Potential clinical applications of placental stem cells for use in fetal therapy of birth defects

Placental stem cells are of growing interest for a variety of clinical applications due to their multipotency and ready availability from otherwise frequently discarded biomaterial. Stem cells derived from the placenta have been investigated in a number of disease processes, including wound healing, ischemic heart disease, autoimmune disorders, and chronic lung or liver injury. Fetal intervention for structural congenital defects, such as spina bifida, has rapidly progressed as a field due to advances in maternal-fetal medicine and improving surgical techniques. In utero treatment of structural, as well as non-structural, congenital disorders with cell-based therapies is of particular interest given the immunologic immaturity and immunotolerant environment of the developing fetus. A comprehensive literature review was performed to assess the potential utilization of placenta-derived stem cells for in utero treatment of congenital disorders. Most studies are still in the preclinical phase, utilizing animal models of common congenital disorders. Future research endeavors may include autologous transplantation, gene transfers, induced pluripotent stem cells, or cell-free therapies derived from the stem cell secretome. Though much work still needs to be done, placental stem cells are a promising therapeutic agent for fetal intervention for congenital disease.     

The granulocyte colony-stimulating factor (G-CSF) upregulates metalloproteinase-2 and VEGF through PI3K/Akt and Erk1/2 activation in human trophoblast Swan 71 cells

IntroductionAlthough the expression of the granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in placental tissues suggests that the cytokine could play a role in placental development, the relevance of G-CSF:G-CSFR interaction in trophoblast cells remains to be studied. Thus, the possible functional role of G-CSF was examined in a human trophoblast cell line (Swan 71 cells).Methods and resultsThe expression of G-CSFR was detected by immunocytochemistry and Western blot assays. G-CSF treatment exerted neither a proliferative nor a protective effect on H₂O₂-mediated cell death in trophoblast cells. Gelatin zymography of supernatants collected from G-CSF-treated cells showed an increment of metalloproteinase-2 (MMP-2) activity. We also found higher MMP-2 and VEGF expression levels in conditioned medium from cells exposed to G-CSF. In addition, it was demonstrated that G-CSF induced the activation of PI3K/Akt and Erk1/2 pathways, which in turn activated NF-kB. By using selective pharmacological inhibitors, it was showed that these pathways are mediating the biological effects produced by G-CSF in Swan 71 cells.Discussion and conclusionWe have demonstrated for the first time that G-CSF increases MMP-2 activity and VEGF secretion in Swan 71 cells through activation of PI3K/Akt and Erk signaling pathways, both contributing to the translocation of NF-kB to the nucleus. These data suggest that G-CSF is involved in the regulation of trophoblast function, and should be considered as a locally produced cytokine probably contributing to embryo implantation and the development of a functional placenta.     

Pentoxifylline inhibits lipopolysaccharide-induced inflammatory mediators in human second trimester placenta explants

BackgroundIntrauterine infection and inflammation during pregnancy, which leads to up-regulation of inflammatory cytokines and prostaglandin synthesis, has been implicated in the pathogenesis of preterm delivery and other pregnancy complications. Effective preventive and therapeutic strategies to reduce these outcomes are lacking to date. Pentoxifylline (PTX) is a non-specific phosphodiesterase inhibitor which raises intracellular cyclic adenosine monophosphate and decreases production of pro-inflammatory mediators while enhancing anti-inflammatory cytokines. We hypothesized that pentoxifylline will decrease lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production in human placental explants.MethodsPlacental explants derived from normal second trimester human placentas were treated with PTX, stimulated with LPS and cultured at 37 °C in 5% CO₂. Conditioned media were assayed for pro- and anti-inflammatory mediators with multiplex immunoassays or ELISA, and explant tissues for mRNA with real time PCR. Means of PTX-treated and untreated samples were compared using paired t tests and Wilcoxon-signed rank tests.ResultsPTX preferentially inhibited placental expression and production of LPS-induced pro-inflammatory cytokines including TNF-α (25461 vs. 1908 pg/ml, p < 0.001), IL-1β (2921 vs. 1067 pg/ml, p < 0.001) and IFN-γ (2190 vs 427 pg/ml, p < 0.001) with relative preservation of anti-inflammatory mediators. The suppressive effects on LPS-induced placental inflammation were independent of the timing of PTX administration in relation to LPS-induced stimulation.ConclusionOur study suggests that PTX attenuates the LPS-induced pro-inflammatory milieu in human placental explants. We speculate that PTX may have utility as a candidate anti-inflammatory agent for prophylaxis and/or treatment of human placental inflammation.     

