Placental Amnion Research Articles

Efficacy of Amniotic and Chorionic Membrane in Facial Wound Healing: A Comparative Study.

Background Advancements in regenerative techniques have been utilized in placental amnion and chorion for a variety of purposes. Their ability to regenerate tissues has led to their usage in tissue engineering, wound healing, and other therapeutic applications. This study aims to evaluate and compare the efficacy of amnion and chorion in facial tissue wound healing. Methodology The study was an observational comparative study conducted in the Department of Oral and Maxillofacial Surgery, involving 20 participants divided into two groups (Group I and Group II).Study groups were selected according to the inclusion and exclusion criteria. A dehydrated human amnion/ chorion membrane was applied to the affected site of each group respectively. Its efficacy in wound healing was analyzed in the first, third, seventhday, and secondweek. Statistical analysis was done using SPSS software(IBM Corp., Armonk, NY). Results Patients treated with amnion membrane showed a decrease in wound size and the wound was completely healed by secondweek with mean scores of wound sizes of 0.00whereas the wound remained unhealed by secondweek with mean of 1.70to those treated with chorion membrane. Conclusion Amnion showed superior efficacy in wound healing at two-week intervals when compared to the chorion. Hence, this could be used in regenerative medicine as a graft to induce healing in facial wounds.

УЛЬТРАСТРУКТУРНАЯ ОРГАНИЗАЦИЯ АМНИОТИЧЕСКОГО ЭПИТЕЛИЯ БЕЛЫХ КРЫС

With the amniotic membrane, many authors, not without reason, associate the development of such forms of obstetric pathology as hydramnios, oligohydramnios, and late toxicosis of pregnancy. In this regard, interest in the study of the amnion-amniotic fluid system does not weaken. It is known that the amnion is involved in the regeneration of the skin, the cornea of the eye, it is used in surgery. In many clinical cases, the use of the amniotic membrane is an alternative to already known methods of treatment. At the same time, ultrastructural changes in the amniotic epithelium during pregnancy remain insufficiently studied. The aim of this work was to elucidate the dynamics of the restructuring of the ultrastructure of the amniotic epithelium of albino rats during pregnancy. The work was performed on 33 white rats weighing 200.0–250.0 g. Ultrastructural examination of the amniotic membrane was carried out from the 13th to the 22nd day of pregnancy of the animals. We studied the extraplacental amnion, as well as the amnion covering the placental disc. The material was fixed with 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH = 7.2) followed by additional fixation with 1% osmium tetroxide solution. The material was poured into a mixture of epon-araddite. Ultrathin sections were obtained on an Ultracut ultratome, contrasted with 2% uranyl acetate and lead citrate. The preparations were studied using an EVM-100B microscope. Our electron microscopy studies made it possible to give clear criteria for differentiating the extraplacental and placental amnion of albino rats. The dynamics of ultrastructural reorganization of the amniotic epithelium of white rats during pregnancy and, in connection with this, the formation of the processes of secretion and resorption of amniotic fluid are shown.

From insulin injections to islet transplantation: An overview of the journey.

When, in 1869, Paul Langerhans detected the "islands of tissue" in the pancreas, he took the first step on a journey towards islet transplantation as a treatment for type 1 diabetes. The route has embraced developments across biosciences, surgery, gene therapy and clinical research. This review highlights major milestones along that journey involving whole pancreas transplantation, islet transplantation, the creation of surrogate insulin-secreting cells and novel islet-like structures using genetic and bio-engineering technologies. To obviate the paucity of human tissue, pluripotent stem cells and non-β-cells within the pancreas have been modified to create physiologically responsive insulin-secreting cells. Before implantation, these can be co-cultured with endothelial cells to promote vascularisation and with immune defence cells such as placental amnion cells to reduce immune rejection. Scaffolds to contain grafts and facilitate surgical placement provide further opportunities to achieve physiological insulin delivery. Alternatively, xenotransplants such as porcine islets might be reconsidered as opportunities exist to circumvent safety concerns and immune rejection. Thus, despite a long and arduous journey, the prospects for increased use of tissue transplantation to provide physiological insulin replacement are drawing ever closer.

