Membrane Of Human Term Placenta Research Articles

B Lymphocytes as Targets of the Immunomodulatory Properties of Human Amniotic Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) from the amniotic membrane of human term placenta (hAMSC), and the conditioned medium generated from their culture (CM-hAMSC) offer significant tools for their use in regenerative medicine mainly due to their immunomodulatory properties. Interestingly, hAMSC and their CM have been successfully exploited in preclinical disease models of inflammatory and autoimmune diseases where depletion or modulation of B cells have been indicated as an effective treatment, such as inflammatory bowel disease, lung fibrosis, would healing, collagen-induced arthritis, and multiple sclerosis. While the interactions between hAMSC or CM-hAMSC and T lymphocytes, monocytes, dendritic cells, and macrophages has been extensively explored, how they affect B lymphocytes remains unclear. Considering that B cells are key players in the adaptive immune response and are a central component of different diseases, in this study we investigated the in vitro properties of hAMSC and CM-hAMSC on B cells. We provide evidence that both hAMSC and CM-hAMSC strongly suppressed CpG-activated B-cell proliferation. Moreover, CM-hAMSC blocked B-cell differentiation, with an increase of the proportion of mature B cells, and a reduction of antibody secreting cell formation. We observed the strong inhibition of B cell terminal differentiation into CD138+ plasma cells, as further shown by a significant decrease of the expression of interferon regulatory factor 4 (IRF-4), PR/SET domain 1(PRDM1), and X-box binding protein 1 (XBP-1) genes. Our results point out that the mechanism by which CM-hAMSC impacts B cell proliferation and differentiation is mediated by secreted factors, and prostanoids are partially involved in these actions. Factors contained in the CM-hAMSC decreased the CpG-uptake sensors (CD205, CD14, and TLR9), suggesting that B cell stimulation was affected early on. CM-hAMSC also decreased the expression of interleukin-1 receptor-associated kinase (IRAK)-4, consequently inhibiting the entire CpG-induced downstream signaling pathway. Overall, these findings add insight into the mechanism of action of hAMSC and CM-hAMSC and are useful to better design their potential therapeutic application in B-cell mediated diseases.

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.

Human amniotic mesenchymal stromal cells (hAMSCs) as potential vehicles for drug delivery in cancer therapy: an in vitro study.

IntroductionIn the context of drug delivery, mesenchymal stromal cells (MSCs) from bone marrow and adipose tissue have emerged as interesting candidates due to their homing abilities and capacity to carry toxic loads, while at the same time being highly resistant to the toxic effects. Amongst the many sources of MSCs which have been identified, the human term placenta has attracted particular interest due to its unique, tissue-related characteristics, including its high cell yield and virtually absent expression of human leukocyte antigens and co-stimulatory molecules. Under basal, non-stimulatory conditions, placental MSCs also possess basic characteristics common to MSCs from other sources. These include the ability to secrete factors which promote cell growth and tissue repair, as well as immunomodulatory properties. The aim of this study was to investigate MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) as candidates for drug delivery in vitro.MethodsWe primed hAMSCs from seven different donors with paclitaxel (PTX) and investigated their ability to resist the cytotoxic effects of PTX, to upload the drug, and to release it over time. We then analyzed whether the uptake and release of PTX was sufficient to inhibit proliferation of CFPAC-1, a pancreatic tumor cell line sensitive to PTX.ResultsFor the first time, our study shows that hAMSCs are highly resistant to PTX and are not only able to uptake the drug, but also release it over time. Moreover, we show that PTX is released from hAMSCs in a sufficient amount to inhibit tumor cell proliferation, whilst some of the PTX is also retained within the cells.ConclusionTaken together, for the first time our results show that placental stem cells can be used as vehicles for the delivery of cytotoxic agents.

