Human Amnion Epithelial Cells Research Articles

The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation.

Lung inflammation and impaired alveolarization are hallmarks of bronchopulmonary dysplasia (BPD). We hypothesize that human amnion epithelial cells (hAECs) are anti-inflammatory and reduce lung injury in preterm lambs born after antenatal exposure to inflammation. Pregnant ewes received either intra-amniotic lipopolysaccharide (LPS, from E.coli 055:B5; 4mg) or saline (Sal) on day 126 of gestation. Lambs were delivered by cesarean section at 128 d gestation (term ~150 d). Lambs received intravenous hAECs (LPS/hAECs: n = 7; 30x106 cells) or equivalent volumes of saline (LPS/Sal, n = 10; or Sal/Sal, n = 9) immediately after birth. Respiratory support was gradually de-escalated, aimed at early weaning from mechanical ventilation towards unassisted respiration. Lung tissue was collected 1 week after birth. Lung morphology was assessed and mRNA levels for inflammatory mediators were measured. Respiratory support required by LPS/hAEC lambs was not different to Sal/Sal or LPS/Sal lambs. Lung tissue:airspace ratio was lower in the LPS/Sal compared to Sal/Sal lambs (P<0.05), but not LPS/hAEC lambs. LPS/hAEC lambs tended to have increased septation in their lungs versus LPS/Sal (P = 0.08). Expression of inflammatory cytokines was highest in LPS/hAECs lambs. Postnatal administration of a single dose of hAECs stimulates a pulmonary immune response without changing ventilator requirements in preterm lambs born after intrauterine inflammation.

HUMAN AMNION EPITHELIAL CELLS REDUCE BLOOD PRESSURE AND PREVENT AORTIC INFLAMMATION AND COLLAGEN EXPRESSION DURING EXPERIMENTAL HYPERTENSION

Objective: Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease. Current treatments for hypertension may reduce blood pressure but do not target the associated pathological changes and end-organ damage. Cell therapy has great therapeutic potential for many conditions as, unlike single pharmacological agents, cells can deliver multiple mediators which could more effectively target complex disease mechanisms. Human amnion epithelial cells (hAECs) have many properties (eg. anti-inflammatory, anti-fibrotic, regenerative and immunologically inert) that make them attractive candidates for a cell-based therapy for vascular pathology. This study aimed to test the potential of hAECs to treat vascular pathology in angiotensin II-induced hypertension. Design and method: Male C57Bl6 mice (8–12 weeks) were administered vehicle (saline; n = 35) or angiotensin II (0.7 mg/kg/d, n = 37) for 14 d via a subcutaneous osmotic minipump. After minipump implantation, a subset of mice were injected with 10^6 of hAECs intravenously. Systolic blood pressure was measured using tail-cuff; inflammation was assessed using flow cytometry and markers of fibrosis using quantitative PCR. Results: Angiotensin II infusion increased systolic blood pressure and promoted accumulation of aortic leukocytes, specifically macrophages and monocytes, which were elevated by ∼3-fold compared to vehicle-infused mice (n = 9–11, P &lt; 0.05). Angiotensin II also increased aortic mRNA expression of collagen type 1 alpha 1 (Col1a1) by ∼4-fold and collagen type 5 alpha 1 (Col5a1) by ∼7-fold compared to vehicle (n = 6–8, P &lt; 0.05). Co-administration of hAECs limited the development of hypertension by angiotensin II (185 ± 5 mmHg vs 165 ± 4 mmHg; n = 9–11, P &lt; 0.05), as well as the aortic infiltration of macrophages and monocytes (n = 9–11, P &lt; 0.05) and expression of Col1a1 and Col5a1 (n = 6–8, P &lt; 0.05). Conclusions: Intravenous administration of hAECs blunted angiotensin II-induced hypertension, aortic inflammation and collagen expression in male mice. This study suggests that hAECs or their cellular products could be explored as treatments for vascular pathology during hypertension.