Histological aspects of remodeling of the placental complex in pregnancy complicated by late preeclampsia with different options for its correction

Annotation. The study of the problem of preeclampsia is still relevant because it is one of the most common complications of pregnancy and is one of the causes of maternal mortality, ranking second after obstetric bleeding. Perinatal mortality rates vary widely and depend on the severity and duration of late preeclampsia. The aim of the study was to investigate the histological aspects of placental complex remodeling in patients whose pregnancy was complicated by late preeclampsia on the background of metabolic syndrome. Women, pregnancy complicated by late gestosis on the background of metabolic syndrome, were divided into two groups: patients of group I received standard protocol therapy group (31 women), women of group II received preventive, modified, pathogenetically justified therapy – protocol treatment + vitamin D in dose of 2000 IU per day, L-arginine, calcium, magnesium sulfate and salicylates 1 sachet for 10 days (38 patients), group III consisted of 40 women whose physiological pregnancy was without complications and extragenital pathology. The obtained histological specimens were examined using a Nikon Eclipse Ci-E light microscope. The algorithm of histological examination was regulated by the generally accepted protocol of pathological examination of manure. In the first study group there were histological changes, which should be regarded as morphological markers of functional disorders in the system “mother-placenta-foetus”, accompanying late preeclampsia. Metaplasia of the cylindrical epithelium of the foetal surface into a multi-layered flat one was observed in the placental amnion, and in some places increased proliferative activity of epitheliocytes was observed, which formed plaque-like structures and growths. In the second study group, which received a modified treatment, the tissue structure of the manure was also characterized by deviations from the histological picture of the control group. However, the intensity and prevalence of pathological changes were significantly lower than in the first study group. In most of the studied histological sections, the cell structure of the amniotic epithelium was preserved, desquamation was not observed. Circulatory disorders in the villous chorion were present, but the structure of most vessels remained unchanged. Based on the results of histological examination, it can be concluded that a comprehensive, modified therapy of late preeclampsia on the background of metabolic syndrome, allows you to normalize the structure of the placenta.

Evaluating the efficacy of human placental amnion membrane in combination with modified coronally advanced flap technique in the management of gingival recession: An interventional study

Aims and objectives: A common problem experienced by adults is the recession of the gingiva resulting in unpleasant appearance of their teeth. There are various techniques to achieve the gingival recession coverage (GRC). But, only few techniques such as connective tissue grafts, coronally advanced flap technique (CAF) have been proved effective in achieving 100% recession coverage. The present study aims at evaluating the efficacy of amnion membrane along with CAF in the management of GRC. Materials and methods: A total number of 10 patients with class I or class II gingival recession were included in the study. The clinical parameters such as Probing depth (PD), clinical attachment level (CAL), thickness of attached gingiva (TAG), width of keratinized gingiva (WKG), height of gingival recession (HGR), percentage of recession coverage (PRC) and recession coverage esthetic score (RCES) were measured at baseline, 3 months and 6 months post-operatively. The patients were treated using amnion membrane in combination with modified coronally advanced flap technique (MCAF). Results: The patients showed statistically significant improvement in all the clinical parameters at the 3 months and 6 months follow-up visit. 97.8% CRC was observed by the end of 6 months. Moreover, the RCES showed remarkable esthetic outcome at the post-operative 6 months visit. Conclusion: Modified coronally advanced flap technique produces excellent clinical outcomes in terms of GRC. The use of amnion membrane aids in faster healing and improves the TAG which is an important aspect for the long term stability of the results obtained. Keywords Amnion membrane, Coronally advanced flap, Gingival recession, Guided tissue regeneration, Recession coverage

Effects of Preservation Methods in the Composition of the Placental and Reflected Regions of the Human Amniotic Membrane