Uptake mechanism of an estriol precursor, 16α-hydroxy dehydroepiandrosterone sulfate, at the basal plasma membrane of human term placenta

Uptake mechanism of an estriol precursor, 16α-hydroxy dehydroepiandrosterone sulfate, at the basal plasma membrane of human term placenta

Feasibility and potential of in utero foetal membrane-derived cell transplantation

Cells isolated from foetal membranes of human term placenta display multiple properties, including some features of stem/progenitor cells, together with immunomodulatory actions and the ability to secrete bioactive soluble factors. Whilst such properties support the potential applicability of these cells in transplantation settings aimed at regenerating/repairing tissues in adults, theoretically, using these cells in prenatal treatment strategies may also be achievable. To assess the feasibility of a foetal membrane-derived cell-based therapeutic treatment during foetal development, we firstly addressed the question of whether in utero transplantation using these cells was possible. To this end, we assessed postnatal microchimerism after transplantation of amniotic membrane-derived cells (a mixture of both mesenchymal stromal/stem cells and epithelial cells) in foetal sheep. Transplantation was performed with or without human umbilical cord blood mononuclear cells and chorionic membrane-derived mesenchymal stromal/stem cells, and was followed by a postnatal booster cell injection. Lambs were euthanized 2-4 months postnatally and their organs/tissues were analysed for microchimerism through detection of human DNA. Human DNA was found in almost all tissues of all of the lambs, with the seemingly random appearance of human cells in some of the analysed tissues suggesting long-term human microchimerism and donor cell migration after in utero/postnatal booster xenotransplation. Differences in microchimerism tissue distribution between animals transplanted with different cell types are discussed. This pilot study adds to ongoing efforts by different investigators to explore the potential of in utero cellular transplantation, and warrants further investigation of using foetal membrane-derived cells for prenatal cell therapies.

Amniotic membrane‐derived cells inhibit proliferation of cancer cell lines by inducing cell cycle arrest

Cells derived from the amniotic foetal membrane of human term placenta have drawn particular attention mainly for their plasticity and immunological properties, which render them interesting for stem-cell research and cell-based therapeutic applications. In particular, we have previously demonstrated that amniotic mesenchymal tissue cells (AMTC) inhibit lymphocyte proliferation in vitro and suppress the generation and maturation of monocyte-derived dendritic cells. Here, we show that AMTC also significantly reduce the proliferation of cancer cell lines of haematopoietic and non-haematopoietic origin, in both cell–cell contact and transwell co-cultures, therefore suggesting the involvement of yet-unknown inhibitory soluble factor(s) in this ‘cell growth restraint’. Importantly, we provide evidence that the anti-proliferative effect of AMTC is associated with induction of cell cycle arrest in G0/G1 phase. Gene expression analyses demonstrate that AMTC can down-regulate cancer cells' mRNA expression of genes associated with cell cycle progression, such as cyclins (cyclin D2, cyclin E1, cyclin H) and cyclin-dependent kinase (CDK4, CDK6 and CDK2), whilst they up-regulate cell cycle negative regulator such as p15 and p21, consistent with a block in G0/G1 phase with no progression to S phase. Taken together, these findings warrant further studies to investigate the applicability of these cells for controlling cancer cell proliferation in vivo.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation

Possible cardiac repair by adult stem cell transplantation is currently hampered by poor cell viability and delivery efficiency, uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, positron emission tomography, and immunohistochemistry, we show that injection of a hyaluronan mixed ester of butyric and retinoic acid (HBR) into infarcted rat hearts afforded substantial cardiovascular repair and recovery of myocardial performance. HBR restored cardiac [18F]fluorodeoxyglucose uptake and increased capillary density and led to the recruitment of endogenous Stro-1-positive stem cells. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay demonstrated that HBR-treated hearts exhibited a decrease in the number of apoptotic cardiomyocytes. In isolated rat cardiomyocytes and Stro-1 stem cells, HBR enhanced the transcription of vascular endothelial growth factor, hepatocyte growth factor, kdr, akt, and pim-1. HBR also increased the secretion of vascular endothelial growth factor and hepatocyte growth factor, suggesting that the mixed ester may have recruited both myocardial and Stro-1 cells also. An increase in capillarogenesis was induced in vitro with medium obtained from HBR-exposed cells. In the infarcted myocardium, HBR injection increased histone H4 acetylation significantly. Acetyl-H4 immunoreactivity increased in rat cardiomyocytes and Stro-1 cells exposed to HBR, compared with untreated cells. In conclusion, efficient cardiac regenerative therapy can be afforded by HBR without the need of stem cell transplantation or vector-mediated gene delivery.