Regulating cell fate of human amnion epithelial cells using natural compounds: an example of enhanced neural and pigment differentiation by 3,4,5-tri-O-caffeoylquinic acid

Over the past years, Human Amnion Epithelial Cells (hAECs), a placental stem cell, are gaining higher attention from the scientific community as they showed several advantages over other types of stem cells, including availability, easy accessibility, reduced rejection rate, non-tumorigenicity, and minimal legal constraint. Recently, natural compounds are used to stimulate stem cell differentiation and proliferation and to enhance their disease-treating potential. A polyphenolic compound 3,4,5-Tri-O-Caffeoylquinic Acid (TCQA) has been previously reported to induce human neural stem cell differentiation and may affect melanocyte stem cell differentiation as well. In this study, TCQA was tested on 3D cultured hAECs after seven days of treatment, and then, microarray gene expression profiling was conducted of TCQA-treated and untreated control cells on day 0 and day 7. Analyses revealed that TCQA treatment significantly enriched pigment and neural cells sets; besides, genes linked with neurogenesis, oxidation–reduction process, epidermal development, and metabolism were positively regulated. Interestingly, TCQA stimulated cell cycle arrest-related pathways and differentiation signaling. On the other hand, TCQA decreased interleukins and cytokines expression and this due to its anti-inflammatory properties as a polyphenolic compound. Results were validated to highlight the main activities of TCQA on hAECs, including differentiation, cell cycle arrest, and anti-inflammatory. This study highlights the important role of hAECs in regenerative medicine and the use of natural compounds to regulate their fate.4ntr2z8GsC-Lz3nsa9SfYxVideo abstract

Exposure to 50 Hz Extremely-Low-Frequency Magnetic Fields Induces No DNA Damage in Cells by Gamma H2AX Technology.

The current results for extremely-low-frequency magnetic fields (ELF-MF) on DNA damage are still debated. A sensitive indicator and systematic research are needed to assess the effects of ELF-MF. In this study, we used γH2AX as an early and sensitive molecular marker to evaluate the DNA damage effects of ELF-MF in vitro. Human amnion epithelial cells (FLs), human skin fibroblast cells (HSFs), and human umbilical vein endothelial cells (HUVECs) were exposed to 50 Hz ELF-MF at 0.4, 1, and 2 mT for 15 min, 1 h, and 24 h, respectively. After exposure, cells were subjected to γH2AX immunofluorescence and western blot. The results showed no significant difference in the average number of foci per cell, the percentage of γH2AX foci-positive cells, or the expression of γH2AX between the sham and 50 Hz ELF-MF exposure groups (P > 0.05). In conclusion, 50 Hz ELF-MF did not induce DNA damage in FLs, HSFs, or HUVECs, which was independent of the intensity or duration of the exposure.

Preventing Brain Injury in the Preterm Infant-Current Controversies and Potential Therapies.

Preterm birth is associated with a high risk of morbidity and mortality including brain damage and cerebral palsy. The development of brain injury in the preterm infant may be influenced by many factors including perinatal asphyxia, infection/inflammation, chronic hypoxia and exposure to treatments such as mechanical ventilation and corticosteroids. There are currently very limited treatment options available. In clinical trials, magnesium sulfate has been associated with a small, significant reduction in the risk of cerebral palsy and gross motor dysfunction in early childhood but no effect on the combined outcome of death or disability, and longer-term follow up to date has not shown improved neurological outcomes in school-age children. Recombinant erythropoietin has shown neuroprotective potential in preclinical studies but two large randomized trials, in extremely preterm infants, of treatment started within 24 or 48 h of birth showed no effect on the risk of severe neurodevelopmental impairment or death at 2 years of age. Preclinical studies have highlighted a number of promising neuroprotective treatments, such as therapeutic hypothermia, melatonin, human amnion epithelial cells, umbilical cord blood and vitamin D supplementation, which may be useful at reducing brain damage in preterm infants. Moreover, refinements of clinical care of preterm infants have the potential to influence later neurological outcomes, including the administration of antenatal and postnatal corticosteroids and more accurate identification and targeted treatment of seizures.

A protocol for cell therapy infusion in neonates

Cell therapies for neonatal morbidities are progressing to early phase clinical trials. However, protocols for intravenous (IV) delivery of cell therapies to infants have not been evaluated. It has been assumed the cell dose prescribed is the dose delivered. Early in our clinical trial of human amnion epithelial cells (hAECs), we observed cells settling in the syringe and IV tubing used to deliver the suspension. The effect on dose delivery was unknown. We aimed to quantify this observation and determine an optimal protocol for IV delivery of hAECs to extremely preterm infants. A standard pediatric infusion protocol was modeled in the laboratory. A syringe pump delivered the hAEC suspension over 60 minutes via a pediatric blood transfusion set (200‐μm filter and 2.2 mL IV line). The infusion protocol was varied by agitation methods, IV‐line volumes (0.2‐2.2 mL), albumin concentrations (2% vs 4%), and syringe orientations (horizontal vs vertical) to assess whether these variables influenced the dose delivered. The influence of flow rate (3‐15 mL/h) was assessed after other variables were optimized. The standard infusion protocol delivered 17.6% ± 9% of the intended hAEC dose. Increasing albumin concentration to 4%, positioning the syringe and IV line vertically, and decreasing IV‐line volume to 0.6 mL delivered 99.7% ± 13% of the intended hAEC dose. Flow rate did not affect dose delivery. Cell therapy infusion protocols must be considered. We describe the refinement of a cell infusion protocol that delivers intended cell doses and could form the basis of future neonatal cell delivery protocols.

