Chorionic Villi Of Human Placenta Research Articles

The Effect of Gestational Diabetes Mellitus on the Chorionic Villi of Human Placenta Among Saudi Arabian Mothers: A Quantitative and Comparative Study.

BackgroundThe placenta is a temporary pivotal organ - the gate between the mother and the fetus. It has multiple functions such as nutrient uptake, elimination of waste products, gas exchange, and production of some vital hormones. However, the pregnancy state is a diabetogenic condition caused by insulin resistance, resulting from physiological variations. Gestational diabetes mellitus (GDM) can have an impact on both the mother and the fetus by causing numerous complications. In our research study, we aim to study and compare the quantitative effect of GDM at the microscopical level within the chorionic villi of the placenta of both mothers diagnosed with GDM and healthy mothers as well as the clinical correlation.MethodsAfter applying the inclusion and exclusion criteria, we collected 84 placental samples from February 2017 until May 2017, which were composed of 42 GDM samples and 42 healthy samples. All of these samples have been studied under a light microscope for measuring different parameters.ResultsWe found that some of the measured parameters among diabetic villi were lower than those of healthy villi with a p-value < .05 being significant. These include the surface area of the blood vessels (P = .008), the perimeters of the blood vessels (P = .002), the placental barrier thickness/perimeters of the villous blood vessels ratio (P ≤ .001), the placental barrier thickness/surface area of the blood vessels ratio (P ≤ .001), the number of Hofbauer cells/surface area of the villous ratio (P ≤ .001), the number of the blood vessels/surface area of the villous ratio (P = .001), the surface area of the blood vessels/surface area of the villous ratio (P = .004), and the perimeters of the blood vessels/surface area of the villous ratio (P ≤ .001). These parameters have significant effects on fetal development as well as the mother’s status.ConclusionsGDM is associated with multiple changes in the placenta. Moreover, these changes can impact the fetoplacental circulation and cause multiple complications for the mother and the fetus. Therefore, identifying pregnant women with GDM and controlling hyperglycemia will improve the outcomes of the pregnancy.

Clonal isolation of endothelial colony-forming cells from early gestation chorionic villi of human placenta for fetal tissue regeneration.

Endothelial colony-forming cells (ECFCs) have been implicated in the process of vascularization, which includes vasculogenesis and angiogenesis. Vasculogenesis is a de novo formation of blood vessels, and is an essential physiological process that occurs during embryonic development and tissue regeneration. Angiogenesis is the growth of new capillaries from pre-existing blood vessels, which is observed both prenatally and postnatally. The placenta is an organ composed of a variety of fetal-derived cells, including ECFCs, and therefore has significant potential as a source of fetal ECFCs for tissue engineering. To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi (CV-ECFCs) of the placenta, and assess their potential for tissue engineering. The early gestation chorionic villus tissue was dissociated by enzyme digestion. Cells expressing CD31 were selected using magnetic-activated cell sorting, and plated in endothelial-specific growth medium. After 2-3 wks in culture, colonies displaying cobblestone-like morphology were manually picked using cloning cylinders. We characterized CV-ECFCs by flow cytometry, immunophenotyping, tube formation assay, and Dil-Ac-LDL uptake assay. Viral transduction of CV-ECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector, and transduction efficiency was tested by fluorescent microscopy and flow cytometry. Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved, small intestinal submucosa extracellular matrix scaffold. After four passages in 6-8 wks of culture, we obtained a total number of 1.8 × 107 CV-ECFCs using 100 mg of early gestational chorionic villus tissue. Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31, CD144, CD146, CD105, CD309, only partially expressed CD34, and did not express CD45 and CD90. CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation, similar to cord blood-derived ECFCs (CB-ECFCs). CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%. Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold. In summary, we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs. A substantial number of CV-ECFCs can be obtained within a short time frame, representing a promising novel source of ECFCs for fetal treatments.

