Human Amniotic Epithelial Cells Improve Uterine Spiral Artery Remodeling to Ameliorate Preeclampsia in a Rat Model.

Preeclampsia (PE) is a multisystem pregnancy disorder characterized by impaired remodeling of placental spiral arteries, which leads to the release of pro-inflammatory cytokines and anti-angiogenic agents. However, treatment options for PE are limited, with termination of pregnancy being the only curative option. In this work, we investigated the effects of human amniotic epithelial cells (hAECs) in PE rat model. The rats were induced with Lipopolysaccharide (LPS) on gestational day (GD) 14.5 followed by injection of hAECs and human umbilical cord mesenchymal stem cells (hUC-MSCs) 24 hours later. The hAECs treatment resulted in a reduction in blood pressure and proteinuria in the PE rat model. Futhermore, hAECs treatmentdecreased levels of pro-inflammatory cytokines, reduced inflammatory cells aggregation, and alleviated the damage to placental spiral arteries by downregulating the expression of anti-angiogenic factor and upregulating proangiogenic factor. In vitro experiments comfirmed that hAECs treatment restored the proliferation, migration, and angiogenesis of LPS-damaged human umbilical vein endothelial cells (hUVECs). Additionally, hAECs treatmenthad positive effects on fetal weight and neurological development in the PE group, with no negative effects onthe physical development or fertility of offspring rats. These results suggested that hAECs transplantation may be a novel adjuvant therapeutic strategy for PE by reducing the inflammatory andenhancing placental spiral artery angiogenesis.

The administration of human amniotic epithelial cells in premature ovarian insufficiency: From preclinical to clinical.

Premature ovarian insufficiency (POI) or premature ovarian failure (POF) is a multifactorial disorder occurring in reproductive-age women, characterized by elevated levels of follicle-stimulating hormone (FSH) and irregular or absent menstrual cycles, often accompanied by perimenopausal symptoms and infertility. While assisted reproductive technology can address the reproductive aspirations of some POI-affected women, it is hindered by issues such as exorbitant expenses, substantial risks, and poor rates of conception. Encouragingly, extensive research is exploring novel approaches to enhance fertility, particularly in the realm of stem cell therapy, showcasing both feasibility and significant potential. Human amniotic epithelial cells (hAECs) from discarded placental tissues are crucial in regenerative medicine for their pluripotency, low immunogenicity, non-tumorigenicity, accessibility, and minimal ethical concerns. Preclinical studies highlight the underlying mechanisms and therapeutic effects of hAECs in POI treatment, and current research is focusing on innovative interventions to augment hAECs' efficacy. However, despite these strides, overcoming application challenges is essential for successful clinical translation. This paper conducted a comprehensive analysis of the aforementioned issues, examining the prospects and challenges of hAECs in POI, with the aim of providing some insights for future research and clinical practice.

NETs promote ROS production to induce human amniotic epithelial cell apoptosis via ERK1/2 signaling in spontaneous preterm birth.

PROBLEM: Premature birth is a common obstetric complication but its pathogenesis is unclear. Inflammation at the maternal-fetal interface in preterm labor leads to the infiltration of neutrophils, which promotes inflammatory responses and induces the degradation of extracellular matrix and cell apoptosis, thus contributing to preterm labor. It is unclear whether neutrophil extracellular traps (NETs), a functional form of neutrophils, are involved in preterm labor. METHODS OF STUDY: After collecting amniotic membranes from research objects, we localized NETs by immunofluorescence and evaluated the expression of matrix metalloproteinase (MMP)-9 and MMP-2 by western blotting. Primary human amniotic epithelial cells (hAECs) subjected to treatment with NETs, 5-ethynyl-20-deoxyuridine cell proliferation assay, lactate dehydrogenase (LDH) assay, western blotting, and flow cytometry apoptosis assay were used to determine the effects of NETs on hAECs. We also elucidated possible mechanisms underlying the effects. RESULTS: Compared with normal term women, NETs infiltration and MMP-9 expression in the amniotic membrane from preterm women had increased. Thereafter, NETs might suppress the proliferation and promote the apoptosis of hAECs. Furthermore, after NETs treatment, the mitochondrial membrane potential was significantly decreased, ERK1/2 phosphorylation expression was upregulated and reactive oxygen species (ROS) production was increased in hAECs. Changes in cell proliferation, LDH release, and cell apoptosis level due to NETs could be reversed by ROS inhibitor or ERK phosphorylation inhibitors. CONCLUSIONS: NETs can promote the apoptosis of hAECs via ERK1/2 pathways dependent on ROS release.

