Human amnion mesenchymal stem cells promote endometrial repair via paracrine, preferentially than transdifferentiation.

BACKGROUND: Intrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA. METHODS: Human amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-ß1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection. RESULTS: qPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC. CONCLUSIONS: Our data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.

IUA is the crucial cause of infertility in women of childbearing age, and no satisfactory treatment measures have been found in the clinic. hAMSCs can effectively treat intrauterine adhesions through paracrine and transdifferentiation mechanisms. This study confirmed in vitro and in vivo that amniotic mesenchymal stem cells preferentially inhibited endometrial fibrosis and promoted epithelial repair through paracrine, thus effectively treating intrauterine adhesions. The level of fibrosis marker proteins in IUA-THESCs decreased significantly after co-culturing with hAMSCs for 2 days in vitro. However, the level of epithelial marker proteins in hAMSCs increased significantly, requiring at least 6 days of co-culture. hAMSCs-CM had the same efficacy as hAMSCs in inhibiting fibrosis and promoting endometrial repair in IUA rats, supporting the idea that hAMSCs promoted endometrial remodeling through paracrine in vivo. In addition, GFP-labeled hAMSCs continuously colonized the endometrial stroma instead of the epithelium and gradually underwent apoptosis. These findings prove that hAMSCs ameliorate endometrial fibrosis of IUA via paracrine, preferentially than transdifferentiation, providing the latest insights into the precision treatment of IUA with hAMSCs and a theoretical basis for promoting the "cell-free therapy" of MSCs.

3D-bioprinted GelMA/gelatin/amniotic membrane extract (AME) scaffold loaded with keratinocytes, fibroblasts, and endothelial cells for skin tissue engineering.

Gelatin-methacryloyl (GelMA) is a highly adaptable biomaterial extensively utilized in skin regeneration applications. However, it is frequently imperative to enhance its physical and biological qualities by including supplementary substances in its composition. The purpose of this study was to fabricate and characterize a bi-layered GelMA-gelatin scaffold using 3D bioprinting. The upper section of the scaffold was encompassed with keratinocytes to simulate the epidermis, while the lower section included fibroblasts and HUVEC cells to mimic the dermis. A further step involved the addition of amniotic membrane extract (AME) to the scaffold in order to promote angiogenesis. The incorporation of gelatin into GelMA was found to enhance its stability and mechanical qualities. While the Alamar blue test demonstrated that a high concentration of GelMA (20%) resulted in a decrease in cell viability, the live/dead cell staining revealed that incorporation of AME increased the quantity of viable HUVECs. Further, gelatin upregulated the expression of KRT10 in keratinocytes and VIM in fibroblasts. Additionally, the histological staining results demonstrated the formation of well-defined skin layers and the creation of extracellular matrix (ECM) in GelMA/gelatin hydrogels during a 14-day culture period. Our study showed that a 3D-bioprinted composite scaffold comprising GelMA, gelatin, and AME can be used to regenerate skin tissues.

A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged ß cells.

Stem-Cell-Driven Chondrogenesis: Perspectives on Amnion-Derived Cells.

Regenerative medicine harnesses stem cells' capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

Low-level mosaic trisomy 21 at amniocentesis in a pregnancy associated with cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes, perinatal progressive decrease of the trisomy 21 cell line and a favorable fetal outcome.

OBJECTIVE: We present low-level mosaic trisomy 21 at amniocentesis in a pregnancy with a favorable fetal outcome. CASE REPORT: A 38-year-old, gravida 2, para 1, woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XY,+21[4]/46,XY[34]. Prenatal ultrasound findings were normal. At 27 weeks of gestation, she was referred for genetic counseling, and the cultured amniocytes had a karyotype of 47,XY,+21[2]/46,XY[26]. Quantitative fluorescent polymerase chain reaction (QF-PCR) analysis on the DNA extracted from uncultured amniocytes and parental bloods excluded uniparental disomy (UPD) 21. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes revealed 30% (30/100 cells) mosaicism for trisomy 21. Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed the result of arr 21q11.2q22.3 × 2.25, consistent with 20%-30% mosaicism for trisomy 21. The parental karyotypes were normal. The woman was advised to continue the pregnancy, and a 3510-g phenotypically normal male baby was delivered at 39 weeks of gestation. Cytogenetic analysis of the cord blood, umbilical cord and placenta revealed the karyotypes of 47,XY,+21[1]/46,XY[39], 47,XY,+21[2]/46,XY[38] and 46,XY in 40/40 cells, respectively. When follow-up at age 1 year and 2 months, the neonate was normal in phenotype and development. The peripheral blood had a karyotype of 46,XY in 40/40 cells, and interphase FISH analysis on uncultured buccal mucosal cells showed 6.4% (7/109 cells) mosaicism for trisomy 21. CONCLUSION: Low-level mosaic trisomy 21 at amniocentesis can be associated with cytogenetic discrepancy between cultured amniocytes and uncultured amniocytes, perinatal progressive decrease of the trisomy 21 cell line and a favorable fetal outcome.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Human amniotic mesenchymal stem cells inhibit immune rejection injury from allogeneic mouse heart transplantation: A preliminary study on the microRNA expression.

