Human Term Placenta Research Articles

Isolation and Purification of Villous Cytotrophoblast Cells from Term Human Placenta.

The placenta is a key element during pregnancy for the health of the fetus and the mother, which justifies why placental studies are so important. One of the best models for placental studies is the primary cell culture of cytotrophoblast cells from human term placentas. In this chapter, we will detail firstly the isolation of cytotrophoblast cells, with tissue preparation, digestion, Percoll gradient, and cell freezing, and secondly the cell immunopurification and seeding.

Human term placenta as source of stem cells for regenerative medicine

Human term placenta as source of stem cells for regenerative medicine

Invited commentary

Invited commentary

Clinical course of chronic abruption-oligohydramnios sequence: A report of four cases

Placenta-derived mesenchymal stromal cells (MSC) have often been considered to linger behind their equivalents from other tissues, such as MSC from bone marrow, in many aspects including their therapeutic potential in regenerative medicine. Nowadays however, it is clear that certain aspects make placental MSC attractive as a cellular therapy, such as their lack of ethical concerns and ease of isolation from human term placenta, a material long regarded as biological waste. Moreover, placental MSC virtually lack expression of human leukocyte antigens and co-stimulatory molecules, making them very attractive for transplantation in allogeneic settings.In the context of cancer, cell therapy remains an area of intense investigation whereby MSC have been shown to play opposing roles, and placental MSC are no exception. In this review, we will discuss dichotomy of placental MSC that underscores the challenges in understanding their therapeutic potential in oncology.

DICER1 is present in the syncytiotrophoblast of the human full-term placenta

DICER1 is present in the syncytiotrophoblast of the human full-term placenta

Two Distinct Myeloid Subsets at the Term Human Fetal-Maternal Interface.

During pregnancy, immune cells infiltrate the placenta at different stages of fetal development. NK cells and macrophages are the most predominant cell types. These immune cells play pleiotropic roles, as they control spiral artery remodeling to ensure appropriate blood supply and maintain long-term tolerance to a true allograft; yet, they must be able to mount appropriate immune defenses to pathogens that may threaten the fetus. Whether the same cell type accomplishes all these tasks or if there are dedicated subsets remains controversial. Here, we identify and characterize two distinct subsets of myeloid cells that differ in their pro-inflammatory/regulatory capacity. While one subset predominantly produces the immune-modulating cytokine IL-10, the second subset has superior capacity to secrete pro-inflammatory mediators, such as IL-1β and IL-6. The putative regulatory myeloid cells also express high levels of inhibitory receptors and their ligands, including programmed cell death 1 (PD1) ligands. Importantly, a large fraction of CD8 and CD4 cells in normal term human placenta are PD1 positive, suggesting that the PD1/PD1 ligands axis might be critical to maintain tolerance during pregnancy.

Sexually Dimorphic Impact of Chromium Accumulation on Human Placental Oxidative Stress and Apoptosis.

Environmental contamination with hexavalent chromium (CrVI) is a growing problem both in the United States and developing countries. Hexavalent chromium is widely used in numerous industries. Environmental exposure to CrVI adversely affects pregnancy outcomes and subsequent health of 2 generations, resulting in higher pregnancy loss, spontaneous abortion and low birth rate. Pregnant women exposed to CrVI through occupational settings experience increased risk of spontaneous abortion, stillbirth, preterm birth, and neonatal death. Children of the CrVI exposed women experience respiratory problems, perinatal jaundice, and increased birth defects. Because placental dysfunction may have a role in such adverse pregnancy outcome, we tested the hypothesis that environmental Cr exposure in pregnant women results in Cr accumulation in the human placenta, which could increase placental oxidative stress by disrupting antioxidant machinery and inducing apoptosis. Studies using frozen, deidentified human term placenta samples indicated that: (1) Cr accumulates in human term placenta tissues and (2) increase in Cr accumulation is positively correlated with oxidative stress and apoptotic markers, and altered antioxidants levels. Interestingly, there was a sexual dimorphism in the correlation between Cr accumulation and oxidative stress, and expression of apoptotic and antioxidant markers. Mechanistic in vitro studies using human trophoblast cells BeWo confirmed the detrimental effects of Cr in altering antioxidant genes. For the first time, this study provides evidence in support of a positive correlation between Cr accumulation in the human placenta and accelerated oxidative stress, with a gender bias toward the male sex.