The human amniotic membrane (AM) is emerging as an interesting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors produced by local cells and stored in the stromal matrix. However, the search for a preservation method capable of preserving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical regions of the human AM (reflected and placental amnion) after different preservation methods. For this purpose, human placentas were harvested and processed for AM isolation and storage at 2 different conditions: room temperature for 18 h in DMEM (fresh AM) and −80°C in DMEM/glycerol solution for 30 days (cryopreserved AM). After the storage period, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular viability analysis was performed using the MTT assay, and the soluble proteins were quantified with the Qubit Protein Assay Kit. Both preservation protocols reduced the cell viability, mainly in the placental amnion region of the AM, but preserved the morphology of epithelial and stromal layers, as well as the organization and distribution of collagen fibers. There was a reduction in soluble proteins only in fresh AM. Importantly, the cryopreserved AM group presented the same concentration as the control group. In conclusion, the cryopreservation using DMEM/glycerol was ideal for preserving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.

145 To determine if Vitamin D prophylaxis decreases the incidence of maternal and neonatal infection

To determine if Vitamin D prophylaxis decreases the incidence of maternal and neonatal infection. A secondary analysis assessing maternal and neonatal infection was performed on a RCT in which pregnant women received Vitamin D3 3000 IU daily or no supplement. Women with a confirmed intrauterine pregnancy 12-16 weeks without a history of Vitamin D use or malabsorption disorder were included. Maternal 25-hydroxyvitamin D (25(OH)D) levels were assessed at the time of enrollment and admission for delivery. Neonatal 25(OH)D level was assessed via cord blood. Outcomes collected included clinical chorioamnionitis (temperature > 38.0°C + maternal leukocytosis, purulent cervical discharge, or fetal tachycardia), endometritis (temperature > 38.0°C + uterine tenderness on exam), neonatal infection (as documented by NICU staff), neonatal sepsis (+ blood culture) and histologic chorioamnionitis (polymorphonucleocytes in the placental amnion and chorion). Parametric and nonparametric statistics were used to compare groups. A P value of <0.05 was statistically significant. Of the 412 women enrolled, 392 (95.1%) had completed pregnancies for analysis. Demographic characteristics, including baseline 25(OH)D levels, were similar between groups. Table 1. Compared to women who did not receive Vitamin D prophylaxis, those who did had a significantly higher 25(OH)D delivery (29.18 +/- 11.87 ng/mL vs. 23.79 +/- 9.29 ng/mL; P<0.001) and cord blood levels (33.73 +/- 13.68 ng/mL vs. 26.06 +/- 9.72 ng/mL; P<0.001). Vitamin D prophylaxis did not decrease the incidence of clinical chorioamnionitis, endometritis, neonatal infection, and neonatal sepsis. Table 2. Stratification by Vitamin D deficiency status revealed a higher incidence of clinical chorioamnionitis in those who did not receive intervention and remained Vitamin D deficient at delivery (9.7% vs. 1.6%; RR 5.9 95% CI 1.3-21.7; P=0.010). Vitamin D prophylaxis does not decrease the incidence of maternal or neonatal infection. Persistent Vitamin D deficiency in pregnancy may be associated with a risk for clinical chorioamnionitis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Sub-Regional Differences of the Human Amniotic Membrane and Their Potential Impact on Tissue Regeneration Application.

For more than 100 years, the human amniotic membrane (hAM) has been used in multiple tissue regeneration applications. The hAM consists of cells with stem cell characteristics and a rich layer of extracellular matrix. Undoubtedly, the hAM with viable cells has remarkable properties such as the differentiation potential into all three germ layers, immuno-modulatory, and anti-fibrotic properties. At first sight, the hAM seems to be one structural entity. However, by integrating its anatomical location, the hAM can be divided into placental, reflected, and umbilical amniotic membrane. Recent studies show that cells of these amniotic sub-regions differ considerably in their properties such as morphology, structure, and content/release of certain bioactive factors. The aim of this review is to summarize these findings and discuss the relevance of these different properties for tissue regeneration. In summary, reflected amnion seems to be more immuno-modulatory and could have a higher reprogramming efficiency, whereas placental amnion seems to be pro-inflammatory, pro-angiogenic, with higher proliferation and differentiation capacity (e.g., chondrogenic and osteogenic), and could be more suitable for certain graft constructions. Therefore, we suggest that the respective hAM sub-region should be selected in consideration of its desired outcome. This will help to optimize and fine-tune the clinical application of the hAM.

