted between the liver lobules of SCID mice. After a period of in vivo differentiation, the scaffolds were retrieved and analyzed using reverse transcription polymerase chain reaction, immunofluorescent staining and scanning electron microscopy. RESULTS A proportion of the hESCs within the scaffolds differentiated into cells that produced proteins characteristic of specific tissues, including endoderm and pancreatic markers glucogon-like peptide-1 receptor, islet amyloid polypeptide and Insulin. Markers of hepatic and neuronal lineages were also investigated. Major matrix proteins abundant in multiple tissue types, including collagen I, laminin and collagen IV, were found to be profuse within the scaffold pores. Transplantation of the seeded scaffolds between liver lobules also resulted in extensive vascularization both from host blood vessel incursion and the differentiation of hESCs into endothelial progenitor cells. An investigation of teratoma-forming potential demonstrated that transplantation of 3D scaffolds seeded with hESCs will, under certain conditions, lead to the growth of teratomas. DISCUSSION Transplantation of 3D scaffolds seeded with hESCs between liver lobules resulted in the development of surrogate tissue containing cells that produced proteins representing the pancreatic, hepatic and neuronal lineages, the assembly of an extracellular matrix structure and the formation of a vasculature. hESCs seeded within 3D scaffolds and transplanted into SCID mice were capable of forming teratomas. However, the formation and progression of teratoma growth is shown to be dependant on both the site of transplantation and the treatment of cells prior to transplantation.

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta

Tumor-homing peptides have been exploited to create nanocarriers for targeted delivery of therapeutic agents to the placenta. The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics.

Detrimental Effects of Ethanol and Its Metabolite Acetaldehyde, on First Trimester Human Placental Cell Turnover and Function

Fetal alcohol spectrum disorder (FASD) describes developmental issues from high maternal alcohol intake, which commonly results in fetal growth restriction and long term morbidity. We aimed to investigate the effect of alcohol and acetaldehyde, on the first trimester placenta, the period essential for normal fetal organogenesis. Normal invasion and establishment of the placenta during this time are essential for sustaining fetal viability to term. We hypothesise that alcohol (ethanol) and acetaldehyde have detrimental effects on cytotrophoblast invasion, turnover and placental function. Taurine is an important amino acid for neuronal and physiological development, and so, its uptake was assayed in cells and placental explants exposed to alcohol or acetaldehyde. First trimester villous explants and BeWo cells were treated with 0, 10, 20, 40 mM ethanol or 0, 10, 20, 40 µM acetaldehyde. The invasive capacity of SGHPL4, a first trimester extravillous cytotrophoblast cell line, was unaffected by ethanol or acetaldehyde (p>0.05; N = 6). The cells in-cycle were estimated using immunostaining for Ki67. Proliferating trophoblast cells treated with ethanol were decreased in both experiments (explants: 40% at 20 mM and 40 mM, p<0.05, N = 8–9) (cell line: 5% at 20 mM and 40 mM, p<0.05, N = 6). Acetaldehyde also reduced Ki67-positive cells in both experiments (explants at 40 µM p<0.05; N = 6) (cell line at 10 µM and 40 µM; p<0.05; N = 7). Only in the cell line at 20 µM acetaldehyde demonstrated increased apoptosis (p<0.05; N = 6). Alcohol inhibited taurine transport in BeWo cells at 10 mM and 40 mM (p<0.05; N = 6), and in placenta at 40 mM (p<0.05; N = 7). Acetaldehyde did not affect taurine transport in either model (P<0.05; N = 6). Interestingly, system A amino acid transport in placental explants was increased at 10 µM and 40 µM acetaldehyde exposure (p<0.05; N = 6). Our results demonstrate that exposure to both genotoxins may contribute to the pathogenesis of FASD by reducing placental growth. Alcohol also reduces the transport of taurine, which is vital for developmental neurogenesis.