Analysis of correlations between the placental expression of glucose transporters GLUT-1, GLUT-4 and GLUT-9 and selected maternal and fetal parameters in pregnancies complicated by diabetes mellitus

Purpose: The aim of the study was to analyze the correlations between the expression of glucose transporters GLUT-1, GLUT-4, and GLUT-9 in human term placenta and selected maternal and fetal parameters in pregnancies complicated by diabetes mellitus (DM). Materials and methods: Placental samples were obtained from healthy control (n = 25) and diabetic pregnancies, including diet-controlled gestational diabetes mellitus (GDMG1) (n = 16), insulin-controlled gestational diabetes mellitus (GDMG2) (n = 6), and pregestational DM (PGDM) (n = 6). Computer-assisted quantitative morphometry of stained placental sections was performed to determine the expression of selected glucose transporter proteins. For the purposes of correlation analysis, the following parameters were selected: type of diabetes, gestational age, maternal prepregnancy body mass index (BMI), gestational weight gain, third trimester glycated hemoglobin concentration, placental weight, fetal birth weight (FBW) as well as ultrasonographic indicators of fetal adiposity, including subscapular (SSFM), abdominal (AFM), and midthigh (MTFM) fat mass measurements. Results: In the PGDM group, the analysis demonstrated positive correlations between the placental expression of GLUT-1, GLUT-4, and GLUT-9 and FBW, AFM, and SSFM measurements (p < .05). Similarly in the GDMG2 patients positive correlations between GLUT-4 expression, FBW and SSFM were observed (p < .05). In the multivariate regression analysis, only the type of diabetes and FBW were significantly associated with GLUTs expression (p < .001). In addition, maternal prepregnancy BMI significantly contributed to GLUT-1 expression (p < .001). Conclusions: The study results revealed that placental expression of GLUT-1, GLUT-4, and GLUT-9 may be involved in the intensification of the fetal growth in pregnancies complicated by GDM/PGDM.

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Leptin upregulates aquaporin 9 expression in human placenta in vitro

Aquaporins are integral membrane proteins that have permeability functions in many tissues. Aquaporin 9 may transport not only water but also small molecules, such as glycerol, monocarboxylates, purines and pyrimidines. Aquaporin 9 is expressed in syncytiotrophoblast of human term placenta, and it may contribute to the embryonic/fetal growth and survival. We have previously found that Aquaporin 9 expression levels seem to be increased in placenta from gestational diabetes. Since leptin plasma levels and leptin expression are increased in placenta from gestational diabetes, we aimed to study the possible role of leptin on Aquaporin 9 expression in human placenta in vitro. The present work shows that leptin produces a dose-dependent increase of Aquaporin 9 expression, resulting in an increase in Aquaporin-9 protein in human trophoblast explants.

PP-6 Integration and validation of the ex vivo human placenta perfusion model

Background Pregnant women and their fetuses are or-phan populations with respect to knowledge on safety and efficacy of drugs. It is estimated that over 90% of pregnant women uses over-the-counter or prescription medication.1 Albeit, data on transplacental transfer or fetal effects are still lacking for most of the medicines and food supplements. The ex vivo human placenta perfusion model is an effective and non-invasive method to study transplacental passage of drugs and environmental com-pounds in humans.2 It is the only method that retains the full structure of a full term human placenta, making it possible to study transplacental passage without harm-ing the foetus or the mother.3 Due to many challenges and its high complexity it remains difficult to incorporate it routinely into laboratories. Methods A step-by-step protocol for the implementa-tion and validation of a closed-closed ex vivo perfusion model was developed. Different quality controls were implemented to ensure the integrity, viability and func-tionality of the method: (i). Antipyrine is a small drug molecule that does not bind to proteins and that passes the placental barrier by passive diffusion; It was used here to determine ‘overlap’ (solute exchange) between foetal and maternal circulation; (ii) the pressure and the flow rate in the foetal circulation as a marker for leakage; (iii) pH and glucose consumption were implemented as a marker for tissueviability. Results In total 89 placentas were collected of which 34 placentas were successfully perfused with antipyrine and fulfilled all quality control measurements. A foe-tal/maternal antipyrine concentration ratio of 0.75 was reached within 89±21 min, while 210 min were required to achieve equilibrium. The foetal pressure remained un-der 70 mmHg during the entire experiment. The end foe-tal flow was 98% of the foetal starting flow. The average glucose consumption was 0.30±0.15 µmol/min/g. Every 30 min the maternal pH declined to 7.29±0.06 and was adjusted to 7.4. The foetal pH stayed stable at 7.30±0.05. Conclusion Based on the multiple quality control mea-surements, the described method of a closed human ex-vivo placenta perfusion model was validated. The success rate (38%) was more than twice the success rate reported in literature (15%).