Global Gene Expression Profiling Reveals Isorhamnetin Induces Hepatic-Lineage Specific Differentiation in Human Amniotic Epithelial Cells.

Human amnion epithelial cells (hAECs), derived from discarded term placenta, is anticipated as a new stem cell resource because of their advantages over embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), such as no risk of tumorigenicity and minimal ethical issue. hAECs have been reported to differentiate into hepatic-like cells (HLCs) with variable functionalities suitable for cell-based therapy of end-stage liver diseases, drug screening, and drug toxicity tests. On the other hand, a new research stream has been evolving to use natural compounds as stimulants of stem cell differentiation because of their high availability and minimum side effects. Isorhamnetin is a naturally occurring flavonoid commonly found in fruits and vegetables and has been reported to improve hepatic fibrosis and steatosis. In this present study, we have screened the differentiation potential of isorhamnetin in hAECs. The cells were grown on 3D cell culture and were treated with 20 μM of synthesized isorhamnetin for 10 days without adding any additional growth factors. DNA microarray global gene expression analysis was conducted for differentially expressed genes between isorhamnetin-treated and untreated control cells, gene expression validation was carried out using RT-qPCR method, and finally, several hepatic functions were assessed. Microarray analysis showed that isorhamnetin could activate essential biological processes, molecular functions, and signaling pathways for hepatic differentiation. Hepatic progenitor markers, EPCAM and DLK1, were upregulated in the isorhamnetin-treated hAECs. AFP was downregulated, while ALB was upregulated on Day 10. Furthermore, isorhamnetin-treated cells could show increased CYP enzyme mRNA levels, ICG uptake and release, glycogen storage activity, and urea secretion. Additionally, isorhamnetin-treated cells did not show any trace of transdifferentiation evident by significant downregulation of several colon- and cholangiocyte-specific markers. However, longer treatment with isorhamnetin did not promote hepatic maturation. Altogether, our findings indicate that isorhamnetin has a promising effect on directing the hepatic-lineage specific differentiation in hAECs.

Window of opportunity for human amnion epithelial stem cells to attenuate astrogliosis after umbilical cord occlusion in preterm fetal sheep.

There is increasing evidence that administration of many types of stem cells, including human amnion epithelial cells (hAECs), can reduce hypoxic‐ischemic injury, including in the perinatal brain. However, the therapeutic window for single dose treatment is not known. We compared the effects of early and delayed intracerebroventricular administration of hAECs in fetal sheep at 0.7 gestation on brain injury induced by 25 minutes of complete umbilical cord occlusion (UCO) or sham occlusion. Fetuses received either 1 × 106 hAECs or vehicle alone, as an infusion over 1 hour, either 2 or 24 hours after UCO. Fetuses were killed for brain histology at 7 days post‐UCO. hAEC infusion at both 2 and 24 hours had dramatic anti‐inflammatory and anti‐gliotic effects, including significantly attenuating the increase in microglia after UCO in the white and gray matter and the number of astrocytes in the white matter. Both protocols partially improved myelination, but had no effect on total or immature/mature numbers of oligodendrocytes. Neuronal survival in the hippocampus was increased by hAEC infusion at either 2 or 24 hours, whereas only hAECs at 24 hours were associated with improved neuronal survival in the striatum and thalamus. Neither protocol improved recovery of electroencephalographic (EEG) power. These data suggest that a single infusion of hAECs is anti‐inflammatory, anti‐gliotic, and neuroprotective in preterm fetal sheep when given up to 24 hours after hypoxia‐ischemia, but was associated with limited white matter protection after 7 days recovery and no improvement in the recovery of EEG power.

Inflammation, but not infection, induces EMT in human amnion epithelial cells.