Differential expression of angiotensin II type 1 and type 2 receptors at the maternal–fetal interface: potential roles in early placental development

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal-fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal-fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

MORPHOLOGICAL AND FUNCTIONAL CHANGES TO THE UTERUS OF LIZARDS WITH DIFFERENT PLACENTAL COMPLEXITIES

ABSTRACT We compared the uterine component of the placentae of each placental region of two species of lizards, Eulamprus tympanum and Pseudemoia entrecasteauxii, to identify potential routes and mechanisms of nutrient transport. We focused on the uterine epithelium because nutrients must pass though it to reach the embryo. At this point, we have only been able to study two species in detail, but each has a placenta of different complexity and quantitatively different placentotrophy. The uterine epithelium of the chorioallantoic placenta has a different morphology from the omphaloplacenta, and the chorioallantoic placenta of P. entrecasteauxii, the most placentotrophic of the two species, is further differentiated in placentomal and paraplacentomal regions. The chorioallantoic region of E. tympanum, and the paraplacentomal region of P. entrecasteauxii have morphologies indicative of gas exchange epithelia. The blood-uterine lumen distance in P. entrecasteauxii, however, is about one tenth that in E. tympanum, suggesting a greater diffusive capacity for gases in P. entrecasteauxii. The omphaloplacenta of both species has a similar structure, with hypertrophied cells, electron-dense granules in the apical cytoplasm and vesicles budding off into the uterine lumen. This result is particularly interesting because E. tympanum exhibits little net nutrient transfer to the embryo during pregnancy. Nutrient provision across the omphaloplacenta in both species occurs by histotrophy. The placentome of P. entrecasteauxii also has a morphology suggestive of nutrient provision, but by some non-histotrophic mechanism and may be very important in the evolution of substantial placentotrophy.

Transcriptomic profiling of human choriodecidua during term labor: inflammation as a key driver of labor.

Inflammation is a driver of labor in myometrium and cervix; however, the involvement of decidua is poorly defined. We have reported decidual leukocyte infiltration prior to and during labor; the regulators of these inflammatory processes are unknown.

A role for the mitochondrial-associated protein p32 in regulation of trophoblast proliferation

p32 is a conserved eukaryotic protein which is primarily expressed in the mitochondria and regulates cell proliferation, migration and metabolism in various tissues. In this study, we sought to examine the expression and function of p32 in the human placenta. p32 was highly expressed in the syncytiotrophoblast, the underlying cytotrophoblast (CTB), the vascular endothelium and by a proportion of cells in the villous stroma in first trimester and term placenta. p32 mRNA and protein expression was significantly higher in the first trimester of pregnancy than at term, and expression in the trophoblast was significantly reduced in placentas from women with fetal growth restriction (FGR). Small interfering RNA (siRNA)-mediated knockdown of p32 in term placental explants significantly reduced the number of Ki67-positive CTB, but did not alter CTB apoptosis or necrosis. p32 knockdown increased lactate production, reduced glucose extraction from culture medium and was associated with reduced MitoTracker dye accumulation in trophoblast mitochondria. p32 knockdown was also associated with a significant reduction in expression of the mitochondrial respiratory complexes I and IV. These data suggest that p32 expression is important for CTB proliferation, via a mechanism involving regulation of normal mitochondrial function. As p32 expression is reduced in FGR placentas, this may contribute to some of the observed placental pathology, such as reduced CTB proliferation and mitochondrial dysfunction.

The consequence of matrix dysfunction on lung immunity and the microbiome in COPD

The pulmonary extracellular matrix (ECM) is a complex network of proteins which primarily defines tissue architecture and regulates various biochemical and biophysical processes. It is a dynamic system comprising two main structures (the interstitial matrix and the basement membrane) which undergo continuous, yet highly regulated, remodelling. This remodelling process is essential for tissue homeostasis and uncontrolled regulation can lead to pathological states including chronic obstructive pulmonary disease (COPD). Altered expression of ECM proteins, as observed in COPD, can contribute to the degradation of alveolar walls and thickening of the small airways which can cause limitations in airflow. Modifications in ECM composition can also impact immune cell migration and retention in the lung with migrating cells becoming entrapped in the diseased airspaces. Furthermore, ECM changes affect the lung microbiome, aggravating and advancing disease progression. A dysbiosis in bacterial diversity can lead to infection, inducing epithelial injury and pro-inflammatory reactions. Here we review the changes noted in the different ECM components in COPD and discuss how an imbalance in microbial commensalism can impact disease development. A summary of the changes observed in the extracellular matrix and microbiome of the COPD lung http://ow.ly/Yj5730kuLbg