ID: 1054 Comparison between fresh and cryopreserved Human Amnion Mesenchymal Stem Cells (HAMCs) in terms of serial passaging, morphology and differentiation potential during long term culture

The therapeutic efficiency of Human Amnion Mesenchymal stem cells (HAMCs) is significantly promising. However, its sustainability in long term sub-cultivation has yet to be identified, especially post-cryopreservation. There are concerns whether stem cells which are cryopreserved will be able to retain its functions and differentiation potential efficiently. The aim of this study is to determine the effects of cryopreservation on the serial passaging, morphology and differentiation potential of HAMCs culture post-cryopreservation compared to fresh HAMCs culture. HAMCs was isolated through serial enzymatic digestion of the amnion membrane of human term placenta after delivery. Fresh HAMCs cultures were sub-cultivated until passage 15 while cryopreserved HAMCs samples was preserved at passage 2 of the cell culture and revived after 3 months of cryopreservation period. Through microscopic observation, the cryopreserved HAMCs started to flatten and become larger in size, losing it fibroblastic feature as early as passage 6. The enlarged and flatten morphological features of the HAMCs indicate that the cells began to lose its proliferative activity at this point. In comparison, the HAMCs normally began to enlarge when reaching passage 15 in non-cryopreserved HAMCs cultures. Fresh HAMCs cultures were able to be sustained up to 15 passages while cryopreserved HAMCs were not able to survive pass 10 passages of cell culture. Furthermore, it was observed that both fresh and cryopreserved HAMCs were able to retain its differentiation potential through osteogenesis and adipogenesis medium induction. This was experimentally visualized through positive staining of the calcium composite and lipid droplet in the induced HAMCs culture using Von Kossa stain solution and Oil Red-O stain solution, respectively. Despite retaining it differentiation potential, cryopreserved HAMCs were only able to survive a few passages after being revived. The result suggests that fresh HAMCs is a more suitable candidate to be used in cellular therapy and various clinical application as it were able to retain all its function in long term culture.

Salt (Sodium Chloride) regulates lipopolysaccharide induced IL-1 family cytokine production in the term human placenta and choriodecidua

Salt (Sodium Chloride) regulates lipopolysaccharide induced IL-1 family cytokine production in the term human placenta and choriodecidua

Quantification of S-adenosylmethionine and S-adenosylhomocysteine in human placenta and placental explants under homocysteine treatment

S-Adenosylmethionine (SAM) and S-adenosylhomocysteine(SAH) are essential compounds in methionine cycle. SAM is a universal donor in majority of methyltransferase reactions while SAH is a universal product and a potent inhibitor of methyltransferase reactions. The reactions of methionine cycle and connected with them folate-related reactions and transsulfuration pathway are implicated in various complications of pregnancy. Their role in placental metabolism and responsiveness to hyperhomocysteinemia is scarcely investigated though hyperhomocysteinemia is a risk factor in several obstetric pathologies. In this paper, a simple and fast method based on LC/MS technology is applied for simultaneous SAM and SAH quantification in human placenta and placental explants. The developed method proved to be highly sensitive, inter-day repeatable and intra-day reproducible. The content of SAM and SAH in term human placenta is in the range 8.33–18.73 and 5.20–8.74nmol/g wet tissue, respectively. SAM concentration doubles while SAH decreases under treatment of cultivated placental explants with clinically relevant 20μM homocysteine in comparison with placental tissue with ultimate impact on velocity of numerous methyltransferase reactions.