Preconditioning human natural killer cells with chorionic villous mesenchymal stem cells stimulates their expression of inflammatory and anti-tumor molecules

BackgroundMesenchymal stem cells derived from the chorionic villi of human placentae (pMSCs) produce a unique array of mediators that regulate the essential cellular functions of their target cells. These properties make pMSCs attractive candidates for cell-based therapy. Here, we examined the effects of culturing human natural killer (NK) cells with pMSCs on NK cell functions.MethodspMSCs were cultured with IL-2-activated and non-activated NK cells. NK cell proliferation and cytolytic activities were monitored. NK cell expression of receptors mediating their cytolytic activity against pMSCs, and the mechanisms underlying this effect on pMSCs, were also investigated.ResultsOur findings show that IL-2-activated NK cells, but not freshly isolated NK cells, efficiently lyse pMSCs and that this response might involve the activating NK cell receptor CD69. Interestingly, although pMSCs expressed HLA class I molecules, they were nevertheless lysed by NK cells, suggesting that HLA class I antigens do not play a significant role in protecting pMSCs from NK cell cytolytic activity. Co-culturing NK cells with pMSCs also inhibited NK cell expression of receptors, including CD69, NKpG2D, CD94, and NKp30, although these co-cultured NK cells were not inhibited in lysing cancer cells in vitro. Importantly, co-cultured NK cells significantly increased their production of molecules with anti-tumor effects.ConclusionsThese findings suggest that pMSCs might have potential applications in cancer therapy.

Phenotypic and Functional Characterization of Mesenchymal Stem Cells from Chorionic Villi of Human Term Placenta

Bone marrow derived mesenchymal stem cells (BM-MSCs) are used extensively in transplantation but their use is associated with many problems including low abundance in BM, low overall number, decreased differentiation potential with age and the invasive isolation procedures needed to obtain BM. We report a novel method of isolating placental MSCs (pMSCs) from chorionic villi, which exhibit the phenotypic and functional characteristics that will make them an attractive source of MSCs for cell-based therapy. A novel explant approach was used to isolate pMSCs from chorionic villi of human placentae. These pMSCs were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation medium as demonstrated by cytochemical staining. The gene and protein expression profiles of pMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by pMSCs was also analysed using sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, the migration and proliferation potentials of pMSCs were also determined. pMSCs were isolated from fetal part of the chorionic villi and these pMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these pMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed several adhesion molecules, chemokines/receptors, growth factor receptors and cytokines/receptors. Moreover, they secreted many cytokines (IL-1Ra, IL6, IL8, IL10, IL11 and IL15) and they were able to proliferate. Furthermore, they migrated in response to chemotactic factors including stromal cell-derived factor-1 (SDF-1), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1). We devised a novel explant method of isolating pMSCs that expressed many biological factors responsible for mediating cellular processes such as migration/homing, immune modulation and angiogenesis. Therefore, we suggest that pMSCs prepared from human term placental chorionic villous explants are an attractive source of MSCs for cell therapy.

Abstract 231: Allogeneic Mesenchymal Stromal Cells Derived from the Chorionic Villi of Human Placentae Are a Superior Source of Progenitor Cells for Angiogenic Therapies

Introduction: Autologous bone marrow mesenchymal stromal cells (ABMSCs) have been the focus for the treatment of myocardial and limb ischemia. The utility of ABMSCs is limited because, in addition to requiring an invasive procedure to harvest, the number and the regenerative capacity of ABMSCs decrease with age and comorbid conditions. A redirected focus on alternative sources of MSCs is warranted. Here we compare MSCs from adipose tissue (ASCs) and human placenta (pMSCs) for angiogenic therapies. Methods: ASCs and pMSCs were characterized using culture assays, flow cytometry and immunofluorescent staining. Immunodulatory properties were assessed using mixed lymphocyte reactions (MLR). Inflammatory and angiogenic cytokines were quantified using ELISA. The ability of ASCs and pMSCs to restore perfusion in a murine model of hindlimb ischemia was compared. Results: ASCs and pMSCs expressed cell surface antigens consistent with MSCs. In permissive cultures, ASCs and pMSCs stained positive for Oil Red O (adipogenic), Alcian Blue (chondrogenic) and alkaline phosphatase (osteogenic) in addition to neural and myogenic lineages. Immunofluorescent staining demonstrated that PMSCs but not ASCs express embryonic stem cell antigens Oct-3/4, Nanog, SSEA-3 and SSEA-4. ASCs and pMSCs demonstrated immunosuppressive properties in MLRs (n=3). There was a significant reduction of MNC proliferation when cultured with pMSCs (96.5 ± 3.7 % reduction; p &lt; 0.001) or ASCs (93.27 ± 2.3 % reduction; p &lt; 0.001). When compared to ASCs, pMSCs (n=3) secrete significantly greater quantities of VEGF (4.4 ± 0.13 vs. 2.5 ± 0.031 ng/mL; p &lt; 0.001), HGF (2.4 ± 0.114 vs. 1.6 ± 0.173 ng/mL; p &lt; 0.001), and Ang-1 (4.6 ± .23 vs. 1.6 ± 0.016 ng/mL; p &lt; 0.001) in hypoxia. In NOD/SCID mice with femoral artery ligation (n=6), those treated with pMSCs demonstrated a significant improvement in perfusion compared to those treated with ASCs at 35 days post-treatment (56.18 vs. 40.82; p &lt; 0.001). Conclusion: In conclusion, while both ASCs and pMSCs have potent immunosuppressive properties, pMSCs are superior in the production of angiogenic cytokines and restoration of perfusion in ischemic tissue. PMSCs can be banked and thus made readily available for the treatment of acute ischemic syndromes.