Porphyromonas gingivalis induces IL-8 and IFN-gamma secretion and apoptosis in human extravillous trophoblast derived HTR8/SVneo cells via activation of ERK1/2 and p38 signaling pathways

IntroductionPreterm birth is a major cause for infant mortality and morbidity. A large number of studies have suggested a link between periodontal disease and preterm birth. The purpose of this study was to investigate the interaction between a periodontopathic bacterium Porphyromonas gingivalis and human extravillous trophoblast derived HTR8/SVneo cells.MethodsProduction of cytokines in HTR8 cells was measured via ELISA. Annexin V/PI flow cytometry was performed to assess apoptosis. Protein expression was measured by western blot. Specific pharmacological inhibitors were used to inactivate relevant signaling pathways (p38 MAPK, SB203580; ERK1/2, U0126; JNK, SP600125; NF-κB, JSH-23) to determine their roles in inflammation and apoptosis.ResultsHTR8 cells released significant amounts of IL-8 and IFN-γ during exposure to P. gingivalis. Meanwhile, the percentages of both early and late apoptotic cells increased significantly in response to P. gingivalis. The most significant effect on inflammation was found using SB203580 and U0126, followed by SP600125 and JSH-23. Moreover, U0126 and SB203580 both partially but significantly suppressed P. gingivalis-induced apoptosis, with a large effect by U0126. Additionally, both heat-killed P. gingivalis and P. gingivalis lipopolysaccharide significantly induced IL-8 production.ConclusionP. gingivalis induces inflammation and apoptosis in HTR8 cells, and we demonstrated for the first time that activation of ERK1/2 and p38 MAPK pathways participates in P. gingivalis-induced inflammation and apoptosis. The abnormal regulation of inflammation and apoptosis in human trophoblasts by P. gingivalis infection may give new insights into how maternal periodontal disease and periodontal pathogens might be linked to preterm birth.     

Transendothelial migration of human umbilical mesenchymal stem cells across uterine endothelial monolayers: Junctional dynamics and putative mechanisms

IntroductionDuring pregnancy, fetal stem cells can transfer to the maternal circulation and participate in tissue repair. How they transmigrate across maternal endothelial barriers and whether they can subsequently influence maternal endothelial integrity is not known.MethodsMesenchymal stem cells (WJ-MSC) were isolated from Wharton's jelly and their interactions with human uterine microvascular endothelial cell (HUtMEC) monolayers, junctional occupancy and expression/phosphorylation of vascular endothelial (VE)- cadherin and vascular endothelial growth factor (VEGF-A) secretion was studied over 48h by real time, confocal microscopy, immunoblotting and ELISA.ResultsWJ-MSC displayed exploratory behaviour with interrogation of paracellular openings and spreading into the resultant increased gaps followed by closing of the endothelium over the WJ-MSC. 62% of added cells crossed within 22h to sub-endothelial niches. There was a concomitant loss of junctional VE-cadherin in HUtMEC followed by a full return and increased VE-cadherin expression after 22h. During early hours, VE-cadherin showed a transient phosphorylation at Tyrosine (Tyr)-685 when VEGF-A secretion were high. From 16 to 22h, there was increased de-phosphorylation of Tyr-731. Anti-VEGF-A blocked Tyr-685 phosphorylation but not the decrease in P-Tyr731; this partially inhibited WJ-MSC transmigration.DiscussionFetal WJ-MSC can traverse uterine endothelial monolayers by mediating a non-destructive paracellular pathway. They can promote junctional stability of uterine endothelium from the sub-endothelial niche. Mechanistically, WJ-MSC induces VEGF-dependent phosphorylation events linked with paracellular permeability and VEGF-independent de-phosphorylation events associated with leukocyte extravasation. Our data also allows consideration of a possible role of fetal MSC in mature functioning of the uterine vasculature needed for optimal utero-placental perfusion.     

Vesicular uptake of macromolecules by human placental amniotic epithelial cells

IntroductionStudies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy.MethodsPlacental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%–50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1–30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris^® software.ResultsVesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions.ConclusionsIn human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.     

YAP mediates human decidualization of the uterine endometrial stromal cells

IntroductionThe decidualization of uterine endometrial stromal cells (ESCs) is critical for the successful establishment and maintenance of pregnancy and involves extensive cell proliferation and differentiation. A newly established signaling pathway, the Hippo/Yes-associated protein (YAP) pathway, plays a critical role in these proliferation processes. Our previous study demonstrated that YAP is expressed in human ESCs. However, its role in decidualization remains unclear. The objective of the present study was to explore the role of YAP in the decidualization of human ESCs.MethodsThe expression of YAP was first investigated in the endometrium of non-pregnant women and in the decidua of pregnant women. The role of YAP was investigated by transfecting ESCs with mRNA silencing constructs and observing the negative effects of this action upon decidualization induced in vitro.ResultsOur results revealed that the expression of YAP was higher in decidual cells from early pregnant decidua compared with ESCs from non-pregnant endometrium. The expression levels of YAP and TEA domain 1 (TEAD1) were both increased in ESCs during in vitro decidualization and the knockdown of YAP in ESCs caused negative effects upon decidualization in vitro.DiscussionOur study suggests that YAP is upregulated in human decidual cells compared with ESCs and influences the decidualization of ESCs.     

Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation.     