Amniotic Membrane Patching Promotes Ischemic Rat Heart Repair

The amniotic membrane has long been applied for wound healing and treatment of ophthalmological disorders, even though the mechanisms underlying its actions remain to be clarified. Recently, cells derived from fetal membranes of human term placenta have raised strong interest in regenerative medicine for their stem cell potential and immunomodulatory features. Our study aimed to investigate the possible utility of amniotic membrane to limit postischemic cardiac injury. A fragment of human amniotic membrane was applied onto the left ventricle of rats that had undergone ischemia through left anterior descending coronary artery ligation. Echocardiographic assessment of morphological and functional cardiac parameters was then performed over a 3-month period. We demonstrated that application of an amniotic membrane fragment onto ischemic rat hearts could significantly reduce postischemic cardiac dysfunction. The amniotic membrane-treated rats showed higher preservation of cardiac dimensions and improved cardiac contractile function in terms of higher left ventricle ejection fraction, fractional shortening, and wall thickening. These improvements were apparent by day 7 after application of the amniotic membrane, persisted for at least 2 months, and occurred independently of cardiac injury severity. No engraftment of amniotic cells was detected into host cardiac tissues. Our results suggest that use of amniotic membrane may constitute a convenient vehicle for supplying cells that produce cardioprotective soluble factors, and reinforce the notion that this tissue constitutes a cell source with clinical potential that has yet to be completely revealed.

Amniotic Mesenchymal Tissue Cells Inhibit Dendritic Cell Differentiation of Peripheral Blood and Amnion Resident Monocytes

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G(0) phase and abolishes the production of inflammatory cytokines such as TNF-alpha, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Placenta-derived stem cells: new hope for cell therapy?

An urgent current need in regenerative medicine is that of identifying a plentiful, safe and ethically acceptable stem cell source for the development of therapeutic strategies to restore functionality in damaged or diseased organs and tissues. In this context, human term placenta represents a prime candidate, as it is available in nearly unlimited supply, is ethically problem-free and easily procured. Placental cells display differentiation capacity toward all three germ layers, while also displaying immunomodulatory effects, therefore supporting the possibility that they could be applied in an allogeneic transplantation setting. Although promising data have been reported to date, further study is required to fully characterize the differentiation potential of placenta-derived cells and to identify their possible clinical applications. Here, we provide a snapshot of current knowledge regarding the potential of cells from the amniotic membrane of human term placenta to address current shortcomings in the field of regenerative medicine.

Use of Phospholipid Transfer Protein as a Probe to Study the Lipid Dynamics and Alkaline Phosphatase Activity in the Brush Border Membrane of Human Term Placenta

Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver-Burk plot, which showed decreased V(max) and K(M). The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreased in the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences.

Structural analysis and tissue localization of human C4.4A: a protein homologue of the urokinase receptor.

C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor (uPAR), was originally identified as a metastasis-associated membrane protein, but little is known about its structural and functional properties. Therefore, we expressed, purified and characterized a soluble truncated form of human C4.4A, and used this protein to produce specific polyclonal anti-C4.4A antibodies. By immunohistochemistry we observed a pronounced surface staining for C4.4A in suprabasal keratinocytes of chronic human wounds and found C4.4A expression markedly upregulated in migrating keratinocytes during re-epithelisation of incisional skin wounds. Phorbol-ester-induced hyperplasia of mouse skin is also accompanied by a significant induction of C4.4A expression in the multilayered, suprabasal keratinocytes. C4.4A contains two Ly-6 (leucocyte antigen 6)/uPAR/alpha-neurotoxin modules. Our recombinant human C4.4A is extensively modified by post-translational glycosylation, which include 5-6 N-linked carbohydrates primarily located in or close to its second Ly-6/uPAR/alpha-neurotoxin module and approximately 15 O-linked carbohydrates clustered in a Ser/Thr/Pro-rich region at the C-terminus. A highly protease-sensitive region (Tyr200-Arg204) is located between these two clusters of N- and O-linked carbohydrates. The natural, glycolipid-anchored C4.4A from amnion membranes of human term placenta exhibits similar properties. Using recombinant, soluble C4.4A or MCF 7 cells, which express significant amounts of GPI-anchored C4.4A, we find no evidence for an interaction between C4.4A and uPA, a property suggested previously for rat C4.4A. Collectively these data indicate that C4.4A, although being a structural homologue of uPAR, is unlikely to have a functional overlap with uPAR.