Expression of glucose transporters in the human amnion derived mesenchymal stromal cells under normoglycemic and hyperglycemic conditions

Glucose is vital for the proliferation and differentiation of mesenchymal stromal cells. The presence and function of Glucose Transporter (GLUT) proteins in human placental amnion derived mesenchymal stromal cells (hAMSCs) is unknown. We aimed to investigate, the presence of GLUT1, GLUT3, GLUT4 proteins and mRNAs in hAMSCs and their level of expression in normal and hyperglycemic conditions. hAMSCs isolated from amniotic membranes were characterized by flow cytometry. The expression of GLUT1, GLUT3 and GLUT4 proteins was detected in hAMSCs by immunofluorescence. Characterized cells were cultured in normoglycemic and hyperglycemic conditions for 24 and 48 h and the presence of GLUT1, GLUT3 and GLUT4 proteins and mRNAs were identified by Western blot and quantitative real-time PCR, respectively. Isolated cells were positive with MSC markers and negative with hematopoietic markers. Normoglycemic and hyperglycemic culture of hAMSCs did not change the expression of GLUT1, GLUT3 and GLUT4 protein expression in a time and dose-dependent manner. We showed that GLUT1, GLUT3, GLUT4 proteins and mRNAs were expressed in hAMSCs. Proliferation and differentiation of hAMSCs in vitro are still not optimized. The clarification of this relationship is necessary for clinical applications of stem cells and for optimization of culture conditions. Disclosure of this relationship may provide a better understanding of glucose-related pathologies during pregnancy.

Effects of Human Placental Amnion Derived Mesenchymal Stem Cells on Proliferation and Apoptosis Mechanisms in Chronic Kidney Disease in the Rat.

Background and ObjectivesThe feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed. Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure.Methods and ResultsIn this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson’s Trichrome and Sirius Red staining showed fibrosis reduction in kidney.ConclusionsAccording to our results hAMSCs can be ameliorate renal failure.

Pluripotency markers in tissue and cultivated cells in vitro of different regions of human amniotic epithelium

Studies have described the presence of pluripotent markers in vivo and in vitro in human amnion. However, the amnion can be divided into reflected, placental and umbilical regions that are anatomically and functionally heterogeneous. Here, we evaluated the expression of pluripotency markers in tissue and cultivated cells in vitro of different regions of human amnion. To this end, we determined the presence of the core pluripotency factors OCT-4, NANOG and SOX-2 by immunofluorescence and RT-PCR and also performed transcriptome analysis of the different regions of amnion tissue. We identified the mRNA and protein of the pluripotency factors in the different regions of human amnion tissue. However, the OCT-4 and NANOG immunolocalization was cytoplasmic, whereas SOX-2 immunolocalization was nuclear regardless of the region analyzed. Moreover, we found three subpopulations of cells in the in vitro cultures of reflected and placental amnion: cells with immunostaining only in the nucleus, only in the cytoplasm, or in both compartments. Yet no statistically significant differences were found between the reflected and placental amnion. These results suggest a homogeneous distribution of the pluripotency transcription factors of the different regions of human amnion to isolate stem cells that can be used in regenerative medicine.

MiR-21 Contributes to Human Amniotic Membrane-Derived Mesenchymal Stem Cell Growth and Human Amniotic Membrane-Derived Mesenchymal Stem Cell-Induced Immunoregulation.

