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Effects of Cryogenic Storage on Human Amnion Epithelial Cells.

ABSTRACT: 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.

SUBMITTER: Srinivasan RC

PROVIDER: S-EPMC7407665 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Effects of Cryogenic Storage on Human Amnion Epithelial Cells.

Srinivasan Raghuraman C RC Strom Stephen C SC Gramignoli Roberto R

Cells 20200715 7

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 t ...[more]

PMID: 32679793

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Project description: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.

Project description:BackgroundFetal endocrine signals are generally considered to contribute to the timing of birth and the initiation of labor. Fetal tissues under oxidative stress release inflammatory mediators that lead to sterile inflammation within the maternal-fetal interface. Importantly, these inflammatory mediators are packaged into exosomes, bioactive cell-derived extra cellular vesicles that function as vectors and transport them from the fetal side to the uterine tissues where they deposit their cargo into target cells enhancing uterine inflammatory load. This exosome-mediated signaling is a novel mechanism for fetal-maternal communication.ObjectiveThis report tested the hypothesis that oxidative stress can induce fetal amnion cells to produce exosomes, which function as a paracrine intermediary between the fetus and mother and biochemically signal readiness for parturition.Study designPrimary amnion epithelial cells were grown in normal cell culture (control) or exposed to oxidative stress conditions (induced by cigarette smoke extract). Exosomes were isolated from cell supernatant by sequential ultracentrifugation. Exosomes were quantified and characterized based on size, shape, and biochemical markers. Myometrial, decidual, and placental cells (BeWo) were treated with 2 × 105, 2 × 107, and 2 × 109 control or oxidative stress-derived amnion epithelial cell exosomes for 24 hours. Entry of amnion epithelial cell exosomes into cells was confirmed by confocal microscopy of fluorescent-labeled exosomes. The effect of amnion epithelial cell exosomes on target cell inflammatory status was determined by measuring production of interleukin-6, interleukin-8, interleukin-1β, tumor necrosis factor-α, and prostaglandin E2 by enzyme-linked immunosorbent assay and inflammatory gene transcription factor (nuclear factor-κβ) activation status by immunoblotting for phosphorylated RelA/p65. Localization of NANOG in term human myometrium and decidua obtained from women before labor and during labor was performed using immunohistochemistry. Data were analyzed by Wilcoxon-Mann-Whitney test to compare effects of exosomes from control and oxidative stress-treated amnion epithelial cells on inflammatory status of target cells.ResultsAmnion epithelial cells released ∼125 nm, cup-shaped exosomes with ∼899 and 1211 exosomes released per cell from control and oxidative stress-induced cells, respectively. Amnion epithelial cell exosomes were detected in each target cell type after treatment using confocal microscopy. Treatment with amnion epithelial cell exosomes increased secretion of interleukin-6, interleukin-8, and PGE2 and activation of NF-κβ (each P < .05) in myometrial and decidual cells. Exosome treatments had no effect on interleukin-6 and PGE2 production in BeWo cells. NANOG staining was higher in term labor myometrium and decidua compared to tissues not in labor.ConclusionIn vitro, amnion epithelial cell exosomes lead to an increased inflammatory response in maternal uterine cells whereas placental cells showed refractoriness. Fetal cell exosomes may function to signal parturition by increasing maternal gestational cell inflammation.

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Effects of Cryogenic Storage on Human Amnion Epithelial Cells.

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Effects of Cryogenic Storage on Human Amnion Epithelial Cells.

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