Delineating differential regulatory signatures of the human transcriptome in the choriodecidua and myometrium at term labor

Abstract Preterm deliveries remain the leading cause of neonatal morbidity and mortality. Current therapies target only myometrial contractions and are largely ineffective. As labor involves multiple coordinated events across maternal and fetal tissues, identifying fundamental regulatory pathways of normal term labor is vital to understanding successful parturition and consequently labor pathologies. We aimed to identify transcriptomic signatures of human normal term labor of two tissues: in the fetal-facing choriodecidua and the maternalmyometrium. Microarray transcriptomic data from choriodecidua and myometrium following term labor were analyzed for functional hierarchical networks, using Cytoscape 2.8.3. Hierarchically high candidates were analyzed for their regulatory casual relationships using Ingenuity Pathway Analysis. Selectedmaster regulators were then chemically inhibited and effects on downstream targets were assessed using real-time quantitative PCR (RT-qPCR). Unbiased network analysis identified upstream molecular components in choriodecidua including vimentin, TLR4, and TNFSF13B. In the myometrium, candidates included metallothionein 2 (MT2A), TLR2, and RELB. These master regulators had significant differential gene expression during labor, hierarchically high centrality in community cluster networks, interactions amongst the labor gene set, and strong causal relationships with multiple downstream effects. In vitro experiments highlighted MT2A as an effective regulator of labor-associated genes. We have identified unique potential regulators of the term labor transcriptome in uterine tissues using a robust sequence of unbiased mathematical and literature-based in silico analyses. These findings encourage further investigation into the efficacy of predicted master regulators in blocking multiple pathways of labor processes across maternal and fetal tissues, and their potential as therapeutic approaches. Summary Sentence Combined in silico and in vitro analyses have identified novel transcriptomic regulators of term labor in human choriodecidua and myometrium with further therapeutic potential.

PLD.24 Gene expression profiling of human decidua during term labour: inflammation as a key driver of labour

Background There is accumulating evidence that sterile inflammation is a driver of labour in myometrium and cervix; however the involvement of the decidua is poorly defined. Our previous studies report decidual leukocyte infiltration prior to and during labour. The current study aimed to characterise the inflammatory gene profile in the decidua during term labour. Methods Decidua was obtained from two groups (no labour and term labour); pooled RNA was applied to Human Genome U133 Plus 2.0 Affymetrix GeneChips. Bioinformatic and pathway analysis was performed using Ingenuity pathway analysis (IPA), with gene validation by real time PCR. Results Affymetrix analysis identified widespread changes in decidual gene expression following labour. The most highly altered biological functions included: haematological system function, cell-cell signalling, immune cell trafficking, cellular movement and inflammatory responses; however those involved in tissue morphology, metabolism and cell survival were also altered. PCR validation was selected based on fold change and functional significance: ICAM (p = 0.006), CXCR4 (p = 0.017), CD44 (p = 0.02), TLR 4 (p = 0.035), SOCS3 (p = 0.02), BCL2A (p = 0.018) and IDO1 (p = 0.029) were upregulated following labour. Transcription factors predicted, from the gene array, as potential upstream regulators were: NFkB, RELA, STAT1 (Signal transducer and activator of transcription 1) and MEOX2 (mesenchyme homeobox 2) Conclusions This study provides further evidence of non-infection related inflammatory pathways contributing to term labour, in particular identifying inflammatory changes in the decidua similar to those in myometrium and cervix. Ongoing studies focus on identifying potential targets to suppress decidual inflammation as a potential means of arresting labour.