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are considered a new and favorable source of stem cells for cell replacement-based therapy. Some microRNAs (miRNAs) have been reported to participate in the regulation of immune responses. Our aim was to investigate the effects of miR-21 on the biological characteristics, immunoregulatory properties, and potential mechanisms of hAM-MSCs. hAM-MSCs were isolated from the placental amnion membrane of a newborn. Cell proliferation, cell cycle, apoptosis, and expressions of cell surface markers were measured by CCK-8 and flow cytometric assays in hAM-MSCs. The expression of mesenchymal-specific antigens vimentin and stage-specific embryonic antigen-4 (SSEA-4) were identified by immunofluorescence staining. Tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10) expressions in the cocultured supernatant of hAM-MSCs and peripheral blood mononuclear cells (PBMC) were detected via enzyme-linked immunosorbent assays (ELISA). Flow cytometric analyses revealed that the positive expression rates of the cell surface markers CD29, CD44, CD73, and CD90 in hAM-MSCs were 97.3%, 96.3%, 97.8%, and 98.2%, respectively, while the rates of CD34 and CD45 expression were only 0.6% and 0.84%, respectively. The immunofluorescent staining results showed that vimentin and SSEA-4 were positive in hAM-MSCs. CCK-8 assays revealed that miR-21 overexpression significantly promoted hAM-MSC proliferation. Cell cycle analyses revealed that the number of hAM-MSCs-miR-21+ cells during the synthesis phase (S phase) was significantly increased. miR-21 overexpression also significantly inhibited apoptosis in hAM-MSCs. The ELISA analyses revealed that miR-21 overexpression enhanced the inhibitory effect of hAM-MSCs on the secretion of TNF-α and MCP-1 as well as the promotive effect on the secretion of IL-10 in PBMC cocultured with miR-21-hAM-MSCs. In addition, miR-21 downregulation reduced the inhibitory effect of hAM-MSCs on the secretion of TNF-α, MCP-1, and the promotive effect on the secretion of IL-10 in PBMC cocultured with anti-miR-21-hAM-MSCs. Our data showed that miR-21 promoted hAM-MSCs proliferation, inhibited apoptosis, and was involved in controlling the immunoregulatory capacity of hAM-MSCs.

Evaluation of Placental Mesenchymal Stem Cell Sheets for Myocardial Repair and Regeneration.

This work explores placental tissue as a cell source for fabrication of tissue-engineered surgical patches for myocardial repair of congenital heart defects. This study demonstrates promising findings for the clinically driven evaluation of the cell source as defined by potential cardiac benefit, compatibility, cell source availability, and implant deliverability. It documents methods for the isolation of mesenchymal stem cells from human placental amnion and chorion tissues, characterization of these cells, and eventual cell sheet growth that can be leveraged going forward for patch fabrication. It establishes support to continue pursuing the placenta as a valuable cell source for myocardial repair.

Effects of maternal western-style diet on amniotic fluid volume and amnion VEGF profiles in a nonhuman primate model.

During pregnancy, high fat diet (HFD) induces maternal obesity, insulin resistance, and placental inflammatory responses that compromise placental and fetal development. Whether maternal HFD would adversely affect amniotic fluid volume (AFV) has not been explored. Vascular endothelial growth factor (VEGF) is expressed in the amnion and has been proposed as a regulator of AFV. Our aim was to investigate the effects of HFD on AFV and the associated changes in VEGF and soluble VEGF receptor 1 (sFlt‐1) expression profiles in three amnion regions of a nonhuman primate model. Further, we examined the relationships between VEGF expression and HFD‐induced changes in maternal metabolic status. Japanese macaques were maintained on control or HFD and amniotic fluid index (AFI) was measured as an ultrasonic estimate of AFV. Amniotic fluid VEGF concentrations were determined by ELISA and amnion VEGF and sFlt‐1 mRNA levels by real‐time RT‐qPCR. HFD increased maternal plasma triglyceride while glucose levels were unchanged. Maternal weight gain was found in diet‐sensitive animals whereas amniotic fluid VEGF concentration was reduced in diet‐resistant animals. HFD did not alter AFI and there was no correlation between AFI and maternal weight or amniotic fluid VEGF concentrations. VEGF mRNA levels were lowest in secondary placental amnion while sFlt‐1 mRNA were lowest in the primary placental amnion. HFD did not affect amnion VEGF or sFlt‐1 mRNA expression. These findings suggest that although maternal HFD increased maternal weight in diet‐sensitive and reduced amniotic fluid VEGF concentrations in diet‐resistant phenotype, AFV as indicated by the AFI, was not significantly affected.

Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds

ObjectiveMesenchymal stem/stromal cells derived from human term placentas (PMSCs) are novel therapeutic agents and more topical than ever. Here we evaluated the effects of three types of PMSCs on wound healing in an in vivo mouse model: Amnion-derived MSCs (AMSCs), blood vessel-derived MSCs (BV-MSCs) from the chorionic plate and Wharton's jelly-derived MSCs (WJ-MSCs) from the umbilical cord. MethodsWe topically applied PMSCs onto skin wounds in mice using the dermal substitute Matriderm® as carrier and evaluated wound healing parameters. In addition, we investigated the effects of all PMSC types under co-application with placental endothelial cells (PLECs). After 8 days, we compared the percent of wound closure and the angiogenic potential between all groups. ResultsAMSCs, BV-MSCs and WJ-MSCs significantly induced a faster healing and a higher number of blood vessels in the wound when compared to controls (Matriderm®-alone). PLECs did not further improve the advantageous effects of PMSC-treatment. Quantitative data and 3D analysis by high resolution episcopic microscopy confirmed a lower density of vessels in Matriderm®/PMSCs/PLECs co-application compared to Matriderm®/PMSCs treatment. ConclusionResults indicate that all three PMSC types exert similar beneficial effects on wound closure and neovascularization in our mouse model. PracticeUsing Matriderm® as carrier for PMSCs propagates rapid cell migration towards the wound area that allows a fast and clinically practicable method for stem cell application. ImplicationsThese promising effects warrant further investigation in clinical trials.

Effects of Δ(9)-tetrahydrocannabinol (THC) on human amniotic epithelial cell proliferation and migration.

BackgroundThe deleterious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) is able to cross the placenta barrier and cause alterations in fetal growth, low birth weight and preterm labor. However, the effects of THC on the human placenta amnion are still unknown. MethodsThe distributions of CB1R and CB2R in human amnion tissues were observed by immunohistochemistry (IHC). Human amniotic epithelial cell proliferation and migration in response to THC treatment were measured by MTS and transwell assays, respectively. The PCR array was performed to study the key regulators involved in the cell migration. The protein levels of CB1R, CB2R in amnion tissues and MMP2, MMP9 in cells were detected by western blotting. Small interfering RNAs (siRNAs) were used to knockdown MMP2 and MMP9 in WISH cells. ResultsOur results indicated that both CB1R and CB2R primarily identified in the epithelial layer of human placental amnion tissue. The CB1R expression in the amnion tissue was higher in the preterm group than normal control. High-dose of THC (30uM, but not 20 and 10uM) significantly inhibited (p<0.01) human amniotic epithelial cell lines (WISH) proliferation. Meanwhile, THC at both 10uM and 20uM (p<0.05) significantly suppressed cells migration in both WISH and primary human amniotic epithelial cells. The PCR array data and siRNA experiments demonstrated that MMP2/9 were tightly involved in the regulation of THC-inhibited cell migration in WISH cells. ConclusionThese results suggested that THC inhibited the migration of human amniotic epithelial cell through the regulation of MMP2 and MMP9, which in turn altered the development of the amnion during the gestation and partially resulted in preterm labor and other adverse pregnancy outcomes.

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.

Antifibrotic Activity of Human Placental Amnion Membrane-Derived CD34+ Mesenchymal Stem/Progenitor Cell Transplantation in Mice With Thioacetamide-Induced Liver Injury.

In this study, a CD34+ subpopulation of stem/progenitor cells derived from neonatal placental amnion membrane, denoted as CD34+ AMSPCs, were identified, enriched, and characterized. These cells are highly proliferative, express mesenchymal stromal cells and pluripotent stem cell markers, and demonstrate multidirectional differentiation potentials, indicating their promising application in clinical regenerative therapies. CD34+ AMSPC transplantation ameliorated liver fibrosis in mice with drug-induced liver injury. These cells represent a potential therapeutic agent for treating liver diseases associated with fibrosis.