Influence of advanced maternal age on histology of full term human placenta in Basrah

Advanced maternal age defines as age over 35 years at estimated date of delivery.The aims of this research were to determine the impact of maternal age on placental barrier thickness, fetal and placental weight .The study was conducted one total of 25 human full term placentas of multiparous healthy pregnant women obtained from department of Obstetrics and Gynecology unit in Bin Gazwan hospital in Basrah .The examined women were divided into 2 groups ;the control group of consisted of 10 placentas from pregnant women between age of( 20 -34) years and experimental group consisted of 15 placenta from pregnant women of 35years old and older .Stereological and histological study were applied to determine the effect of advanced maternal age on placental barrier and fetal and placental weight . The results showed:1. Increase in the mean of placental barrier thickness of the placenta of mother &gt;35 years than that of placenta of control group (20-34) .2. Increase in the main placental weight of mothers &gt;35 years in comparison to that of the control . .3. Decrease in mean birth weight of mothers &gt; 35 years in comparison to that of control.4. Extensive morphological changes in the structural component of terminal villi (capillaries, stroma and trophoblast). The results showed that there is an increase in the thickness of placental barrier of placenta, decrease in vascularization of the terminal villi and increase in stoma and fibrin deposition in the placenta of mothers more than 35 years of age in comparison to the control group .

Micro- and Nano-vesicles from First Trimester Human Placentae Carry Flt-1 and Levels Are Increased in Severe Preeclampsia.

Preeclampsia is a life-threatening hypertensive disease affecting 3-5% of pregnancies. While the pathogenesis of preeclampsia remains unclear, it is known that placenta-derived factors trigger the disease by activating maternal endothelial cells prior to the onset of clinical symptoms. Extracellular vesicles (EVs) of different sizes extruded by the placenta may be one factor. The truncated/secreted form of Flt-1 (sFlt-1) has also been implicated in the pathogenesis of preeclampsia. We investigated whether placental EV production is altered in preeclampsia such that they induce endothelial cell activation, and whether (s)Flt-1 is involved. Macro-, micro-, and nano-vesicles were collected from normal and preeclamptic (PE) placental explants, and separated by differential centrifugation. The number and size of micro- and nano-vesicles was measured by nanoparticle tracking analysis and their ability to activate endothelial cells was quantified by endothelial cell intercellular adhesion molecule 1 expression and monocyte adhesion. The levels of Flt-1 were measured by western blots and ELISA. PE placentae extruded significantly more micro- and nano-vesicles than control placentae and the extruded micro-vesicles were larger than those from control placentae. Micro- and nano-vesicles from both first trimester and term human placentae carried Flt-1 and levels were significantly increased in EVs from severe, but not mild, PE compared to normotensive placentae. All fractions of EVs from PE placentae activated endothelial cells, and for micro- and nano-vesicles, activation was reduced in the presence of exogenous vascular endothelial growth factor (VEGF), a Flt-1 neutralizing antibody, or by pre-treatment with VEGF. While EV-bound VEGF constituted over 20% of the total detected VEGF secreted by PE and normotensive placentae, EV-bound Flt-1 did not significantly contribute to the total level of sFlt-1/Flt-1 released by human third trimester placentae. Micro- and nano-vesicles extruded by human placentae carry Flt-1 across gestation and in severe preeclampsia, the levels of vesicle-bound Flt-1 are upregulated. All fractions of PE placental EVs activated endothelial cells and for micro- and nano-vesicles, this was in part due to the ability of EV-bound Flt-1 to sequester VEGF. That placental EVs can activate endothelial cells supports the contention that EVs are one placental toxin contributing to the pathogenesis of preeclampsia.