A non-reversible state of epithelial to mesenchymal transition (EMT) at term accumulates proinflammatory mesenchymal cells and predisposes fetal membrane to weakening prior to delivery at term. We investigated the induction of EMT in amnion epithelial cells (AEC) in response to inflammation and infection associated with spontaneous preterm birth (SPTB). For this, membranes from SPTB were screened for EMT markers. Primary AEC in culture were treated with TNF-α (10 and 50 ng/mL) and LPS (50 and 100 ng/mL) for 72 h. Cell shape index (SI) was determined based on morphological shift (microscopy followed by ImageJ software analysis). Immunocytochemistry and Western blot assessed changes in epithelial markers (cytokeratin-18 and E-cadherin) and mesenchymal markers (vimentin and N-cadherin). Involvement of transforming growth factor beta (TGF-β) in EMT induction and EMT associated inflammation was tested using specific markers (Western blot) and by measuring MMP9 (ELISA), respectively. We report that PTB is associated with fetal membrane EMT. TNF-α produced dose- and time-dependent induction of EMT; within 24 h by 50 ng/mL and after 72 h by 10 ng/mL. AEC showed mesenchymal morphology, lower E-cadherin, higher vimentin and N-cadherin and higher MMP9 compared to control. TNF-α-induced EMT was not associated with canonical TGF-β pathway. LPS, regardless of dose or time, did not induce EMT in AEC. We conclude that PTB with intact membranes is associated with EMT. Our data suggest that inflammation, but not infection, is associated with non-canonical activation of EMT and inflammation that can predispose membrane to undergo weakening.

Human amniotic epithelial cells ameliorate kidney damage in ischemia-reperfusion mouse model of acute kidney injury

BackgroundAcute kidney injury (AKI) is a common clinical disease with complex pathophysiology and limited therapeutic choices. This prompts the need for novel therapy targeting multiple aspects of this disease. Human amnion epithelial cell (hAEC) is an ideal stem cell source. Increasing evidence suggests that exosomes may act as critical cell–cell communicators. Accordingly, we assessed the therapeutic potential of hAECs and their derived exosomes (hAECs-EXO) in ischemia reperfusion mouse model of AKI and explored the underlying mechanisms.MethodsThe hAECs were primary cultured, and hAECs-EXO were isolated and characterized. An ischemic-reperfusion injury-induced AKI (IRI-AKI) mouse model was established to mimic clinical ischemic kidney injury with different disease severity. Mouse blood creatinine level was used to assess renal function, and kidney specimens were processed to detect cell proliferation, apoptosis, and capillary density. Macrophage infiltration was analyzed by flow cytometry. hAEC-derived exosomes (hAECs-EXO) were used to treat hypoxia-reoxygenation (H/R) injured HK-2 cells and mouse bone marrow-derived macrophages to evaluate their protective effect in vitro. Furthermore, hAECs-EXO were subjected to liquid chromatography-tandem mass spectrometry for proteomic profiling.ResultsWe found that systematically administered hAECs could improve mortality and renal function in IRI-AKI mice, decrease the number of apoptotic cells, prevent peritubular capillary loss, and modulate kidney local immune response. However, hAECs showed very low kidney tissue integration. Exosomes isolated from hAECs recapitulated the renal protective effects of their source cells. In vitro, hAECs-EXO protected HK-2 cells from H/R injury-induced apoptosis and promoted bone marrow-derived macrophage polarization toward M2 phenotype. Proteomic analysis on hAECs-EXO revealed proteins involved in extracellular matrix organization, growth factor signaling pathways, cytokine production, and immunomodulation. These findings demonstrated that paracrine of exosomes might be the key mechanism of hAECs in alleviating renal ischemia reperfusion injury.ConclusionsWe reported hAECs could improve survival and ameliorate renal injury in mice with IRI-AKI. The anti-apoptotic, pro-angiogenetic, and immunomodulatory capabilities of hAECs are at least partially, through paracrine pathways. hAECs-EXO might be a promising clinical therapeutic tool, overcoming the weaknesses and risks associated with the use of native stem cells, for patients with AKI.

Human Amnion Epithelial Cells Impair T Cell Proliferation: The Role of HLA-G and HLA-E Molecules.