Characterization and expansion of mesenchymal progenitor cells from first-trimester chorionic villi of human placenta

Mesenchymal stromal cells (MSC) have been identified in a variety of fetal and adult tissues, including bone marrow (BM), fetal blood and liver. We report on the isolation, expansion and differentiation in vitro of MSC-like cells from chorionic villi (CV). We evaluated 10 samples of CV collected at the first trimester (gestational age 11-13 weeks). We only used cells taken from back-up culture after a successful karyotype analysis. CV cells were characterized by morphologic, immunophenotypic and molecular analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions. Cell expansion was assessed after plating cells at different densities in different media, supplemented with animal and human serum. CV cells showed a homogeneous population of spindle-shaped cells after the first passage. Cells expressed CD90, CD105, CD73, CD44, CD29 and CD13 but not CD45, CD14, CD34 and CD117. They expressed Oct-4, Rex-1, GATA-4 and nestin, which characterize the undifferentiated stem cell state. They differentiated into osteocytes, adipocytes, chondrocytes and neuronal cells. Cell expansion was greater than that of adult BM-derived MSC, 9 logs with fetal bovine serum and 6 logs with human serum. Despite their high proliferative capacity, we did not observe any karyotypic abnormalities after culture. Our study shows that CV cells have better potential for expansion than adult stem cells. They can proliferate in a medium with human allogeneic serum and can differentiate into mesenchymal and neural lineages. CV cells may be an excellent cell source for therapeutic applications.

Mesenchymal progenitor cells derived from chorionic villi of human placenta for cartilage tissue engineering

Human mesenchymal stem cells are currently being studied extensively because of their capability for self-renewal and differentiation to various connective tissues, which makes them attractive as cell sources for regenerative medicine. Herein we report the isolation of human placenta-derived mesenchymal cells (hPDMCs) that have the potential to differentiate into various lineages to explore the possibility of using these cells for regeneration of cartilage. We first evaluated the chondrogenesis of hPDMCs in vitro and then embedded the hPDMCs into an atelocollagen gel to make a cartilage-like tissue with chondrogenic induction media. For in vivo assay, preinduced hPDMCs embedded in collagen sponges were subcutaneously implanted into nude mice and also into nude rats with osteochondral defect. The results of these in vivo and in vitro studies suggested that hPDMCs can be one of the possible allogeneic cell sources for tissue engineering of cartilage.

3-D Ultrastructural Distribution of Collagen in Human Placental Villi at Term in Relation to Vascular Tree

In order to understand the 3-D distribution of collagen in relation to vascularization, chorionic villi of human placentae, belonging to normal pregnancies at term, were studied by scanning electron microscopy (SEM) after alkali maceration techniques, and by transmission electron microscopy (TEM). The villous tree appeared made of an uninterrupted structure of collagen fibres. The collagen fibres connected the chorionic villi axis with their basal plates and organised differently according to the various levels of villous branching. The collagen of stem villi showed copious fibres. The external fibres (facing the villous surface) were arranged mainly longitudinally. The central core of the villi (inner fibres) were arranged concentrically around the wall of the fetal vessels. Both external and internal fibres formed stratified lamellae or small parallel bundles. The inner core of stem villi showed small holes housing capillary spaces. Mature intermediate and terminal villi showed a scarce amount of collagen arranged in thin concentric layer within the villous core, surrounding numerous dilated capillary and sinusoid spaces.These observations demonstrated that the extracellular matrix of human chorionic villi is highly compartmentalised and shows a variable structural 3-D distribution depending on the branching level of the villous tree, such a distribution ensures the most favourable microenvironment for feto-maternal exchanges and it is likely able to provide a modulated support to the developing chorionic fetal vessels and trophoblastic layer as well.

Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta

BM-derived mesenchymal stem cells (MSC) are attractive sources for autotransplantation with no risk of rejection, but the use of these cells bas problems, including the necessity of harvesting BM from donors, the donors' age-dependency, limitation to autologous use and difficulty of use for patients with hereditary diseases. We report a method of isolating placenta-derived mesenchymal progenitor cells (PDMPC) that can be used as an alternative source of MSC. We isolated PDMPC from human fetal chorionic villi using the explant culture method, from placentas collected after neonatal delivery (38-40 weeks of gestation). The PDMPC were characterized by morphologic and immunophenotypic analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions and determined by morphology, reverse transcription(RT)-PCR, histochemical staining and immunocytostaining. The PDMPC all originated from fetal chorionic villi, as confirmed by fluorescence in situ hybridization analysis. The PDMPC population consisted of spindle-shaped cells and large flat cells. The PDMPCexpressed CD13, CD44, CD73, CD90, CDIO5 and HLA class I as surface epitopes, but not CD31, CD34, CD45 and HLA-DR. These cells differentiated into osteocytes, chondrocytes and adipocytes under specific culture conditions, and were also induced to form neural-like cells. Our study shows that PDMPC can differentiate into mesenchymal lineages and be induced to form neural-like cells. Thus, PDMPCisolated from chorionic villi of placenta may provide a novel source for the research of stem and progenitor cells in placenta, cell therapy and regenerative medicine, particularly as a source of allogenic mesenchymal stem and progenitor cells with little ethical conflict and various advantages

Immortalized Human Placenta Derived Mesenchymal Cells by Transfection with Bmi-1 Lost the Abilitiy To Differencitate into Cell Types of Osteocytic, Condoricytic and Neural Lineages.

Introduction: We previously reported that mesenchymal cells derived from chorionic villi of human placenta could differentiate into various cell types including osteocytic, condoricytic and neural lineages. However, these cells can slowly proliferate with limited life span. In the present study, we transduced human placenta-derived mesenchymal cells (PDMCs) with cDNA encoding Bmi-1 and telomerase reverse transferase (hTert), respectively or simultaneously by lentiviral vector, and tested transduced cells for their ability to differentiate into multi-lineages and to support expansion of CD34+ cells in cord blood as feeder cells in the presence of cytokine cocktails.Method: The lentivairal vector expressing either Bmi-1, hTert or hrGFP was prepared as previously. The human PDMCs were isolated from chorionic villi of human placenta using the explant culture, and were cultured in DMEM (low glucose) with 10% FBS. The established cells were infected with either of the three viruses or a combination of Bmi-1 and hTert virus at the multiplicity of infection that allowed almost 100% of infection efficiency based on hrGFP fluorescence by flow cytometry. Those infected cells and uninfected cells served as control were then continuously propagated. These cells were then transferred into the specific culture media to differentiate them to osteocyte, condorocyte and neural cells as reported previously.Results: The control and PDMC/hTert cells showed a broad and flat shape suggesting that these cells were senescence and ceased proliferation. Cells infected with Bmi-1 and Bmi-1/hTert escaped the crisis and continued proliferation. These cells have been maintained in culture for more than 1 year and continued to proliferate near to 100PD. We found that Bmi-1 over-expression could induce endogenous telomerase activity and elongate telomere length in PDMCs. The expression of p16 mRNA and protein were down-regulated in prolonged culture cells, but p14ARF was not. The differentiation potential of transduced cells was gradually lost as culture was prolonged as well as the ability to support the expansion of CB CD34+ cells. Gene expression analysis on Bmi-1- infected cells at different PD revealed that more than 100 genes were induced or repressed in several different functional categories.Extensive increase in expression of the IGFBP2 mRNA and higher concentration of IGFBP2 was observed.Conclusion: Bmi-1 induced Telomerase activity and immorlized human placenta derived mesenchymal cells. The ability of transduced cells to differentiate into various cell types and support expansion of CB CD34+ cells were lost with propagation suggesting that subtle transformation has occurred in the Bmi-1 transduced cells.

