Human amniotic epithelial stem cells: Hepatic differentiation and regenerative properties in liver disease treatment.

The placenta and the extraembryonic tissues represent a valuable source of cells for regenerative medicine. In particular, the amniotic membrane possesses cells with stem cells characteristics that have attracted research attention. Human amniotic epithelial cells (hAECs) have unique and desirable features that position them over other stem cells, not only because of the unlimited potential supplied of, the easy access to placental tissues, and the minimal ethical and legal barriers associated, but also due to the embryonic stem cells markers expression and their ability to differentiate into the three germ layers. In addition, they are non-tumorigenic and have immunomodulatory and anti-inflammatory properties. Hepatic failure is one of the major causes of morbidity and mortality worldwide. Organ transplantation is the best way to treat acute and chronic liver failure, but there are several associated obstacles. Stem cells have been highlighted as alternative hepatocytes source because of their potential for hepatogenic differentiation. HAECs, in particular, have some properties that make them suitable for hepatocyte differentiation. In this work, we review the general characteristics of the epithelial stem cells isolated from human amniotic membrane as well as their ability to differentiate to hepatic cells. We also revise their regenerative properties, with the focus on their potential application in the liver disease treatment.

Stem cells and COVID-19: are the human amniotic cells a new hope for therapies against the SARS-CoV-2 virus?

A new coronavirus respiratory disease (COVID-19) caused by the SARS-CoV-2 virus, surprised the entire world, producing social, economic, and health problems. The COVID-19 triggers a lung infection with a multiple proinflammatory cytokine storm in severe patients. Without effective and safe treatments, COVID-19 has killed thousands of people, becoming a pandemic. Stem cells have been suggested as a therapy for lung-related diseases. In particular, mesenchymal stem cells (MSCs) have been successfully tested in some clinical trials in patients with COVID-19. The encouraging results positioned MSCs as a possible cell therapy for COVID-19. The amniotic membrane from the human placenta at term is a valuable stem cell source, including human amniotic epithelial cells (hAECs) and human mesenchymal stromal cells (hAMSCs). Interestingly, amnion cells have immunoregulatory, regenerative, and anti-inflammatory properties. Moreover, hAECs and hAMSCs have been used both in preclinical studies and in clinical trials against respiratory diseases. They have reduced the inflammatory response and restored the pulmonary tissue architecture in lung injury in vivo models. Here, we review the existing data about the stem cells use for COVID-19 treatment, including the ongoing clinical trials. We also consider the non-cellular therapies that are being applied. Finally, we discuss the human amniotic membrane cells use in patients who suffer from immune/inflammatory lung diseases and hypothesize their possible use as a successful treatment against COVID-19.

Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells.

The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.

Proliferation and survival of human amniotic epithelial cells during their hepatic differentiation.

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.

Involvement of leptin in the molecular physiology of the placenta.

Leptin is a homeostatic regulator in the placenta where it promotes proliferation, protein synthesis and the expression of tolerogenic maternal response molecules such as HLA-G. Leptin also exerts an anti-apoptotic action in placenta controlling the expression of p53 master cell cycle regulator under different stress conditions. On the other hand, leptin is an integrative target of different placental stimuli. The expression of leptin in placenta is regulated by hCG, insulin, steroids, hypoxia and many other growth hormones, suggesting that it might have an important endocrine function in the trophoblastic cells. The leptin expression is induced involving the cAMP/PKA or cAMP/Epac pathways which have profound actions upon human trophoblast function. The activation of PI3K and MAPK pathways also participates in the leptin expression. Estrogens play a central role during pregnancy, particularly 17ß-estradiol upregulates the leptin expression in placental cells through genomic and non-genomic actions. The leptin promoter analysis reveals specific elements that are active in placental cells. The transcription factors CREB, AP1, Sp1, NFκB and the coactivator CBP are involved in the placental leptin expression. Moreover, placental leptin promoter is a target of epigenetic marks such as DNA methylation and histone acetylation that regulates not only the leptin expression in placenta during pregnancy but also determines the predisposition of acquiring adult metabolism diseases. Taken together, all these results allow a better understanding of leptin function and regulatory mechanisms of leptin expression in human placental trophoblasts, and support the importance of leptin during pregnancy and in programming adult health.

Leptin reduces apoptosis triggered by high temperature in human placental villous explants: The role of the p53 pathway.

Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37 °C, 40 °C and 42 °C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40 °C and 42 °C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40 °C and 42 °C. The BCL2/BAX ratio was also significantly decreased in explants at 40 °C and 42 °C compared with explants incubated at 37 °C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling.

Regulation of placental leptin expression by cyclic adenosine 5'-monophosphate involves cross talk between protein kinase A and mitogen-activated protein kinase signaling pathways.

Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu)(2)cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 microM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)(2)cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 microm PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)(2)cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 microm PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways.

17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions.

The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E(2)) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E(2) effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E(2)-induced leptin expression. Moreover, E(2) treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between -1951 and -1847 bp is both necessary and sufficient to achieve E(2) effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E(2) on leptin expression. Moreover, E(2) action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E(2) could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 microM PD98059 and 0.1 microM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E(2) induction of leptin promoter. On the other hand, E(2) treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E(2) induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways.

Up-regulation of placental leptin by human chorionic gonadotropin.

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.