The immunoprivilege status characteristic of human amnion epithelial cells (hAECs) has been recently highlighted in the context of xenogenic transplantation. However, the mechanism(s) involved in such regulatory functions have been so far only partially been clarified. Here, we have analyzed the expression of HLA-Ib molecules in isolated hAEC obtained from full term placentae. Moreover, we asked whether these molecules are involved in the immunoregulatory functions of hAEC. Human amnion-derived cells expressed surface HLA-G and HLA-F at high levels, whereas the commonly expressed HLA-E molecule has been measured at a very low level or null on freshly isolated cells. HLA-Ib molecules can be expressed as membrane-bound and soluble forms, and in all hAEC batches analyzed we measured high levels of sHLA-G and sHLA-E when hAEC were maintained in culture, and such a release was time-dependent. Moreover, HLA-G was present in extracellular vesicles (EVs) released by hAEC. hAEC suppressed T cell proliferation in vitro at different hAEC:T cell ratios, as previously reported. Moreover, inhibition of T cell proliferation was partially reverted by pretreating hAEC with anti-HLA-G, anti-HLA-E and anti-β2 microglobulin, thus suggesting that HLA-G and -E molecules are involved in hAEC-mediated suppression of T cell proliferation. Finally, either large-size EV (lsEV) or small-size EV (ssEV) derived from hAEC significantly modulated T-cell proliferation. In conclusion, we have here characterized one of the mechanism(s) underlying immunomodulatory functions of hAEC, related to the expression and release of HLA-Ib molecules.

Transplantation of Human Amnion Epithelial Cells Improves Endometrial Regeneration in Rat Model of Intrauterine Adhesions.

Intrauterine adhesions (IUAs) are characterized by the injury of endometrium due to curettage and/or endometritis. The loss of functional endometrium in uterine cavity usually results in hypomenorrhea, amenorrhea, infertility, and/or recurrent pregnancy loss. Recently, stem cell transplantation has been applied to promote the endometrial regeneration. Human amnion epithelial cells (hAECs) have been shown to have stem cell characteristics. In this study, we found that PKH26-labeled hAECs were mainly distributed in the basal layer of endometrium after transplantation, and hAEC transplantation, including uterine injection and tail vein injection, could increase pregnancy rate and the number of embryos in rat model of IUAs. Moreover, hAEC transplantation was demonstrated to increase the endometrial thickness, promote the proliferation of glands and blood vessels, and decrease fibrotic areas in the endometrium. The immunohistochemical and quantitative polymerase chain reaction analysis showed the upregulated expression of growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) after hAEC transplantation; and the downregulated expression of collagen type I alpha 1 (COL1A1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-β (TGF-β), all of which are associated with the extracellular matrix (ECM) deposition after hAEC transplantation. The mRNA sequencing indicated that platelet-derived growth factor-C (PDGF-C), thrombospondin-1 (THBS1), connective tissue growth factor (CTGF), Wnt5a, and Snai2 were significantly modulated in treatment groups. These results indicate that hAEC transplantation promotes endometrial regeneration and the restoration of fertility in rat model of IUAs.

Human Amnion Epithelial Cells (AECs) Respond to the FSL-1 Lipopeptide by Engaging the NLRP7 Inflammasome

Context and Objectives: Inflammation is the leading mechanism involved in both physiological and pathological rupture of fetal membranes. Our aim was to obtain a better characterization of the inflammasome-dependent inflammation processes in these tissues, with a particular focus on the nucleotide-binding oligomerization domain (NOD)–like receptor, pyrin domain containing protein 7 (NLRP7) inflammasome.Methods: The presence of NLRP7 inflammasome actors [NLRP7, apoptosis-associated speck–like protein containing a CARD domain (ASC), and caspase-1] was confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) in human amnion and choriodecidua at the three trimesters and at term. The protein concentrations were then determined by enzyme-linked immunosorbent assay in term tissues, with or without labor. The presence of Mycoplasma salivarium and Mycoplasma fermentans in human fetal membranes was investigated using a PCR approach. Human amnion epithelial cells (AECs) were treated for 4 or 20 h with fibroblast-stimulating lipopeptide-1 (FSL-1), a M. salivarium–derived ligand. Transcripts and proteins quantity was then measured by RT–quantitative PCR and Western blotting, respectively. NLRP7 and ASC colocalization was confirmed by immunofluorescence. Western blots allowed analysis of pro–caspase-1 and gasdermin D cleavage.Results: NLRP7, ASC, and caspase-1 transcripts were expressed in both sheets of human fetal membranes during all pregnancy stages, but only ASC protein expression was increased with labor. In addition, M. salivarium and M. fermentans were detected for the first time in human fetal membranes. NLRP7 and caspase-1 transcripts, as well as NLRP7, ASC, and pro–caspase-1 protein levels, were increased in FSL-1–treated AECs. The NLRP7 inflammasome assembled around the nucleus, and pro–caspase-1 and gasdermin D were cleaved into their mature forms after FSL-1 stimulation.Conclusion: Two new mycoplasmas, M. salivarium and M. fermentans, were identified in human fetal membranes, and a lipopeptide derived from M. salivarium was found to induce NLRP7 inflammasome formation in AECs.