Experimental in vivo and in vitro Formation of Type VI Collagen Periodic Fibrils in Chorionic Villi of Human Placenta

The distribution of type VI collagen in the chorionic villi of normal human term placenta was studied. Since 100 nm periodic fibrils (type VI collagen periodic fibrils) are known to form in the tendon, cornea and cultured fibroblasts by incubation with acidic ATP solution, we incubated the placental tissue with 20 mM ATP, fixed it with Karnovsky's fixative and examined under an electron microscope. Type VI collagen periodic fibrils widely occurred in the interstitial connective tissue, especially close to the basal lamina. By cryo-immunoelectron microscopy, labeling for collagen type VI was found in the area between the trophoblastic basal lamina and the endothelial basal lamina. Furthermore, we extracted type VI collagen from the human placenta and brought both extract and residues into ATP treatment. Periodic structure formation was rare in extracted preparation compared with residues. The results served to clarify the mechanism of fibril formation in the tissue.

Immunohistochemical identification and characterization of a special type of desmin-producing stromal cells in human placenta and other fetal tissues

Abstract An unusual type of stromal cells has been found to be abundantly present in chorionic villi of human placenta of gestational weeks 6, 17, 35–42 and in tissues of early stages of fetal development (gestational weeks 16–21). These mesenchymal cells are loosely arranged throughout the villous interior and contain the intermediate filament (IF) proteins vimentin and desmin; however the smooth muscle (sm) markers sm-α-actin and sm-myosin are absent. Typical myoid stromal cells that are positive for both desmin and sm-α-actin also occur in this tissue but are restricted to certain dispersed cell clusters associated with blood vessels. Similar disperse desmin-positive, sm-α-actin-negative stromal cells have also been identified, although more sparsely, in the chorionic plate of the placenta and in other diverse fetal tissues such as the interstitium of the kidney, of testis and epididymis, and in cells surrounding Hassall bodies of thymus. The biological nature of these desmin-containing but sm-α-actin-negative stromal cells is discussed in relation to myoid cell differentiation. It is emphasized that despite their synthesis of considerable amounts of desmin they cannot be considered myogenic as the occurrence of desmin in the cells may represent an isolated expression of an individual IF protein gene, independent of the synthesis of other muscle proteins.

CAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

The role of adenosine 3',5'-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl- conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100 nM catalytic subunit of PK-A purified from human placenta and 1 mM of the phosphatase resistant adenosine 5'-thiotriphosphate (ATP-gamma-S) and resealed. Cl- conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23 degrees C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([14C]-sucrose) and included in each Cl- flux calculation. In 19 independent experiments, the mean Cl- conductance in placental membranes in the absence of phosphorylation was 3.67 +/- 3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97 +/- 1.75 nmol.sec-1. (mg protein)-1 (mean +/- SD). PK-A dependent phosphorylation reduced the Cl- conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl- channels, confirmed that the PK-A dependent phosphorylation increased in Cl- conductance in 11/13 experiments, from 1.01 +/- 0.61 to 1.85 +/- 0.99 nmol.sec-1.(mg protein)-1 (mean +/- SD). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl- channel(s) of the apical membranes of human placenta.

Chloride conductance in membrane vesicles from human placenta using a fluorescent probe. Implications for cystic fibrosis

Previous evidence suggests that the molecular defect in cystic fibrosis (CF) could reside in an altered chloride conductance of epithelial tissues. Since the brush border of the syncytiotrophoblast of the chorionic villi of human placenta is on abundant source of epithelial membranes and it is unaltered by secondary pathology or treatment we chose to characterize its chloride conductance and to compare it in normal and CF membranes. Chloride transport was studied in microvillar vesicles (MVV) by the quenching of the fluorescent probe 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ).Chloride conductance at 23°C: (a) increased by 39% under a membrane potential change of 70 mV; (b) was inhibited by diphenylamine 2-car☐ylate ( K l × 150 μM); (c) displayed an activation energy of 3.5 kcal · mol −1. The comparison of the chloride conductance for an inwardly directed gradient of 150 mM Cl − at 23° C (membrane potential set at 0 mV) between CF and control membranes was not significantly different. These findings demonstrate the presence of a chloride conductive pathway in microvillar vesicles from human placenta and preliminary results exclude major differences in the conductance of CF derived material in the absence of neurohormonal stimuli.

Cellular junctions on the free surface of human placental syncytium.

Chorionic villi of human placenta at full term have been studied in thin section. On the free surface of syncytium, cellular junctions such as desmosomes, desmosome-like junctions and tight junctions, but not gap junctions, are present. Theories regarding the formation and functions of the junctions on the syncytial free surface are discussed.