BACKGROUND: Mesenchymal stem cell therapy is a new treatment for immune rejection in heart transplantation. The aim of this paper is to investigate the effect of human amniotic mesenchymal stem cells (hAMSCs) on alleviating immune rejection of allogeneic heart transplantation in mice and its possible underlying mechanism. METHODS: We injected hAMSCs into cervical ectopic heart transplantation model mice via tail vein to observe the survival time, the pathological changes of donor myocardium, and the fluorescent distribution of hAMSCs after the transplantation. MicroRNAs (miRs) with significantly differential expression were obtained by RNA sequencing and bioinformatic analysis, and a dual luciferase reporter gene assay together with real-time quantitative PCR (qRT-PCR) was performed to verify the relationship between miRs and their targeting genes. RESULTS: The intervention of hAMSCs prolonged the graft survival time and alleviated the pathological damage of the donor heart. The injected hAMSCs were distributed mainly in the liver, spleen, and kidney, only a very small portion in the donor and recipient hearts. In the allogeneic transplantation models, the expression of miR-34b-5p significantly increased after hAMSC treatment. MiR-34b-5p showed a knockdown effect on gene Fc gamma receptor 2B (FCGR2B). CONCLUSIONS: hAMSCs can reduce the immune rejection injury after allogeneic heart transplantation. This effect may be associated with the upregulation of miR-34b-5p expression to knock down its targeting gene FCGR2B.

Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-ß) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

The administration of amnion-derived multipotent cell secretome ST266 protects against necrotizing enterocolitis in mice and piglets.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily rising in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel prevention or treatment approaches. We and others have shown that NEC arises in part from exaggerated signaling via the bacterial receptor, Toll-like receptor 4 (TLR4) on the intestinal epithelium, leading to widespread intestinal inflammation and intestinal ischemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC development in mouse and piglet models. We now seek to test the hypothesis that a secretome derived from amnion-derived cells can prevent or treat NEC in preclinical models of this disease via a process involving TLR4 inhibition. In support of this hypothesis, we show that the administration of this secretome, named ST266, to mice or piglets can prevent and treat experimental NEC. The protective effects of ST266 occurred in the presence of marked TLR4 inhibition in the intestinal epithelium of cultured epithelial cells, intestinal organoids, and human intestinal samples ex vivo, independent of epidermal growth factor. Strikingly, RNA-seq analysis of the intestinal epithelium in mice reveals that the ST266 upregulates critical genes associated with gut remodeling, intestinal immunity, gut differentiation. and energy metabolism. These findings show that the amnion-derived secretome ST266 can prevent and treat NEC, suggesting the possibility of novel therapeutic approaches for patients with this devastating disease.NEW & NOTEWORTHY This work provides hope for children who develop NEC, a devastating disease of premature infants that is often fatal, by revealing that the secreted product of amniotic progenitor cells (called ST266) can prevent or treat NEC in mice, piglet, and "NEC-in-a-dish" models of this disease. Mechanistically, ST266 prevented bacterial signaling, and a detailed transcriptomic analysis revealed effects on gut differentiation, immunity, and metabolism. Thus, an amniotic secretome may offer novel approaches for NEC.

Aislación y caracterización de las células epiteliales del amnios / Isolation and characterization of amniotic ephitelial cells

RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.

SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.

Effects of Placenta-Derived Human Amniotic Epithelial Cells on the Wound Healing Process and TGF-ß Induced Scar Formation in Murine Ischemic-Reperfusion Injury Model.