Stretch, scratch, and stress: Suppressors and supporters of senescence in human fetal membranes

IntroductionThroughout gestation, amnion membranes undergo mechanical and or physiological stretch, scratch, or stress which is withstood by repairing and remodeling processes to protect the growing fetus. At term, increased oxidative stress (OS) activates p38MAPK, induces senescence, and inflammation contributing to membrane dysfunction to promote labor. However, the signaling initiated by stretch and scratch is still unclear. This study compares the induction of p38MAPK mediated senescence by stretch, scratch, and stress in human amnion epithelial cells (AECs). MethodsPrimary AECs from term, not-in-labor, fetal membranes were cultured using the following conditions (N = 3); 1) CellFlex chambers with or without 20% biaxial stretch, 2) 8-well coverslips with or without scratch, and 3) cells exposed to cigarette smoke extract (CSE) inducing OS. p38MAPK (Western blot or immunocytochemistry), senescence activation, and inflammation (matrix metalloproteinases 9 [MMP9] activity-ELISA) were determined in cells exposed to various conditions. T-test and One-Way ANOVA was used to assess significance. ResultsBiological membrane extension, mimicked by 20% biaxial stretch of AEC, maintained an epithelial morphology and activated P-p38MAPK (P = 0.02) compared to the non-stretch controls, but did not induce senescence or MMP9 activation. AEC scratches were healed within 40-hrs, which included proliferation, migration, and cellular transitions aided by p38MAPK activation but not senescence. CSE induced OS increased p38MAPK (P = 0.018) activation, senescence (P = 0.019), and MMP9 (P = 0.02). ConclusionPhysiologic stretch and scratch experienced during gestation can cause p38MAPK activation without causing senescence or inflammation. This may be indicative of p38MAPK's role in tissue remodeling during pregnancy. Overwhelming OS, experienced at term, results in P-p38MAPK mediated senescence and inflammation to disrupt membrane remodeling.

Effects of Cryogenic Storage on Human Amnion Epithelial Cells.

Perinatal stem cells and epithelial cells isolated from full term amnion membrane, in particular, have attracted interest over the last decade, as a promising source of multipotent cells for cellular therapies. Human amnion epithelial cells (hAEC) have been used to treat monogenetic liver disease such as maple syrup urine disease or fibrosis of the liver in preclinical studies. In most studies xeno-transplants of hAEC were conducted without providing immunosuppression to recipients, reflecting the tolerogenic properties of hAEC. For many cell types, successful cryopreservation is critical for providing a readily available, off-the-shelf product. In this study, hAEC were isolated from full-term human placenta from 14 different donors, cryopreserved using a protocol and reagents commonly adopted for epithelial cell preservation. The cells were analyzed in terms of survival, recovery, and homogeneity, profiled for surface markers characteristic of epithelial, mesenchymal, endothelial, or hematopoietic cells. There were no significant differences observed in the percentage of cells with epithelial cell markers before and after cryopreservation. The relative proportion of stromal and hematopoietic cells was significantly reduced in hAEC preparations after cryopreservation. The expression of stem cell and immunomodulatory molecules were confirmed in the final product. Since multipotent cells are readily available from full-term placenta, this novel cell source might significantly increase the number of patients eligible to receive cellular therapies for liver and other diseases.

The effect of human amniotic epithelial cell on dendritic cell differentiation of peripheral blood monocytes: An experimental study.