Asymmetrical distribution of G proteins in syncytiotrophoblastic brush-border and basal-plasma membranes of human term placenta

In human placental syncytiotrophoblast brush-border (BBM, facing the mother) and basal-plasma membranes (BPM, facing to fetus) we have recently demonstrated the presence of calcaemic hormone-specific receptors for parathyroid hormone and calcitonin, which could be implicated in calcium transport from the mother to the fetus. It is well recognized that signal transducing G proteins (guanosine nucleotide-binding proteins) can associate with various transmembrane receptors and effector proteins, and regulate a variety of second-messenger systems and ion channels. In this present paper, we investigated the presence of a variety of α and β subunits of G proteins in both syncytiotrophoblast, BBM and BPM by Western blot technique. For the first time, we were able to demonstrate the presence of G proteins in the bipolar syncytiotrophoblast membranes, which were evaluated by immunoblotting using affinity purified antiserum raised against the α subunits of G i1, G i1 i2 , G i3, G o, G q, G s, G z and against the β subunits. In BBM, we identified the α subunits of G i1, G i3, G o, G q, G s (42, 46 kDa), G z and β subunits. The same α subunits of G proteins were found in BPM, although α subunits of G i3, G q, G s (46 kDa) were located predominantly in the BBM, and the α subunit of G o was found preferentially in BPM. Moreover, in BBM and BPM, a purified antisera raised against the α subunits of G i1 and G s, detected a 105 kDa protein and a 67 kDa protein, respectively. Interestingly, the 67 kDa protein was preferentially located in BBM, and none of these proteins were detectable in membranes prepared from brain (control). The asymmetrical distribution of the α subunits of G proteins among the two different placental bipolar membranes might reflect the very specialized function of these syncytiotrophoblast membranes in ions and nutrients transport from the mother to the fetus.

Human placental LHRH receptor: agonist and antagonist labeling produces differences in the size of the non-denatured, solubilized receptor.

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kav's indifferent from each other but significantly different from the Kav's of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Histochemical demonstration of the hydroxysteroid oxidoreductases in the umbilical cord and membranes of human term and immature placenta.

Formazan deposition was used as indicator to study the localization of the 3- β- and 17- β-hydroxysteroid oxidoreductases in the umbilical cord and membranes of human term placenta and in the placenta, cord, membranes and skin of a 10-week fetus. The localization of the staining reactions varied with the steroids and pyridine nucleotides used. With 17 β-estradiol and NAD, formazan deposition was localized in the trophoblasts, epithelium of the cord and reflected membranes of the term placenta and 10-week fetal placenta. With 17 β-estradiol and NADP, and with testosterone and NAD and NADP, formazan depositions occurred in the trophoblasts of the 10-week fetal placenta but not in the term placenta. These results suggest the possibility that the trophoblasts lose 17- β- hydroxysteroid oxidoreductase activity with testosterone and with 17 β-estradiol and NADP some time after the 10th week of gestation. The skin of a 10-week fetus gave a positive staining reaction for 17- β-hydroxysteroid oxidoreductase with 17 β-estradiol and NAD. Positive staining reactions for NAD-linked 3- β-hydroxysteroid oxidoreductase were observed in the trophoblasts, amnion, chorion and the connective tissue of the cord of a 10- week fetal placenta and term placenta. (Endocrinology78: 333, 1966)