A Tailored Lipid Supplement Restored Membrane Fatty Acid Composition and Ameliorates In Vitro Biological Features of Human Amniotic Epithelial Cells.

Cell culture conditions influence several biological and biochemical features of stem cells (SCs), including the membrane lipid profile, thus limiting the use of SCs for cell therapy approaches. The present study aims to investigate whether the in vitro culture may alter the membrane fatty acid signature of human Amniotic Epithelial Cells (hAECs). The analysis of the membrane fatty acid composition of hAECs cultured in basal medium showed a loss in polyunsaturated fatty acids (PUFA), in particular in omega-6 (ω-6) content, compared to freshly isolated hAECs. The addition to the basal culture medium of a chemically defined and animal-free tailored lipid supplement, namely Refeed®, partially restored the membrane fatty acid signature of hAECs. Although the amelioration of the membrane composition did not prolong hAECs culture lifespan, Refeed® influenced cell morphology, counteracted the onset of senescence, and increased the migratory capacity as well as the ability of hAECs to inhibit Peripheral Blood Mononuclear Cell (PBMC) proliferation. This study provides new information on hAEC features during culture passages and demonstrates that the maintenance of the membrane fatty acid signature preserved higher cell quality during in vitro expansion, suggesting the use of lipid supplementation for SC expansion in cell-based therapies.

Human Amniotic Epithelial Cells Secretome: Components, Bioactivity, and Challenges.

Human amniotic epithelial cells (hAECs) derived from placental tissue have received significant attention as a promising tool in regenerative medicine. Several studies demonstrated their anti-inflammatory, anti-fibrotic, and tissue repair potentials. These effects were further shown to be retained in the conditioned medium of hAECs, suggesting their paracrine nature. The concept of utilizing the hAEC-secretome has thus evolved as a therapeutic cell-free option. In this article, we review the different components and constituents of hAEC-secretome and their influence as demonstrated through experimental studies in the current literature. Studies examining the effects of conditioned medium, exosomes, and micro-RNA (miRNA) derived from hAECs are included in this review. The challenges facing the application of this cell-free approach will also be discussed based on the current evidence.

Human Amniotic Epithelial Cells and Their Derived Exosomes Protect Against Cisplatin-Induced Acute Kidney Injury Without Compromising Its Antitumor Activity in Mice.

Background: Cisplatin is a widely used chemotherapeutic drug, whereas the clinical application is greatly limited by its nephrotoxic side effect. Currently, there has been no effective treatment to prevent cisplatin-induced acute kidney injury (cisplatin-AKI). Human amniotic epithelial cells (hAECs) and their derived exosomes (EXOs) have been proven to effectively protect against ischemia reperfusion-induced AKI, yet their roles in cisplatin-AKI are still unknown. Methods: C57BL/6J mice were given two doses of cisplatin at 20 or 15 mg/kg of body weight to induce AKI with or without mortality. hAECs or EXOs were injected via tail vein 1 day after cisplatin administration. Serum and kidney tissues were collected on the fourth day after 15 mg/kg cisplatin treatment to explore the nephro-protective effects of hAECs and EXOs on cisplatin-AKI. Lung cancer xenograft model was built by subcutaneous injection of A549 cells into BALB/c nude mice to evaluate the effect of hAECs or EXOs on cisplatin chemotherapy. Results: Cisplatin nephrotoxicity was significantly attenuated by hAECs and EXOs as evidenced by reduced mortality rate and decreased serum creatinine (sCr) and reduced tubular injury score. hAECs or EXOs exerted the nephro-protective effects via suppression of TNF-α/MAPK and caspase signaling pathways. In the A549 lung cancer xenograft mouse model, administration of hAECs or EXOs did not promote tumor growth or compromise the therapeutic effects of cisplatin on tumors. Conclusion: This study is the first to demonstrate that hAECs and their derived exosomes have nephro-protective effects in cisplatin-AKI in vivo. Importantly, neither hAECs nor EXOs compromise the antitumor activity of cisplatin. These results potentially support the use of hAECs and their derived EXOs as nephro-protectors against cisplatin-induced nephrotoxicity clinically.