Background The amniotic membrane plays an important role in maintaining a healthy pregnancy. The main population cells from amniotic membrane include human amnion epithelial cells (hAECs) which have been shown to possess immunomodulatory properties.ObjectiveThe proximity of hAECs with monocyte leads to the generation of tollerogenic dendritic cells.Materials and MethodshAECs were obtained from normal pregnancy. Peripheral blood monocytes were isolated by anti-CD14 MACS method. Co-cultures of monocytes and hAECs were established in Transwell chambers supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the absence and presence of lipopolysaccharide (LPS) to produce immature and mature DCs, respectively. Immunophenotyping of the obtained DCs was done through flow cytometry and the production of cytokines was measured by ELISA. Mixed leukocyte Reaction (MLR) was also performed for the functional assessment of DCs.Results Immunophenotyping of [hAECs - Immature DC (iDC)] and [hAECs - iDC] + LPS cells revealed that the expression of CD1a, CD80, CD86, CD40, HLA-DR, and CD83 markers showed no significant difference as compared with the control group (iDCs and mDCs alone). In the [hAECs-iDCs] + LPS cells, the percentage of CD14 cells at the ratio of 1:2.5 showed significant differences compared to the control group. The production of IL-10 and IL-12 showed no significant difference in any of the cultures as compared to the control groups. Also, co-cultured DCs did not inhibit proliferation of lymphocyte.ConclusionOur findings show that factors secreted from cultured hAECs are unable to generate of tollerogenic dendritic cells. To achieve a better understanding of other mechanisms more investigations are needed.

Ureaplasma parvum induces preterm birth by triggering immune responses in the fetus and at the maternal-fetal interface

Abstract Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. One of every four preterm births is due to intra-amniotic infection, most commonly associated with Ureaplasma species. However, a causal link between Ureaplasma species and adverse pregnancy outcomes, as well as the triggered host immune responses, has not been investigated. Molecular microbiological surveys first revealed that Ureaplasma parvum is the most common bacterium in women with intra-amniotic infection. Ultrasound-guided intra-amniotic injection of U. parvum in pregnant mice resulted in preterm birth and neonatal death. Specific qPCR revealed that U. parvum invaded specific fetal (lung, intestine, spleen, fetal membranes, and placenta) and maternal (decidua, uterus, and cervix) compartments. Consistently, U. parvum induced a strong inflammatory response in the amniotic cavity, fetal membranes, placenta, and fetal lung and spleen, as well as at the maternal-fetal interface. However, U. parvum neither invaded the non-reproductive maternal organs nor induced a maternal systemic inflammatory response. An ex vivo model of intra-amniotic infection showed that U. parvum elicits pro-inflammatory responses in human amnion epithelial cells. Lastly, the treatment of U. parvum-injected mice with clarithromycin, a clinically relevant macrolide, prevented preterm birth and neonatal death. Collectively, this investigation provides a causal link between U. parvum and adverse pregnancy outcomes, and yields insights into the host immune responses triggered by this genital mycoplasma. Importantly, these data support the use of clarithromycin in the treatment of Ureaplasma-associated intra-amniotic infection leading to preterm birth.

Human Amnion Epithelial Cells Reduce Liver Fibrosis Via Effects On Macrophages Phenotype And Liver Progenitor Cells In Murine Liver Injury

Background & Aim Background: Chronic liver disease is characterised by progressive hepatocyte injury, inflammation and accumulation of extracellular matrix. Liver progenitor cells (LPC) are bipotent cells induced during liver injury that can regenerate both hepatocytes and cholangiocytes. In addition, macrophages play a pivotal role in fibrogenesis and fibrosis resolution. Classically, macrophages are classified into M1 (pro-inflammatory macrophages) and M2 (pro-resolution macrophages). Recently, Ly6C expression has been used to identify distinct macrophage populations, Ly6Chi (inflammatory) and Ly6Clow (resolution). We are developing a therapy for chronic liver fibrosis using human amnion epithelial cells (hAECs). The choline-deficient ethionine-supplemented (CDE) diet is a non-invasive and quick method to induce liver injury and LPC proliferation. Here, we aimed to investigate the effect of hAECs on macrophages and LPC during liver injury induced by a CDE diet. Aim Methods, Results & Conclusion Methods: C57/B16J mice were commenced on a CDE diet, then 2 or 4 million hAECs were infused intraperitoneally 24h later and the CDE diet continued. Outcomes were assessed at days 1, 3, 5, 14, 21 and 42 following hAEC administrations. Sirius red staining and immunohistochemistry were used to determine fibrosis and identify LPC in liver sections. Macrophages and LPC were further identified and characterised using flow cytometery sorting and PCR. Results In CDE treated mice, hAEC significantly reduced liver fibrosis compared to untreated mice. hAEC decreased the number of proinflammatory (Ly6Chi) monocyte-derived macrophages (MDM) and increased the number of anti-inflammatory and tissue remodelling (Ly6Clow) MDM. hAEC also enhanced the expression of matrix-metalloproteinase (MMP)12 and MMP13 in Ly6CLow sorted cells. LPC numbers were reduced significantly in hAEC treated mice. Conclusion Our data confirm the potential efficacy of hAECs as an effective therapy in liver fibrosis.