Molecular and cellular characteristics of human and non-human primate multipotent stromal cells from the amnion and bone marrow during long term culture.

IntroductionMultipotent stromal cells (MSCs) are among the key candidates in regenerative medicine. However variety of MSC sources and general heterogeneity lead to controversial data in functional characterization. Furthermore, despite intensive usage as preclinical animal model, little is known about MSCs of the common marmoset monkey.MethodsMSCs derived from placental amnion and bone marrow samples from human and common marmoset were characterized in parallel over 12 passages to monitor similarities and significant differences (p ≤ 0.05, Student’s t-test) in MSC markers and major histocompatibility complex (MHC) class I expression by immunohistochemistry, flow cytometry, real-time PCR, metabolic activity test, with special focus on pluripotency associated genes.ResultsHuman and non-human primate MSCs were characterized for expression of MSC markers and capability of differentiation into mesenchymal lineages. MSCs could be cultured more than 100 days (26 passages), but metabolic activity was significantly enhanced in amnion vs. bone marrow MSCs. Interestingly, MHC class I expression is significantly reduced in amnion MSCs until passage 6 in human and marmoset, but not in bone marrow cells. For MSC markers, CD73 and CD105 levels remain unchanged in amnion MSCs and slightly decline in bone marrow at late passages; CD166 is significantly higher expressed in human MSCs, CD106 significantly lower vs. marmoset. All cultured MSCs showed pluripotency marker expression like Oct-4A at passage 3 significantly decreasing over time (passages 6–12) while Nanog expression was highest in human bone marrow MSCs. Furthermore, human MSCs demonstrated the highest Sox2 levels vs. marmoset, whereas the marmoset exhibited significantly higher Lin28A values. Bisulfite sequencing of the Oct-4 promoter region displayed fewer methylations of CpG islands in the marmoset vs. human.ConclusionsLittle is known about MSC characteristics from the preclinical animal model common marmoset vs. human during long term culture. Studied human and common marmoset samples share many similar features such as most MSC markers and reduced MHC class I expression in amnion cells vs. bone marrow. Furthermore, pluripotency markers indicate in both species a subpopulation of MSCs with true ‘stemness’, which could explain their high proliferation capacity, though possessing differences between human and marmoset in Lin28A and Sox2 expression.

Differential expression and regional distribution of aquaporins in amnion of normal and gestational diabetic pregnancies.

The region of the amnion overlying the placenta plays an active role in fluid exchange between amniotic fluid and fetal blood perfusing the surface of the placenta, whereas little transfer occurs across the reflected amnion that contacts the membranous chorion. Because aquaporins (AQPs) facilitate rapid movement of water across cells, we hypothesized that AQP gene expression in placental amnion is higher than in reflected amnion. Furthermore, because gestational diabetes mellitus (GDM) is often associated with polyhydramnios, we hypothesized that amnion AQP gene expression is reduced when amniotic fluid volume is elevated. Human placental and reflected amnion were obtained at cesarean delivery and subjected to relative quantitation of AQP mRNA by real-time RT-qPCR and proteins by western immunoblot. Amnion mRNA levels of five AQPs differed by up to 400-fold (P < 0.001), with AQP1 and AQP3 most abundant, AQP8 least and AQP9 and AQP11 intermediately expressed. Aquaporin proteins showed a similar profile. Aquaporin mRNA abundance was higher (P < 0.001) in placental than reflected amnion, whereas protein levels were lower (P < 0.01). In GDM pregnancies, neither AQP mRNA nor protein levels were different from normal. There was no correlation between AQP mRNA or protein levels with the amniotic fluid index in normal or GDM subjects. We conclude that there is a strong differential expression profile among individual AQPs and between regions of the amnion. These findings suggest differences in contribution of individual AQPs to water transport in the two regions of the amnion. Furthermore, AQP expression in the amnion is not altered in patients with GDM.