Effects of hyaluronic acid on differentiation of human amniotic epithelial cells and cell-replacement therapy in type 1 diabetic mice.

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the ß cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into ß cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes.

Paracrine effects of human amniotic epithelial cells protect against chemotherapy-induced ovarian damage.

BACKGROUND: Human amniotic epithelial cells (hAECs) are attractive candidates for regenerative medical therapy, with the potential to replace deficient cells and improve functional recovery after injury. Previous studies have demonstrated that transplantation of hAECs effectively alleviate chemotherapy-induced ovarian damage via inhibiting granulose cells apoptosis in animal models of premature ovarian failure/insufficiency (POF/POI). However, the underlying molecular mechanism accounting for hAECs-mediated ovarian function recovery is not fully understood. METHODS: To investigate whether hAECs-secreting cytokines act as molecular basis to attenuate chemotherapy-induced ovarian injury, hAECs or hAEC-conditioned medium (hAEC-CM) was injected into the unilateral ovary of POF/POI mouse. Follicle development was evaluated by H&E staining at 1, 2 months after hAECs or hAEC-CM treatment. In addition, we performed a cytokine array containing 507 human cytokines on hAECs-derived serum-free conditioned medium. Finally, we further investigated whether hAECs could affect chemotherapy-induced apoptosis in primary human granulosa-lutein (hGL) cells and the tube formation of human umbilical vein endothelial cells (hUVECs) via a co-culture system in vitro. RESULTS: We observed the existence of healthy and mature follicles in ovaries treated with hAECs or hAEC-CM, whereas seriously fibrosis and many atretic follicles were found in the contralateral untreated ovaries of the same mouse. To distinguish cytokines involved in the process of hAECs-restored ovarian function, hAEC-CM was analyzed with a human cytokines array. Results revealed that 109 cytokines in hAEC-CM might participate in a variety of biological processes including apoptosis, angiogenesis, cell cycle and immune response. In vitro experiments, hAECs significantly inhibited chemotherapy-induced apoptosis and activated TGF-ß/Smad signaling pathway within primary granulosa-lutein cells in paracrine manner. Furthermore, hAEC-CM was shown to promote angiogenesis in the injured ovaries and enhance the tube formation of human umbilical vein endothelial cells (hUVECs) in co-culture system. CONCLUSIONS: These findings demonstrated that paracrine might be a key pathway in the process of hAECs-mediating ovarian function recovery in animal models of premature ovarian failure/insufficiency (POF/POI).

Amnion-derived stem cell transplantation: A novel treatment for neurological disorders.

In this review, we evaluated the literature reporting the use of amniotic stem cells (ASCs) in regenerative medicine for the treatment of neurological disorders. There is an increasing amount of evidence that indicates the exacerbation of the primary injury by inflammation in neurological disorders characterized by rampant inflammation, thereby increasing damage to the central nervous system (CNS). To address this, we focus on the amnion cells' anti-inflammatory properties, which make their transplantation a promising treatment for these disorders. In addition, we offered insights into new applications of the ASC in the fields of regenerative medicine and tissue engineering.

Using human epithelial amnion cells in human de-epidermized dermis for skin regeneration.

BACKGROUND: Human amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration. OBJECTIVE: To evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin. METHODS: HAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell-cell contact and ultrastructure of BM were examined under the transmission electron microscopy. RESULTS: HAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin. CONCLUSIONS: Our studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine.

Placental stem cells.

The placenta represents a reservoir of progenitor, stem cells and epithelial cells that have been shown to differentiate into various types, including adipogenic, osteogenic, myogenic, hepatogenic, cardiac, pancreatic, endothelial, pulmonary and neurogenic lineages. This review focuses on the properties of placenta-derived cells, and it evaluates their current therapeutic applications in regenerative medicine and cell transplantations. Ongoing clinical and preclinical studies are investigating the safety and efficacy of the human amniotic epithelial cells (hAECs), human amniotic mesenchymal stromal cells (hAMSCs) and chorionic mesenchymal stromal cells (hCMSCs). The establishment of biobanks for placental stem cells will enable the translation of scientific research into the clinic. The advantage of the placenta as a cellular source is that it contains different cell lineages, such as the haematopoietic lineage that originates from the chorion, allantois and yolk sac, and the mesenchymal lineage that originates from the chorion and amnion. In this review, we address advances in placental stem cell characterization, and we explore their possible uses in cell therapy.