Prematurity negatively impacts therapeutic effect of human amnion epithelial cells in experimental bronchopulmonary dysplasia

Background & Aim Extracellular vesicles derived from human amniotic epithelial cells (hAEC-EVs) have been previously shown to be beneficial in a preclinical model of bronchopulmonary dysplasia (BPD) when obtained from full-term, healthy pregnancies. In this study, we compare their therapeutic potential against preterm, complicated pregnancies. Methods, Results & Conclusion Methods: Lipopolysaccharide (LPS) was introduced to C57bl6 mouse fetuses intra-amniotically at E16 prior to exposure to 65% oxygen (hyperoxia) at birth. hAEC-EVs were injected intravenously on postnatal day (PND) 4. One cohort of mice was culled on either postnatal day PND7 or PND14, while another cohort were kept in hyperoxia until PND 28, following which they were recovered under normoxia conditions for a further 2 weeks for cardiovascular assessment. Results: The EVs isolated from either term or preterm hAEC had distinct cup shaped morphology with average size of 40-120nm. All hAEC-EV samples presented epithelial marker CD326, and EV markers CD9, CD63, CD81 and CD105. Term EVs had significant higher level of CD142 and CD133 compared to preterm EV. Mass spectrometry showed that all samples contained CD9, CD81, Alix and Grp94. Compared to preterm hAEC-EV, 47 proteins were significantly enriched in term hAEC-EVs and 38 proteins significantly reduced by more than 20-fold. In mouse model of BPD-like lung injury, we observed that term but not preterm hAEC-EV improved tissue-to-airspace ratio and septal crest density in a dose-dependent manner. Although both term and preterm hAEC-EV reduced the levels of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α) on PND7, the improvement of lung injury was associated with the increase of the percentage of type II alveolar cells only observed in term EV treatment group. Furthermore, only neonatal term hAEC-EV delivery reduced airway hyper-responsiveness, mitigated pulmonary hypertension and prevented right ventricle hypertrophy that associated with BPD-like lung injury until 6 weeks of age. Conclusions: Both term and preterm hAEC-EVs bore exosome characteristics based on size distribution, morphology under EM and exosomal markers, however, EV cargo and functional potency were significantly different. Term hAEC-EVs were more efficient at repairing the BPD-like lung injury compared to preterm EVs, and this impact of donor criteria should be considered when applying perinatal cells derived EV therapy for clinical use.

Systemic treatment with human amnion epithelial cells after experimental traumatic brain injury

Systemic administration of human amnion epithelial cells (hAECs) was recently shown to reduce neuropathology and improve functional recovery following ischemic stroke in both mice and marmosets. Given the significant neuropathological overlap between ischemic stroke and traumatic brain injury (TBI), we hypothesized that a similar hAEC treatment regime would also improve TBI outcomes. Male mice (12 weeks old, n ​= ​40) were given a sham injury or moderate severity TBI by controlled cortical impact. At 60 ​min post-injury, mice were given a single tail vein injection of either saline (vehicle) or 1 ​× ​106 hAECs suspended in saline. At 24 ​h post-injury, mice were assessed for locomotion and anxiety using an open field, and sensorimotor ability using a rotarod. At 48 ​h post-injury, brains were collected for analysis of immune cells via flow cytometry, or histological evaluation of lesion volume and hAEC penetration. To assess the impact of TBI and hAECs on lymphoid organs, spleen and thymus weights were determined. Treatment with hAECs did not prevent TBI-induced sensorimotor deficits at 24 ​h post-injury. hAECs were detected in the injured brain parenchyma; however, lesion volume was not altered by hAEC treatment. Robust increases in several leukocyte populations in the ipsilateral hemisphere of TBI mice were found when compared to sham mice at 48 ​h post-injury; however, hAEC treatment did not alter brain immune cell numbers. Both TBI and hAEC treatment were found to increase spleen weight. Taken together, these findings indicate that—unlike in ischemic stroke—treatment with hAEC was unable to prevent immune cell infiltration and sensorimotor deficits in the acute stages following controlled cortical impact in mice. Although further investigations are required, our data suggests that the lack of hAEC-induced neuroprotection in the current study may be explained by the differential splenic contributions to neuropathology between these brain injury models.