Whole-transcriptome analysis delineates the human placenta gene network and its associations with fetal growth

BackgroundThe placenta is the principal organ regulating intrauterine growth and development, performing critical functions on behalf of the developing fetus. The delineation of functional networks and pathways driving placental processes has the potential to provide key insight into intrauterine perturbations that result in adverse birth as well as later life health outcomes.ResultsWe generated the transcriptome-wide profile of 200 term human placenta using the Illumina HiSeq 2500 platform and characterized the functional placental gene network using weighted gene coexpression network analysis (WGCNA). We identified 17 placental coexpression network modules that were dominated by functional processes including growth, organ development, gas exchange and immune response. Five network modules, enriched for processes including cellular respiration, amino acid transport, hormone signaling, histone modifications and gene expression, were associated with birth weight; hub genes of all five modules (CREB3, DDX3X, DNAJC14, GRHL1 and C21orf91) were significantly associated with fetal growth restriction, and one hub gene (CREB3) was additionally associated with fetal overgrowth.ConclusionsIn this largest RNA-Seq based transcriptome-wide profiling study of human term placenta conducted to date, we delineated a placental gene network with functional relevance to fetal growth using a network-based approach with superior scale reduction capacity. Our study findings not only implicate potential molecular mechanisms underlying fetal growth but also provide a reference placenta gene network to inform future studies investigating placental dysfunction as a route to future disease endpoints.

Adult venous endothelium is a niche for highly proliferative and vasculogenic endothelial colony-forming cells

ObjectivePostnatal resident endothelium of blood vessels has been proposed to represent terminally differentiated tissue that does not replicate. We previously isolated endothelial colony-forming cells (ECFCs) from human umbilical cord blood (CB) and term placenta by using colony-forming assays and immunocytochemistry. We showed that ECFCs are highly proliferative and form functioning vessels in vivo, the defining characteristics of a true endothelial progenitor cell. This exploratory investigation was conducted to determine whether the endothelium of healthy adult blood vessels contained resident ECFCs. MethodsThe endothelium of great saphenous vein (GSV) obtained from vein stripping procedures was collected with mechanical scraping, and ECFCs were isolated according to established protocols. ResultsGSV ECFCs incorporated acetylated low-density lipoprotein, formed tubules in Matrigel (BD Biosciences, San Jose, Calif) at 24 hours, and expressed endothelial antigens cluster of differentiation (CD) 144, CD31, CD105, and kinase insert domain receptor but not hematopoietic antigen CD45. Using cumulative population doublings and single-cell assays, we demonstrated that GSV ECFCs exhibited comparable proliferative capacities compared with CB ECFCs, including similar numbers of highly proliferative cells. When injected in collagen/fibronectin gels implanted in nonobese diabetic/severe combined immune deficiency mice, GSV ECFCs formed blood vessels with circulating murine red blood cells, demonstrating their vasculogenic potential. ConclusionsThe ECFCs of the GSV contain a hierarchy of progenitor cells with a comparable number of highly proliferative clones as ECFCs of CB. The results of this investigation demonstrate that the adult endothelium contains resident progenitor cells that may have a critical role in vascular homeostasis and repair and could potentially be used as a source of autologous cells for cell therapies focusing on vasculogenesis.

Isolation and characterization of trophoblasts from enzymatic explants of human term placenta.

In the present study, we describe a new method of isolation and culture of human villous and extravillous trophoblasts from term placenta. The cultivation of trypsinized placental villous tissue explants, followed by the isolation of cells from outgrowth islets allows for obtaining a cytotrophoblast subpopulation that is free from contamination by other cell types. Compared to other methods, our protocol is mild, simple and effective, does not request costly reagents and provides isolation of the mononuclear cytotrophoblast cell populations free from contamination by other types of placental cells. The isolated cells proliferated and formed a pleomorphic monolayer, where cells fused into a small number of binuclear or polynuclear syncytiotrophoblasts. Isolated cytotrophoblast cells expressed the specific epithelial intermediate filament cytokeratin 7 (CK7), the epithelium-specific cell-cell adhesion molecule E-cadherin and were CD9-, CD45- and vimentin-negative. Cyto- and syncytiotrophoblasts obtained by this method can be used as a model or tool for the fundamental research of differentiation and function of human placental cells, and can provide a new understanding of drug distribution in placenta. Their combination with other in vitro cell models can be useful for studying a variety of other aspects concerning placental functions, which will provide new knowledge for understanding immunology, endocrinology and development of placenta.