BACKGROUND: Pressure ulcers (PUs), a result of ischemic reperfusion (IR) injuries, are prevalent skin problems which show refractoriness against standard therapeutic approaches. Besides, scar formation is a critical complication of ulcers that affects functionality and the skin's cosmetic aspect. The current study aimed to investigate the effects of placenta-derived human amniotic epithelial cells (hAECs), as important agents of regenerative medicine and stem cell therapy, on accelerating the healing of IR ulcers in mice. We also evaluated the effects of these cells on reducing the TGFß-induced scar formation. METHODS: Male Balb/c mice at the age of 6-8 weeks were subjected to three IR cycles. Afterward, the mice were divided into three experimental groups (n = 6 per group), including the control group, vehicle group, and hAECs treatment group. Mice of the treatment group received 100 µL of fresh hAECs 1 × 106 cell/ml suspension in PBS. Afterward, mice were assessed by histological, stereological, molecular, and western blotting techniques at 3, 7, 14, and 21 days after wounding. RESULTS: The histological and stereological results showed the most diminutive scar formation and better healing in the hAECs treated group compared to control group. Furthermore, our results demonstrated that the expression level of Col1A1 on days 3, 14, and 21 in the hAECs treated group was significantly lower than control. Additionally, injection of hAECs significantly reduced the expression level of Col3A1 on days 3, 7, and 21 while increased Col3A1 on the day 14. Otherwise, in the hAECs treated group, the expression levels of VEGFA on days 7 and 14 were higher, which showed that hAECs could promote angiogenesis and wound healing. Also, cell therapy significantly lowered the protein levels of TGF-ß1 on day 14, while the protein level of TGF-ß3 on day 14 was significantly higher. This data could demonstrate the role of hAECs in scar reduction in IR wounds. CONCLUSION: These results suggest that hAECs can promote re-epithelialization and wound closure in an animal model of PU. They also reduced scar formation during wound healing by reducing the expression of TGF-ß1/ TGF-ß3 ratio.

miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor.

Labor is a process of inflammation and hormonal changes involving both fetal and maternal compartments. MicroRNA-132-3p (miR-132-3p) has been reported to be involved in the development of inflammation-related diseases. However, little is known about its potential role in labor onset. This study aimed to explore the mechanism of miR-132-3p in amnion for labor initiation. In the mouse amnion membranes, the expression of miR-132-3p was found to increase gradually during late gestation. In human amniotic epithelial cell line (WISH), upregulation of miR-132-3p was found to increase proinflammatory cytokines and cyclooxygenase 2 (COX2) as well as prostaglandin E2 (PGE2), which was suppressed by miR-132-3p inhibitor. Dual-specificity phosphatase 9 (DUSP9) was identified as a novel target gene of miR-132-3p, which could be negatively regulated by miR-132-3p. DUSP9 was present in the mouse amnion epithelial cells, with a decrease in its abundance at 18.5 days post coitum (dpc) relative to 15.5 dpc. Silencing DUSP9 was found to facilitate the expression of proinflammatory cytokines and COX2 as well as PGE2 secretion in WISH cells, which could be attenuated by p38 inhibitor SB203580 or JNK inhibitor SP600125. Additionally, intraperitoneal injection of pregnant mice with miR-132-3p agomir not only caused preterm birth, but also promoted the abundance of COX2 as well as phosphorylated JNK and p38 levels, and decreased DUSP9 level in mouse amnion membranes. Collectively, miR-132-3p might participate in inflammation and PGE2 release via targeting DUSP9-dependent p38 and JNK signaling pathways to cause preterm birth.

Human Amnion-Derived Mesenchymal Stromal Cells: A New Potential Treatment for Carbapenem-Resistant Enterobacterales in Decompensated Cirrhosis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. METHODS: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. RESULTS: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. CONCLUSION: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.

Changes in the Transcriptome Profiles of Human Amnion-Derived Mesenchymal Stromal/Stem Cells Induced by Three-Dimensional Culture: A Potential Priming Strategy to Improve Their Properties.

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.

Human Amnion Membrane-Derived Mesenchymal Stem Cells and Conditioned Medium Can Ameliorate X-Irradiation-Induced Testicular Injury by Reducing Endoplasmic Reticulum Stress and Apoptosis.

Today, infertility affects 15% of couples and half of this rate is due to reproductive problems in men. Radiation-induced damage to the testicles causes sterility depending on the dose. Radiation causes endoplasmic reticulum (ER) stress and ER stress induces apoptosis. In this study, the effect of human amniotic membrane-derived mesenchymal stem cells (hAMSCs) and conditioned medium (hAMSCs-CM) on testicular damage induced by ionizing radiation is aimed to be elucidated through ER stress and apoptosis mechanisms. Six gray scrotal irradiation was used to create a testicular injury model. hAMSCs isolated and characterized with immunofluorescence and flow cytometry, while 2.5 × 105 hAMSCs were transplanted into testis and hAMSCs-CM was applied. Fertility assessment was performed. Expressions of ER stress markers GRP78, Ire1, Chop and Caspase-12, and Caspase-3 were determined. TUNEL was performed. Serum FSH, LH, and testosterone were measured. After hAMSC transplantation and administration of hAMSCs-CM, offsprings were obtained. Seminiferous tubule diameter and seminiferous epithelial height increased. The expression of GRP78, IRE1α, CHOP, Caspase-12, and Caspase-3 decreased. Percentages of tunel positive cells decreased. While FSH and LH levels decreased, testosterone increased. After irradiation, both hAMSCs transplantation and paracrine activity of hAMSCs may have a role in reducing ER stress by suppressing the UPR response. Decrease in FSH and LH and increase in testosterone level after MSCs transplantation may have contributed to the improvement of spermatogenesis. Thus, it can be said that MSCs derived from human amniotic membrane can improve ionized radiation-induced testicular damage by reducing ER stress and apoptosis.

Stalling SARS-CoV2 infection with stem cells: can regenerating perinatal tissue mesenchymal stem cells offer a multi-tiered therapeutic approach to COVID-19?

The emergence of COVID-19 has created a major health crisis across the globe. Invasion of SARS-CoV-2 into the lungs causes acute respiratory distress syndrome (ARDS) that result in the damage of lung alveolar epithelial cells. Currently, there is no standard treatment available to treat the disease and the resultant lung scarring is irreversible even after recovery. This has prompted researchers across the globe to focus on developing new therapeutics and vaccines for the treatment and prevention of COVID-19. Mesenchymal stem cells (MSCs) have emerged as an efficient drug screening platform and MSC-derived organoids has found applications in disease modeling and drug discovery. Perinatal tissue derived MSC based cell therapies have been explored in the treatment of various disease conditions including ARDS because of their enhanced regenerative and immunomodulatory properties. The multi-utility properties of MSCs have been described in this review wherein we discuss the potential use of MSC-derived lung organoids in screening of novel therapeutic compounds for COVID-19 and also in disease modeling to better understand the pathogenesis of the disease. This article also summarizes the rationale behind the development of MSC-based cell- and cell-free therapies and vaccines for COVID-19 with a focus on the current progress in this area. With the pandemic raging, an important necessity is to develop novel treatment strategies which will not only alleviate the disease symptoms but also avoid any off-target effects which could further increase post infection sequelae. Naturally occurring mesenchymal stem cells could be the magic bullet which fulfil these criteria.

In vitro cell culture of amniotic fluid keratinocytes on amniotic membrane: the ideal tissue for repairing skin ulcers.

OBJECTIVE: The amniotic fluid contains a large population of stem keratinocytes demonstrating minimal immunological rejection. Recent evidence suggests that stem cells from the amniotic fluid can be employed in the field of tissue engineering. In this work we identified precursors of the epithelial cells and expanded them in vitro. MATERIALS AND METHODS: After collecting samples of amniotic fluid and separating the cells via centrifugation, we seeded a portion of these cells in selection media to analyze the proliferation of epithelial cells. The stem cells precursors of keratinocytes were identified through specific markers. The expression of these markers was evaluated by immunofluorescence and reverse transcription polymerase chain reaction (PCR). RESULTS: The stem cells demonstrated 90% confluence, after undergoing proliferation in the selection medium for 15 days. Most of these cells tested positive for the keratinocyte-specific markers, but negative for stem cell specific markers. Of note, the identity of the keratinocytes was well established even after several subcultures. CONCLUSIONS: These results suggested that it is feasible to isolate and expand differentiated cell populations in the amniotic fluid from precursor cells. Furthermore, amniotic membranes can be utilized as scaffolds to grow keratinocytes, which can be potentially exploited in areas of skin ulcer transplantation and tissue engineering interventions.

Conditioned Medium of Human Amniotic Epithelial Cells Alleviates Experimental Allergic Conjunctivitis Mainly by IL-1ra and IL-10.

Allergic conjunctivitis (AC) is the most prevalent form of mucosal allergy, and the conditioned medium (CM) from mesenchymal stem cells has been reported to attenuate some allergic diseases. However, the therapeutic effects of CM from different tissue stem cells (TSC-CM) on allergic diseases have not been tested. Here, we studied the effects of topical administration of different human TSC-CM on experimental AC (EAC) mice. Only human amniotic epithelial cell-CM (AECM) significantly attenuated allergic eye symptoms and reduced the infiltration of immune cells and the levels of local inflammatory factors in the conjunctiva compared to EAC mice. In addition, AECM treatment decreased immunoglobulin E (IgE) release, histamine production, and the hyperpermeability of conjunctival vessels. Protein chip assays revealed that the levels of anti-inflammatory factors, interleukin-1 receptor antagonist (IL-1ra) and IL-10, were higher in AECM compared to other TSC-CM. Furthermore, the anti-allergic effects of AECM on EAC mice were abrogated when neutralized with IL-1ra or IL-10 antibody, and the similar phenomenon was for the activation and function of B cells and mast cells. Together, the present study demonstrated that AECM alleviates EAC symptoms by multiple anti-allergic mechanisms mainly via IL-1ra and IL-10. Such topical AECM therapy may represent a novel and feasible strategy for treating AC.