Ly49 knockdown in mice results in aberrant uterine crypt formation and impaired blastocyst implantation

Genetic knockdown (KD) of the mouse Ly49 receptor family is reported to result in infertility despite the presence of zona-enclosed blastocysts in the uterus. Ly49 receptors regulate leukocyte functions particularly Natural Killer (NK) cell functions and are analogous to human killer immunoglobulin-like receptors (KIRs). Histological analyses of gd3.5–4.5 B6.Ly49^KD uteri identified hatched but retarded blastocysts with pyknotic nuclei, aberrant endometrial crypt formation and impaired uterine lumen closure accompanied by a lack of primary decidualization These data support peri-implantation roles for leukocytes expressing the Ly49 receptor repertoire and may give insight into KIR-based regulation of human infertility.     

Endometrial androgen signaling and decidualization regulate trophoblast expansion and invasion in co-culture: A time-lapse study

IntroductionTo elucidate whether trophoblast expansion and invasion are modulated by androgen signaling in an in vitro co-culture model system with decidualizing endometrial stromal cells (ESCs).MethodsWe employed an in vitro co-culture model of early embryo implantation, consisting of human ESCs (EtsT499 cells) and spheroids generated by extravillous trophoblast (EVT) derived HTR8/Svneo. The ESCs were decidualized with 8-bromo-cAMP (8-br-cAMP) in the presence or absence of dihydrotestosterone (DHT) at various concentrations for 5 days before co-culture with EVT spheroids. Trophoblast expansion was monitored by fluorescent time-lapse imaging microscopy. ESCs motility was visualized by using CellTracker™ Orange CMRA fluorescent probe. Apoptosis of ESCs was detected by CellEvent™ Caspase-3/7^® green detection reagent. Invasion assays were performed to quantify EVT invasion through a chemotaxis cell membrane.ResultsExpansion of EVT spheroids was significantly enhanced by decidualized compared to undifferentiated ESCs. This process was further stimulated if ESCs were first decidualized in the presence of DHT. In contrast to decidualized ESCs, undifferentiated cells actively migrated away from expanding EVT spheroids. Invasiveness of EVT toward decidualized ESCs was significantly attenuated in comparison to undifferentiated ESCs. DHT had no effect on EVT invasion. However, an inhibitor of intercellular gap junction communication significantly enhanced EVT invasion towards decidualized ESCs.ConclusionsThese results indicate distinct roles for androgen signaling and gap junction formation in decidual cells in regulating trophoblast expansion and invasion.     

Maternal obesity induced by a ‘cafeteria’ diet in the rat does not increase inflammation in maternal,placental or fetal tissues in late gestation

IntroductionObesity during pregnancy can cause serious complications for maternal and infant health. While this has often been attributed to increased inflammation during obese pregnancy, human and animal studies exhibit variable results with respect to the inflammatory status of the mother, placenta and fetus. Cafeteria (CAF) feeding induces more inflammation than standard high-fat feeding in non-pregnant animal models. This study investigated whether maternal obesity induced by a CAF diet increases maternal, fetal or placental inflammation.MethodsMaternal obesity was established in rats by 8 weeks of pre-pregnancy CAF feeding. Maternal plasma inflammatory markers (IL-1β, IL-6, IL-10, IL-12p40, MCP1, GRO/KC, MIP-2 and TNFα) and expression of inflammatory genes (Tnfα, Il-6, Il-1β, Tlr2, Tlr4, Cox2 and Emr1) in maternal, placental and fetal tissues were measured at day 21 of gestation.ResultsDespite CAF animals having 63% more central body fat than controls at day 21 of gestation, plasma inflammatory markers were not increased; indeed, levels of IL-6, IL-12p40 and MIP2 were reduced slightly. Similarly, inflammatory gene expression remained largely unaffected by CAF feeding, except for slight reductions to Tlr4 and Emr1 expression in CAF maternal adipose tissue, and reduced Tlr4 expression in male labyrinth zone (LZ). The junctional zone (JZ) displayed increased Il-6 expression in CAF animals when fetal sexes were combined, but no inflammatory genes were affected by the CAF diet in fetal liver.ConclusionsMaternal obesity induced by a CAF diet before and during pregnancy does not increase the inflammatory status of the mother, placenta or fetus in late gestation.     

Dynamic maternal and fetal Notch activity and expression in placentation

IntroductionMurine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands.MethodsNotch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta.ResultsNotch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells.DiscussionCanonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.     

The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization

In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84–86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H₂-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter.     

Mouse prolyl oligopeptidase plays a role in trophoblast stem cell differentiation into trophoblast giant cell and spongiotrophoblast

IntroductionProlyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X- peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation.MethodsWe cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell.ResultsDuring TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the protein was found mainly in the cytoplasm. The addition of 30 μM and 10 μM SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 μM SUAM-14746 impaired the differentiation into SpTs, and 1 μM SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors.ConclusionThe dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta.