Search

With the increasing demand for exercise, the population of patients with ankle sprain to anterior talofibular ligament injury has the characteristics of a large base and high requirements for returning to sports, and how to promote the repair of damaged ligaments from a microscopic perspective is an urgent problem to be solved. In many studies, human amniotic mesenchymal stem cells have strong differentiation ability, and can be induced to continuously differentiate into ligament cells to achieve the purpose of repairing damaged ligaments. Human amniotic stem cells were extracted and cultured from human amniotic tissues, evaluated by cell identification and other techniques, and evaluated into ligament differentiation by toluidine blue, alizarin red, oil red O staining and detection of ligament cell differentiation, protein detection by Western blot, mRNA level by qPCR, and finally, the targeted binding relationship between miR-16a-5p and mRNA FGF2 was verified by double luciferase reporter assay. The expression of collagen type 1 (COL 1), collagen type 3 (COL3), SCX and MKX was increased by overexpression of mRNA FGF2, respectively, and miR-16a-5p had a targeted effect on FGF2 and regulated the ligamentous differentiation of human amniotic mesenchymal stem cells. We found that the regulatory effect of overexpressed mRNA FGF2 on mesenchymal stem cells could be inhibited by up-regulation of miR-16a-5p, while the knockdown of FGF2 could reverse the regulatory effect of miR-16a-5p inhibition on ligament-forming differentiation of human amniotic mesenchymal stem cells. In this study, we discovered the existence of the miR-16a-5p-FGF2 axis in human amniotic mesenchymal stem cells, and the differentiation of human amniotic mesenchymal stem cells into ligamentous cells can be regulated by regulating various links in this axis., (© 2024. The Author(s).)

Introduction: The human-derived amnion-chorion membrane (ACM) has endogenous antimicrobial properties, which are important for preventing the colonization and survival of oral bacteria on exposed membranes. This project aimed to decipher the underlying mechanism by identifying the components of ACM that confer antibacterial properties. In addition, the antimicrobial efficacy of these identified components on oral bacteria was assessed., Methods: Four antimicrobial proteins, histone H2A/H2B, cathelicidin LL-37, lactoferrin, and lysozyme, were identified via mass spectrometry in ACM. These proteins were then assessed for their efficacy in killing Streptococcus gordonii Challis. Log-phased bacterial cells were cultured with the commercially available proteins that were identified in ACM, either individually or in combination, at different concentrations. After incubation for 8 or 24 hours, the bacteria were stained with a live/dead viability kit and analyzed via confocal microscopy., Results: The combination of these proteins effectively killed S. gordonii in a dose-dependent fashion after 8 or 24 hours of incubation. When each protein was tested individually, it killed S. gordonii at a much lower efficacy relative to the combinations. The synergistic effects of the antimicrobial protein combinations were also observed in both the viable cell count recovery and minimum inhibitory concentration assays., Discussion: By shedding light on the mechanisms in the ACM's antimicrobial property, this study may raise more awareness of the potential benefit of utilization of a membrane with endogenous antimicrobial properties in regeneration surgeries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Brummerhop, Lee, Weltman, Tribble, van der Hoeven, Chiu, Hong and Wang.)

Introduction: Senescence in human amniotic epithelial cells (hAECs) and increased sterile inflammation in the amniotic cavity can lead to the initiation of term labor (TL). We investigated the possible roles of hsa-miR-3928-3p and chemokine ligand 3 (CCL3) in labor initiation and the underlying molecular mechanisms., Methods: Microarray chip screening was used to analyse the differential expression of miRNAs in amniotic fluid exosomes from women in TL and term not-in-labor. The GEO and miRWalk databases were used to identify differential genes, and a dual luciferase assay was used to verify the relationship. Reverse transcription quantitative PCR (RT-qPCR) and immunofluorescence were used to determine the expression and localization of CCL3/CCR5 in fetal membranes. RT-qPCR and western blotting were used to detect the expression of CCL3/CCR5 in hAECs with hsa-miR-3928-3p knockdown/overexpression. Cell counting kit 8, flow cytometry, EdU proliferation, senescence-associated β-galactosidase, and enzyme-linked immunosorbent assays were performed to detect the impact of hsa-miR-3928-3p on hAEC function., Results: hsa-miR-3928-3p expression was downregulated in TL. CCL3 (macrophage inflammatory protein-1α) was identified as a differentially expressed target gene. hsa-miR-3928-3p targeted the 3' UTR of CCL3. Downregulation of hsa-miR-3928-3p expression increased CCL3 expression. CCL3, via its CCR5 receptor, decreased the proliferation, but increased the senescence, apoptosis rate, secretion of inflammatory factors (IL-8, TNF-α, and IL-6), and expression of senescence-associated protein p21 in hAECs., Discussion: hsa-miR-3928-3p negatively regulates CCL3, promoting hAEC senescence through the CCL3-CCR5 axis and inducing signals for labor initiation. These findings provide novel insights for labor initiation in clinical settings., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Placental-derived products have been used since the early 1900s for wound applications and have shown clinical utility in supporting wound healing. A hypothermically stored amniotic membrane (HSAM) was developed using a proprietary process to allow for the retention of the extracellular matrix (ECM), viable cells, and key proteins. To evaluate its utility, we characterized the HSAM and compared it to a native unprocessed amniotic membrane (uAM) and a dehydrated amniotic membrane (dAM), as well as assessing the functionality of the HSAM as a scaffold to promote cell growth. The HSAM, uAM, and dAM were compared using scanning electron microscopy (SEM), histology, and thickness. Scaffold durability was assessed in vitro using mechanical testing and a simulated wound fluid (SWF) model. The ability of the HSAM to act as a scaffold was evaluated using an in vitro attachment model. The HSAM showed similar structural characteristics compared to the uAM; however, the dAM was significantly more compact. There were no significant differences between the HSAM and the uAM following degradation in an SWF model. ECM- and placental-related proteins were shared between the HSAM and uAM, and the HSAM enhanced the attachment and proliferation of fibroblasts in vitro. The HSAM is substantially similar to the uAM by retaining key regulatory proteins, resisting degradation in SWF, and acting as a scaffold for cellular growth and invasion.

Background: Sex steroid hormones, primarily synthesized by gonadal somatic cells, are pivotal for sexual development and reproduction. Mice studies have shown that two transcription factors, steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1), are involved in gonadal development. However, their role in human gonadal somatic differentiation remains unclear. We therefore aimed to investigate the roles of SF-1 and WT1 in human gonadal steroidogenic cell differentiation., Methods: Using a transient lentivirus-mediated gene expression system, we assessed the effects of SF-1 and WT1 expression on the steroidogenic potential of human amniotic membrane-derived mesenchymal stem cells (hAmMSCs)., Results: SF-1 and WT1-KTS, a splice variant of WT1, played distinct roles in human steroidogenic differentiation of hAmMSCs. SF-1 induced hAmMSC differentiation into progesterone- and androgen-producing cell lineages, whereas WT1-KTS promoted hAmMSC differentiation into estrogen-producing cell lineages., Conclusion: Our findings revealed that SF-1 and WT1-KTS play important roles in human gonadal steroidogenic cell differentiation, especially during ovarian development. These findings may pave the way for future studies on human ovarian differentiation and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Miyazaki, Orisaka, Fujita, Mizutani, Yazawa and Yoshida.)

Background: Premature rupture of the membranes (PROM) is a key cause of preterm birth and represents a major cause of neonatal mortality and morbidity. Natural products N-acetyl-d-galactosamine (GalNAc), which are basic building blocks of important polysaccharides in biological cells or tissues, such as chitin, glycoproteins, and glycolipids, may improve possible effects of wound healing., Methods: An in vitro inflammation and oxidative stress model was constructed using tumor necrosis-α (TNF-α) and lipopolysaccharide (LPS) action on WISH cells. Human amniotic epithelial cells (hAECs) were primarily cultured by digestion to construct a wound model. The effects of GalNAc on anti-inflammatory and anti-oxidative stress, migration and proliferation, epithelial-mesenchymal transition (EMT), glycosaminoglycan (GAG)/hyaluronic acid (HA) production, and protein kinase B (Akt) pathway in hAECs and WISH cells were analyzed using the DCFH-DA fluorescent probe, ELISA, CCK-8, scratch, transwell migration, and western blot to determine the mechanism by which GalNAc promotes amniotic wound healing., Results: GalNAc decreased IL-6 expression in TNF-α-stimulated WISH cells and ROS expression in LPS-stimulated WISH cells (P < 0.05). GalNAc promoted the expression of Gal-1 and Gal-3 with anti-inflammatory and anti-oxidative stress effects. GalNAc promoted the migration of hAECs (50% vs. 80%) and WISH cells through the Akt signaling pathway, EMT reached the point of promoting fetal membrane healing, and GalNAc did not affect the activity of hAECs and WISH cells (P > 0.05). GalNAc upregulated the expression of sGAG in WISH cells (P < 0.05) but did not affect HA levels (P > 0.05)., Conclusions: GalNAc might be a potential target for the prevention and treatment of PROM through the galectin pathway, including (i) inflammation; (ii) epithelial-mesenchymal transition; (iii) proliferation and migration; and (iv) regression, remodeling, and healing., (© 2024. The Author(s).)

Introduction: Fetal membrane inflammation is an integral event of parturition. However, excessive pro-inflammatory cytokines can impose threats to the fetus. Coincidentally, the fetal membranes express abundant 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which generates biologically active cortisol to promote labor through induction of prostaglandin synthesis. Given the well-recognized anti-inflammatory actions of glucocorticoids, we hypothesized that cortisol regenerated in the fetal membranes might be engaged in restraining fetus-hazardous pro-inflammatory cytokine production for the safety of the fetus, while reserving pro-labor effect on prostaglandin synthesis to ensure safe delivery of the fetus., Methods: The hypothesis was examined in human amnion tissue and cultured primary human amnion fibroblasts as well as a mouse model., Results: 11β-HSD1 was significantly increased in the human amnion in infection-induced preterm birth. Studies in human amnion fibroblasts showed that lipopolysaccharide (LPS) induced 11β-HSD1 expression synergistically with cortisol. Cortisol completely blocked NF-κB-mediated pro-inflammatory cytokine expression by LPS, but STAT3-mediated cyclooxygenase 2 expression, a crucial prostaglandin synthetic enzyme, remained. Further studies in pregnant mice showed that corticosterone did not delay LPS-induced preterm birth, but alleviated LPS-induced fetal organ damages, along with increased 11β-HSD1, cyclooxygenase 2, and decreased pro-inflammatory cytokine in the fetal membranes., Discussion: There is a feed-forward cortisol regeneration in the fetal membranes in infection, and cortisol regenerated restrains pro-inflammatory cytokine expression, while reserves pro-labor effect on prostaglandin synthesis. This dual role of cortisol regeneration can prevent excessive pro-inflammatory cytokine production, while ensure in-time delivery for the safety of the fetus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ling, Zhou, Zhang, Lei, Li, Lu, Wang, Sun and Ying.)

Purpose: Pterygium is an ocular surface disease characterized by the invasion of fibrovascular tissue from the bulbar conjunctiva to the cornea and is associated with abnormal tear function caused by changes in tear composition and osmolarity. In this study, the effect of two different surgical techniques to remove primary pterygium: conjunctival autograft surgery (CAG) and amniotic membrane transplantation (AMT), on changes in MUC2 and MUC5AC tear mucins concentration were evaluated., Methods: Forty-four patients (>18 years old) with primary unilateral pterygium (> 1.0 mm long, measured from the limbus to the apex on the cornea) were randomly enrolled, and assigned to the AMT or CAG group by using the permuted block technique. Patients with systemic inflammatory diseases or other eye comorbidities were excluded from the study. Tear break-up time (TBUT) and best-corrected visual acuity (BCVA) assessments were performed before surgery and at 1, 3, and 6 months after surgery. Tears were collected concurrently with the clinical evaluations, and MUC2 and MUC5AC concentrations were subsequently measured by means of ELISA., Results: At 6 months after CAG or AMT, TBUT and BCVA were significantly lower (P < 0.05) in comparison with the baseline values in the study subjects. The tear mucin concentrations of both MUC2 and MUC5AC were significantly higher (P < 0.0001) in patients with pterygium before any surgical procedure than in healthy individuals. The concentration of MUC2 increased at 1 and 3 months after CAG surgery and decreased at 6 months; however, the MUC2 concentration decreased on the AMT group in all time point measurements. Interestingly, the MUC5AC concentration significantly increased at 1 month after AMT or CAG and then decreased at 3 and 6 months after surgery. Finally, an inverse correlation was found between both MUC2 and MUC5AC tear mucins concentration and the TBUT., Conclusions: These results suggest that pterygium excision via both CAG or AMT changes the concentrations of the tear mucins MUC2 and MUC5AC during the evaluated times, and these changes could affect tear film stability and clinical recovery after pterygium treatment., Translational Relevance: The tear film stability during pterygium excision was evaluated to determine adequate treatments.

Previous studies showed that the bladder extracellular matrix (B-ECM) could increase the differentiation efficiency of mesenchymal cells into smooth muscle cells (SMC). This study investigates the potential of human amniotic membrane-derived hydrogel (HAM-hydrogel) as an alternative to xenogeneic B-ECM for the myogenic differentiation of the rabbit adipose tissue-derived MSC (AD-MSC). Decellularized human amniotic membrane (HAM) and sheep urinary bladder (SUB) were utilized to create pre-gel solutions for hydrogel formation. Rabbit AD-MSCs were cultured on SUB-hydrogel or HAM-hydrogel-coated plates supplemented with differentiation media containing myogenic growth factors (PDGF-BB and TGF-β1). An uncoated plate served as the control. After 2 weeks, real-time qPCR, immunocytochemistry, flow cytometry, and western blot were employed to assess the expression of SMC-specific markers (MHC and α-SMA) at both protein and mRNA levels. Our decellularization protocol efficiently removed cell nuclei from the bladder and amniotic tissues, preserving key ECM components (collagen, mucopolysaccharides, and elastin) within the hydrogels. Compared to the control, the hydrogel-coated groups exhibited significantly upregulated expression of SMC markers (p ≤ .05). These findings suggest HAM-hydrogel as a promising xenogeneic-free alternative for bladder tissue engineering, potentially overcoming limitations associated with ethical concerns and contamination risks of xenogeneic materials., (© 2024 Federation of American Societies for Experimental Biology.)

During pregnancy, two fetomaternal interfaces, the placenta-decidua basalis and the fetal membrane-decidua parietals, allow for fetal growth and maturation and fetal-maternal crosstalk, and protect the fetus from infectious and inflammatory signaling that could lead to adverse pregnancy outcomes. While the placenta has been studied extensively, the fetal membranes have been understudied, even though they play critical roles in pregnancy maintenance and the initiation of term or preterm parturition. Fetal membrane dysfunction has been associated with spontaneous preterm birth (PTB, < 37 weeks gestation) and preterm prelabor rupture of the membranes (PPROM), which is a disease of the fetal membranes. However, it is unknown how the individual layers of the fetal membrane decidual interface (the amnion epithelium [AEC], the amnion mesenchyme [AMC], the chorion [CTC], and the decidua [DEC]) contribute to these pregnancy outcomes. In this study, we used a single-cell transcriptomics approach to unravel the transcriptomics network at spatial levels to discern the contributions of each layer of the fetal membranes and the adjoining maternal decidua during the following conditions: scheduled caesarian section (term not in labor [TNIL]; n = 4), vaginal term in labor (TIL; n = 3), preterm labor with and without rupture of membranes (PPROM; n = 3; and PTB; n = 3). The data included 18,815 genes from 13 patients (including TIL, PTB, PPROM, and TNIL) expressed across the four layers. After quality control, there were 11,921 genes and 44 samples. The data were processed by two pipelines: one by hierarchical clustering the combined cases and the other to evaluate heterogeneity within the cases. Our visual analytical approach revealed spatially recognized differentially expressed genes that aligned with four gene clusters. Cluster 1 genes were present predominantly in DECs and Cluster 3 centered around CTC genes in all labor phenotypes. Cluster 2 genes were predominantly found in AECs in PPROM and PTB, while Cluster 4 contained AMC and CTC genes identified in term labor cases. We identified the top 10 differentially expressed genes and their connected pathways (kinase activation, NF-κB, inflammation, cytoskeletal remodeling, and hormone regulation) per cluster in each tissue layer. An in-depth understanding of the involvement of each system and cell layer may help provide targeted and tailored interventions to reduce the risk of PTB., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Richardson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes., Competing Interests: Funding The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: Ornella Parolini reports financial support was provided by PRIN 2017 program of Italian Ministry of Research and University (MIUR, Grant No. 2017RSAFK7). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.

Amniotic membrane (AM) is an attractive source for bone tissue engineering because of its low immunogenicity, contains biomolecules and proteins, and osteogenic differentiation properties. Hydroxyapatite is widely used as bone scaffolds due to its biocompatibility and bioactivity properties. The aim of this study is to design and fabricate scaffold based on hydroxyapatite-coated decellularized amniotic membrane (DAM-HA) for bone tissue engineering purpose. So human amniotic membranes were collected from healthy donors and decellularized (DAM). Then a hydroxyapatite-coating was created by immersion in 10X SBF, under variable parameters of pH and incubation time. Hydroxyapatite-coating was characterized and the optimal sample was selected. Human adipose-derived mesenchymal stem cell behaviors were assessed on control, amniotic membrane, and coated amniotic membrane. The results of the SEM, MTT assay, and Live-Dead staining showed that DAM and DAM-HA support cell adhesion, viability and proliferation. Osteogenic differentiation was evaluated by assessment of alkaline phosphatase activity and expression of osteogenic markers. Maximum gene expression values compared to control occurred in 14 days for alkalin phosphatase, while the highest values for osteocalcin and osteopontin in 21 days. These gene expression values in DAM and DAM-HA for alkalin phosphatase is 6.41 and 8.47, for osteocalcin is 3.95 and 5.94 and for osteopontin is 5.59 and 9.9 respectively. The results of this study indicated DAM supports the survival and growth of stem cells. Also, addition of hydroxyapatite component to DAM promotes osteogenic differentiation while maintaining viability. Therefore, hydroxyapatite-coated decellularized amniotic membrane can be a promising choice for bone tissue engineering applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Premature rupture of membranes (PROM) is defined as rupture of fetal membranes before the onset of labor. Prolactin (PRL) is secreted by decidual membranes and accumulated significantly in the amniotic fluid during pregnancy. PRL could ameliorate inflammation and collagen degradation in fetal membranes. However, the role of PRL in amniotic membrane is not well characterized. We isolated human amniotic epithelial stem cells (hAESCs) from human fetal membranes to study the effect of PRL on proliferation, migration, and antioxidative stress. Amniotic pore culture technique (APCT) model was constructed to evaluate the tissue regeneration effect in vitro. The potential targets and pathways of PRL acting in amnion via integrated bioinformatic methods. PRL had a dose-dependent effect on hAESCs in vitro. PRL (500 ng/mL) significantly improved the viability of hAESCs and inhibited cell apoptosis, related to the upregulation of CCN2 expression and downregulation of Bax, Caspase 3, and Caspase 8. PRL accelerated migration process in hAESCs via downregulation of MMP2, MMP3, and MMP9. PRL attenuated the cellular damage and mitochondrial dysfunction induced by hydrogen peroxide in hAESCs. PRL accelerated the healing process in the APCT model significantly. The top 10 specific targets (IGF1R, SIRT1, MAP2K1, CASP8, MAPK14, MCL1, NFKB1, HIF1A, MTOR, and HSP90AA1) and signaling pathways (such as HIF signaling pathway) were selected using an integrated bioinformatics approach. PRL improves the viability and antioxidative stress function of hAESCs and the regeneration of ruptured amniotic membranes in vitro. Thus, PRL has great therapeutic potential for prevention and treatment of ruptured membranes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that bone morphogenetic protein (BMP) signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hr after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification., Competing Interests: NS, JW, AC, LJ, LT, XM, SR, JS, TG, CL, KT No competing interests declared, (© 2023, Sekulovski et al.)

Pathological fibrosis is a significant complication of surgical procedures resulting from the accumulation of excess collagen at the site of repair which can compromise the tissue architecture and severely impede the function of the affected tissue. Few prophylactic treatments exist to counteract this process; however, the use of amniotic membrane allografts has demonstrated promising clinical outcomes. This study aimed to identify the underlying mechanism of action by utilizing relevant models that accurately represent the pathophysiology of the disease state. This study employed a pro-fibrotic in vitro system using TGFβ1 stimulation and macromolecular crowding techniques to evaluate the mechanism by which amniotic membrane allografts regulate collagen biosynthesis and deposition. Following treatment with dehydrated human amnion chorion membrane (DHACM), subsequent RNA sequencing and functional enrichment with Reactome pathway analysis indicated that amniotic membranes are indeed capable of regulating genes associated with the composition and function of the extracellular matrix. Furthermore, macromolecular crowding was used in vitro to expand the evaluation to include both the effects of DHACM and a lyophilized human amnion/chorion membrane (LHACM). DHACM and LHACM regulate the TGFβ pathway and myofibroblast differentiation. Additionally, both DHACM and LHACM modulate the production, secretion, and deposition of collagen type I, a primary target for pathological fibrosis. These observations support the hypothesis that amniotic membranes may interrupt pathological fibrosis by regulating collagen biosynthesis and associated pathways., (© 2024. The Author(s).)

The dental implant surface plays a crucial role in osseointegration. The topography and physicochemical properties will affect the cellular functions. In this research, four distinct titanium surfaces have been studied: machined acting (MACH), acid etched (AE), grit blasting (GBLAST), and a combination of grit blasting and subsequent acid etching (GBLAST + AE). Human amniotic mesenchymal (hAMSCs) and epithelial stem cells (hAECs) isolated from the amniotic membrane have attractive stem-cell properties. They were cultured on titanium surfaces to analyze their impact on biological behavior. The surface roughness, microhardness, wettability, and surface energy were analyzed using interferometric microscopy, Vickers indentation, and drop-sessile techniques. The GBLAST and GBLAST + AE surfaces showed higher roughness, reduced hydrophilicity, and lower surface energy with significant differences. Increased microhardness values for GBLAST and GBLAST + AE implants were attributed to surface compression. Cell viability was higher for hAMSCs, particularly on GBLAST and GBLAST + AE surfaces. Alkaline phosphatase activity enhanced in hAMSCs cultured on GBLAST and GBLAST + AE surfaces, while hAECs showed no mineralization signals. Osteogenic gene expression was upregulated in hAMSCs on GBLAST surfaces. Moreover, α2 and β1 integrin expression enhanced in hAMSCs, suggesting a surface-integrin interaction. Consequently, hAMSCs would tend toward osteoblastic differentiation on grit-blasted surfaces conducive to osseointegration, a phenomenon not observed in hAECs.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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-β) 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., (© 2024 Wiley Periodicals LLC.)

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., (© 2024. The Author(s).)

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged β 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 β cells., (© 2024 John Wiley & Sons Ltd.)

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-β1) 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., (© 2024. The Author(s).)

Danger-associated molecular patterns (DAMPs) are elevated within the amniotic cavity, and their increases correlate with advancing gestational age, chorioamnionitis, and labor. Although the specific triggers for their release in utero remain unclear, it is thought that they may contribute to the initiation of parturition by influencing cellular stress mechanisms that make the fetal membranes (FMs) more susceptible to rupture. DAMPs induce inflammation in many different tissue types. Indeed, they precipitate the subsequent release of several proinflammatory cytokines that are known to be key for the weakening of FMs. Previously, we have shown that in vitro stretch of human amnion epithelial cells (hAECs) induces a cellular stress response that increases high-mobility group box-1 (HMGB1) secretion. We have also shown that cell-free fetal DNA (cffDNA) induces a cytokine response in FM explants that is fetal sex-specific. Therefore, the aim of this work was to further investigate the link between stretch and the DAMPs HMGB1 and cffDNA in the FM. These data show that stretch increases the level of cffDNA released from hAECs. It also confirms the importance of the sex of the fetus by demonstrating that female cffDNA induced more cellular stress than male fetuses. Our data treating hAECs and human amnion mesenchymal cells with HMGB1 show that it has a differential effect on the ability of the cells of the amnion to upregulate the proinflammatory cytokines and propagate a proinflammatory signal through the FM that may weaken it. Finally, our data show that sulforaphane (SFN), a potent activator of Nrf2, is able to mitigate the proinflammatory effects of stretch by decreasing the levels of HMGB1 release and ROS generation after stretch and modulating the increase of key cytokines after cell stress. HMGB1 and cffDNA are two of the few DAMPs that are known to induce cytokine release and matrix metalloproteinase (MMP) activation in the FMs; thus, these data support the general thesis that they can function as potential central players in the normal mechanisms of FM weakening during the normal distension of this tissue at the end of a normal pregnancy.

Purpose: The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties., Methods: Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM)., Results: The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3 days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM., Conclusions: These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations., Translational Relevance: The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.

Human amniotic membranes (hAMs) are widely used as wound management biomaterials, especially as grafts for corneal reconstruction due to the structure of the extracellular matrix and excellent biological properties. However, their fragile nature and rapid degradation rate hinder widespread clinical use. In this work, we engineered a novel self-powered electronic dress (E-dress), combining the beneficial properties of an amniotic membrane and a flexible electrical electrode to enhance wound healing. The E-dress displayed a sustained discharge capacity, leading to increased epidermal growth factor (EGF) release from amniotic mesenchymal interstitial stem cells. Live/dead staining, CCK-8, and scratch-wound-closure assays were performed in vitro . Compared with amniotic membrane treatment alone, the E-dress promoted cell proliferation and migration of mouse fibroblast cells and lower cytotoxicity. In a mouse full-skin defect model, the E-dress achieved significantly accelerated wound closure. Histological analysis revealed that E-dress treatment promoted epithelialization and neovascularization in mouse skin. The E-dress exhibited a desirable flexibility that aligned with tissue organization and displayed maximum bioactivity within a short period to overcome rapid degradation, implying great potential for clinical applications.

Objective: The objective of this study was to assess the characterization of human acellular amniotic membrane (HAAM) using various decellularization methods and their impact on the proliferation and differentiation of human dental pulp stem cells (DPSCs). The goal was to identify scaffold materials that are better suited for pulp regeneration., Methods: Six different decellularization methods were used to generate the amniotic membranes. The characteristics of these scaffolds were examined through hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM), and immunohistofluorescence staining (IHF). The DPSCs were isolated, cultured, and their capacity for multidirectional differentiation was verified. The third generation (P3) DPSCs, were then combined with HAAM to form the decellularized amniotic scaffold-dental pulp stem cell complex (HAAM-DPSCs complex). Subsequently, the osteogenic capacity of the HAAM-DPSCs complex was evaluated using CCK8 assay, live-dead cell staining, alizarin red and alkaline phosphatase staining, and real-time quantitative PCR (RT-PCR)., Results: Out of the assessed decellularization methods, the freeze-thaw + DNase method and the use of ionic detergent (CHAPS) showed minimal changes in structure after decellularization, making it the most effective method. The HAAM-DPSCs complexes produced using this method demonstrated enhanced biological properties, as indicated by CCK8, alizarin red, alkaline phosphatase staining, and RT-PCR., Conclusion: The HAAM prepared using the freeze-thaw + DNase method and CHAPS methods exhibited improved surface characteristics and significantly enhanced the proliferation and differentiation capacity of DPSCs when applied to them. The findings, therefore demonstrate the capacity for enhanced pulp regeneration therapy., (© 2024. The Author(s).)

Scaffolds can be introduced as a source of tissue in reconstructive surgery and can help to improve wound healing. Amniotic membranes (AMs) as scaffolds for tissue engineering have emerged as promising biomaterials for surgical reconstruction due to their regenerative capacity, biocompatibility, gradual degradability, and availability. They also promote fetal-like scarless healing and provide a bioactive matrix that stimulates cell adhesion, migration, and proliferation. The aim of this study was to create a tissue-engineered AM-based implant for the repair of vesicovaginal fistula (VVF), a defect between the bladder and vagina caused by prolonged obstructed labor. Layers of AMs (with or without cross-linking) and electrospun poly-4-hydroxybutyrate (P4HB) (a synthetic, degradable polymer) scaffold were joined together by fibrin glue to produce a multilayer scaffold. Human vaginal fibroblasts were seeded on the different constructs and cultured for 28 days. Cell proliferation, cell morphology, collagen deposition, and metabolism measured by matrix metalloproteinase (MMP) activity were evaluated. Vaginal fibroblasts proliferated and were metabolically active on the different constructs, producing a distributed layer of collagen and proMMP-2. Cell proliferation and the amount of produced collagen were similar across different groups, indicating that the different AM-based constructs support vaginal fibroblast function. Cell morphology and collagen images showed slightly better alignment and organization on the un-cross-linked constructs compared to the cross-linked constructs. It was concluded that the regenerative capacity of AM does not seem to be affected by mechanical reinforcement with cross-linking or the addition of P4HB and fibrin glue. An AM-based implant for surgical repair of internal organs requiring load-bearing functionality can be directly translated to other types of surgical reconstruction of internal organs.

Hyalectan cleavage may play an important role in extracellular matrix remodeling. However, the proteolytic enzyme responsible for hyalectan degradation for fetal membrane rupture at parturition remains unknown. Here, we reveal that versican (VCAN) is the major hyalectan in the amnion, where its cleavage increases at parturition with spontaneous rupture of membrane. We further reveal that ADAMTS4 is a crucial proteolytic enzyme for VCAN cleavage in the amnion. Inflammatory factors may enhance VCAN cleavage by inducing ADAMTS4 expression and inhibiting ADAMTS4 endocytosis in amnion fibroblasts. In turn, versikine, the VCAN cleavage product, induces inflammatory factors in amnion fibroblasts, thereby forming a feedforward loop between inflammation and VCAN degradation. Mouse studies show that intra-amniotic injection of ADAMTS4 induces preterm birth along with increased VCAN degradation and proinflammatory factors abundance in the fetal membranes. Conclusively, there is enhanced VCAN cleavage by ADAMTS4 in the amnion at parturition, which can be reenforced by inflammation., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The use of fresh amniotic membrane (AM) is not a viable option, as it has many disadvantages. Preserving the AM reduces the risk of cross-infection and maintains its effectiveness for a long time. In order to maximize the therapeutic effects of the AM, the basic need is to preserve its vitality and the bioactive molecules it contains. However, the effect of preservation procedures on cell viability and growth factors is a still matter of debate. Optimum preservation method is expected to be cost-effective, easily-accessible, and most importantly, to preserve the effectiveness of the tissue for the longest time. However, each preservation technique has its advantages and disadvantages over the other, and each one compromises the vitality and bioactive molecules of the tissue to some extent. Therefore, the best method of preservation is still controversial, and the question of 'how to preserve the AM best?' has not yet been definitively answered., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Group B streptococcus (GBS) infection is a significant public health concern associated with adverse pregnancy complications and increased neonatal mortality and morbidity. However, the mechanisms underlying the impact of GBS on the fetal membrane, the first line of defense against pathogens, are not fully understood. Here, we propose that GBS induces senescence and inflammatory factors (IL-6 and IL-8) in the fetal membrane through interleukin-1 (IL-1). Utilizing the existing transcriptomic data on GBS-exposed human fetal membrane, we showed that GBS affects senescence-related pathways and genes. Next, we treated primary amnion epithelial cells with conditioned medium from the choriodecidual layer of human fetal membrane exposed to GBS (GBS collected choriodecidual [CD] conditioned medium) in the absence or presence of an IL-1 receptor antagonist (IL-1Ra). GBS CD conditioned medium significantly increased β-galactosidase activity, IL-6 and IL-8 release from the amnion epithelial cells. Cotreatment with IL1Ra reduced GBS-induced β-galactosidase activity and IL-6 and IL-8 secretion. Direct treatment with IL-1α or IL-1β confirmed the role of IL-1 signaling in the regulation of senescence in the fetal membrane. We further showed that GBS CD conditioned medium and IL-1 decreased cell proliferation in amnion epithelial cells. In summary, for the first time, we demonstrate GBS-induced senescence in the fetal membrane and present evidence of IL-1 pathway signaling between the choriodecidua and amnion layer of fetal membrane in a paracrine manner. Further studies will be warranted to understand the pathogenesis of adverse pregnancy outcomes associated with GBS infection and develop therapeutic interventions to mitigate these complications., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Premature rupture of membranes (PROM) is a common pregnancy disorder that is closely associated with structural weakening of fetal membranes. Studies have found that formyl peptide receptor 1 (FPR1) activates inflammatory pathways and amniotic epithelialmesenchymal transition (EMT), stimulates collagen degradation, and leads to membrane weakening and membrane rupture. The purpose of this study was to investigate the anti-inflammatory and EMT inhibitory effects of FPR1 antagonist (BOC-MLF) to provide a basis for clinical prevention of PROM., Methods: The relationship between PROM, FPR1, and EMT was analyzed in human fetal membrane tissue and plasma samples using Western blotting, PCR, Masson staining, and ELISA assays. Lipopolysaccharide (LPS) was used to establish a fetal membrane inflammation model in pregnant rats, and BOC-MLF was used to treat the LPS rat model. We detected interleukin (IL)-6 in blood from the rat hearts to determine whether the inflammatory model was successful and whether the anti-inflammatory treatment was effective. We used electron microscopy to analyze the structure and collagen expression of rat fetal membrane., Results: Western blotting, PCR and Masson staining indicated that the expression of FPR1 was significantly increased, the expression of collagen was decreased, and EMT appeared in PROM. The rat model indicated that LPS caused the collapse of fetal membrane epithelial cells, increased intercellular gaps, and decreased collagen. BOC-MLF promoted an increase in fetal membrane collagen, inhibited EMT, and reduced the weakening of fetal membranes., Conclusion: The expression of FPR1 in the fetal membrane of PROM was significantly increased, and EMT of the amniotic membrane was obvious. BOC-MLF can treat inflammation and inhibit amniotic EMT., (© 2024. Huazhong University of Science and Technology.)

Diabetic wounds, as one of the most important complications of diabetes, face many challenges in treatment. Herein we investigated whether decellularized human amniotic membrane (dAM) loaded with epigallocatechin-3-gallate (EGCG) could promote healing in diabetic rats. Sixty diabetic rats were randomly planned into the untreated group, dAM group, EGCG group, and dAM + EGCG group. On days 7, 14, and 21, five rats from each group were sampled for stereological, molecular, and tensiometrical assessments. Our finding revealed that the wound closure rate, the total volumes of new epidermis and dermis, the numerical densities of fibroblasts, blood vessels, collagen density as well as tensiometrical parameters of the healed wounds were considerably increased in the treated groups than in the untreated group, and these changes were more obvious in the dAM + EGCG ones. Furthermore, the expression of TGF-β, bFGF, and VEGF genes were significantly upregulated in all treated groups compared to the untreated group and were greater in the dAM + EGCG group. This is while expression of TNF-α and IL-1β, as well as cell numerical densities of neutrophils and macrophages decreased more considerably in the dAM + EGCG group in comparison to the other groups. In conclusion, it was found that using both dAM transplantation and EGCG has more effect on diabetic wound healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Tissue Viability Society / Society of Tissue Viability. Published by Elsevier Ltd. All rights reserved.)

Acute graft-versus-host disease (aGVHD) represents a fatal severe complication after allogeneic hematopoietic stem cell transplantation. As a promising cell therapeutic strategy of aGVHD, the mechanism of mesenchymal stem cells (MSC) to ameliorate aGVHD has not been fully clarified, especially in the field of intestinal homeostasis including the intestinal microbiome involved in the pathogenesis of aGVHD. The present study aimed to explore the effect of MSC on intestinal homeostasis including the intestinal barrier and intestinal microbiome and its metabolites, as well as the role of intestinal microbiome in the preventive process of hAMSCs ameliorating aGVHD. The preventive effects of human amniotic membrane-derived MSC (hAMSCs) was assessed in humanized aGVHD mouse models. Immunohistochemistry and RT-qPCR were used to evaluate intestinal barrier function. The 16S rRNA sequencing and targeted metabolomics assay were performed to observe the alternation of intestinal microbiome and the amounts of medium-chain fatty acids (MCFAs) and short-chain fatty acids (SCFAs), respectively. Flow cytometry was performed to analyze the frequencies of T immune cells. Through animal experiments, we found that hAMSCs had the potential to prevent aGVHD. HAMSCs could repair the damage of intestinal barrier structure and function, as well as improve the dysbiosis of intestinal microbiome induced by aGVHD, and meanwhile, upregulate the concentration of metabolites SCFAs, so as to reshape intestinal homeostasis. Gut microbiota depletion and fecal microbial transplantation confirmed the involvement of intestinal microbiome in the preventive process of hAMSCs on aGVHD. Our findings showed that hAMSCs prevented aGVHD in an intestinal microbiome-dependent manner, which might shed light on a new mechanism of hAMSCs inhibiting aGVHD and promote the development of new prophylaxis regimes for aGVHD prevention., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

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 embryos 1-8 . 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 cells 9-17 . 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., (© 2023. The Author(s).)

Human amniotic epithelial cells (hAECs) are novel and promising therapeutic agents for patients suffering from degenerative diseases. Studies have demonstrated that the therapeutic effects of hAECs mainly depend on their paracrine components. Currently, appropriate pretreatment is a widely confirmed strategy for enhancing the repair potential of stem cells; however, the effect of proinflammatory factor pretreatment on hAECs and their secretome is still unclear. In this study, we used the well-characterized proinflammatory factors tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) to stimulate hAECs and analyzed the effect of TNF-α and IFN-γ on hAECs, including gene expression profile, paracrine proteins, and microRNAs (miRNAs) in exosomes. Results showed that TNF-α and IFN-γ pretreatment improved the viability of hAECs but inhibited the proliferation of hAECs. TNF-α and IFN-γ pretreatment altered the gene expression profile of hAECs, and upregulated differentially expressed genes were predominantly enriched in biological adhesion, antioxidant activity, and response to IFN-beta. In addition, TNF-α and IFN-γ pretreatment enhanced the paracrine secretion of cytokines by hAECs. The upregulated differentially expressed proteins were mainly enriched in tissue remodeling proteins and cytokine-cytokine receptor. Notably, the expression of miRNAs in exosomes from hAECs was also changed by TNF-α and IFN-γ pretreatment. The target genes of upregulated exosomal miRNAs substantially contributed to the response to stimulus, metabolic pathways, and PI3K-Akt signaling pathway. Our findings improve our understanding of the biological characteristics of hAECs after proinflammatory factor pretreatment and provide novel insights to strengthen and optimize the therapeutic potential of hAECs and their secretome in regenerative medicine.

Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

Mesenchymal stem cells (MSCs) are a promising tool for treating immune disorders. However, the immunomodulatory effects of canine MSCs compared with other commercialized biologics for treating immune disorders have not been well studied. In this study we investigated the characteristics and immunomodulatory effects of canine amnion membrane (cAM)-MSCs. We examined gene expression of immune modulation and T lymphocytes from activated canine peripheral blood mononuclear cell (PBMC) proliferation. As a result, we confirmed that cAM-MSCs upregulated immune modulation genes (TGF-β1, IDO1 and PTGES2) and suppressed the proliferation capacity of T cells. Moreover, we confirmed the therapeutic effect of cAM-MSCs compared with oclacitinib (OCL), the most commonly used Janus kinase (JAK) inhibitor, as a treatment for canine atopic dermatitis (AD) using a mouse AD model. As a result, we confirmed that cAM-MSCs with PBS treatment groups (passage 4, 6 and 8) compared with PBS only (PBS) though scores of dermatologic signs, tissue pathologic changes and inflammatory cytokines were significantly reduced. In particular, cAM-MSCs were more effective than OCL in the recovery of wound dysfunction, regulation of mast cell activity and expression level of immune modulation protein. Interestingly, subcutaneous injection of cAM-MSCs induced weight recovery, but oral administration of oclacitinib induced weight loss as a side effect. In conclusion, this study suggests that cAM-MSCs can be developed as a safe canine treatment for atopic dermatitis without side effects through effective regeneration and immunomodulation., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The secretome of mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) has been extensively studied for its in vitro immunomodulatory activity as well as for the treatment of several preclinical models of immune-related disorders. The bioactive molecules within the hAMSCs secretome are capable of modulating the immune response and thus contribute to stimulating regenerative processes. At present, only a few studies have attempted to define the composition of the secretome, and several approaches, including multi-omics, are underway in an attempt to precisely define its composition and possibly identify key factors responsible for the therapeutic effect., Methods: In this study, we characterized the protein composition of the hAMSCs secretome by a filter-aided sample preparation (FASP) digestion and liquid chromatography-high resolution mass spectrometry (LC-MS) approach. Data were processed for gene ontology classification and functional protein interaction analysis by bioinformatics tools., Results: Proteomic analysis of the hAMSCs secretome resulted in the identification of 1521 total proteins, including 662 unique elements. A number of 157 elements, corresponding to 23.7%, were found as repeatedly characterizing the hAMSCs secretome, and those that resulted as significantly over-represented were involved in immunomodulation, hemostasis, development and remodeling of the extracellular matrix molecular pathways., Conclusions: Overall, our characterization enriches the landscape of hAMSCs with new information that could enable a better understanding of the mechanisms of action underlying the therapeutic efficacy of the hAMSCs secretome while also providing a basis for its therapeutic translation., (© 2023. The Author(s).)

Tissue regeneration is an essential requirement for wound healing and recovery of organs' function. It has been demonstrated that wound healing can be facilitated by activating paracrine signaling mediated by exosomes secreted from stem cells, since exosomes deliver many functional molecules including growth factors (GFs) and neurotrophic factors (NFs) effective for tissue regeneration. In this study, an exosome-rich conditioned medium (ERCM) was collected from human amniotic membrane stem cells (AMSCs) by cultivating the cells under a low oxygen tension (2% O 2 and 5% CO 2 ). The contents of GFs and NFs including keratinocyte growth factor, epidermal growth factor, fibroblast growth factor 1, transforming growth factor-β, and vascular endothelial growth factor responsible for skin regeneration were much higher (10-30 folds) in the ERCM than in normal conditioned medium (NCM). In was found that CM-DiI-labeled exosomes readily entered keratinocytes and fibroblasts, and that ERCM not only facilitated the proliferation of keratinocytes in normal condition, but also protected against H 2 O 2 cytotoxicity. In cell-migration assay, the scratch wound in keratinocyte culture dish was rapidly closed by treatment with ERCM. Such wound-healing effects of ERCM were confirmed in a rat whole skin-excision model: i.e., the wound closure was significantly accelerated, remaining minimal crusts, by topical application of ERCM solution (4 × 10 9 exosome particles/100 μL) at 4-day intervals. In the wounded skin, the deposition of collagens was enhanced by treatment with ERCM, which was supported by the increased production of collagen-1 and collagen-3. In addition, enhanced angiogenesis in ERCM-treated wounds was confirmed by increased von Willebrand factor (vWF)-positive endothelial cells. The results indicate that ERCM from AMSCs with high concentrations of GFs and NFs improves wound healing through tissue regeneration not only by facilitating keratinocyte proliferation for skin repair, but also activating fibroblasts for extracellular matrix production, in addition to the regulation of angiogenesis and scar tissue formation.

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial-mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies., (© 2023. The Author(s).)

Objectives: Premature ovarian failure (POF) is defined as the cessation of menstrual periods for at least 4-6 months before the age of 40 years, accompanied by FSH values measuring over 40 IU/L for a month. Radiation therapy, one of the cancer treatment methods, is known to accelerate ovarian aging by reducing and eliminating the number of primordial follicles in the ovarian follicle pool. Ionizing radiation has been reported to cause POF. The objective of this study is to investigate the impact of mesenchymal stem cell conditioned medium (hAMSCs-CM), which is isolated from the amniotic membrane of human placenta, on premature ovarian failure (POF) caused by whole-body irradiation. The study will focus on the ER stress and apoptosis mechanisms in the process., Study Disayn: A POF model was created by exposing rats to 7 Gy of whole-body irradiation. Serum-free hAMSCs-CM were then administered via the tail vein. Follicle count was performed on the ovaries, and immunohistochemistry was used to determine the expressions of GRP78, CHOP, IRE-1, caspase-12, caspase-9, caspase-3. TUNEL was also carried out, and levels of serum FSH, LH, E2, AMH, and oxidative stress marker 8-OHdG were measured., Results and Conclusion: The application of hAMSCs-CM has been found to have a positive impact on follicles affected by radiation. After treatment, the number of primordial, primary, secondary, and graafian follicles, which had previously decreased due to radiation, showed an increase. Furthermore, the number of atretic follicles, which had been increasing due to radiation, showed a decrease. ER is one of the targets affected by ionizing radiation. After ionizing radiation, the expressions of ER stress-related markers and apoptosis markers increased in the ovary. After hAMSCs-CM administration, the expressions of these markers and number of TUNEL-positive cells decreased. Following irradiation, anti-mullerian hormone (AMH) and estradiol (E2) levels decreased, while follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels increased. After administration of hAMSCs-CM, AMH and E2 levels increased, while FSH and LH levels decreased. Amnion membrane-derived mesenchymal stem cell conditioned medium can play a therapeutic role in ionizing radiation-induced premature ovarian failure by reducing endoplasmic reticulum stress and apoptosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Testicular torsion is considered a urological disorder that requires immediate detorsion surgery. Ischemia/reperfusion (I/R) injury after testicular torsion detorsion causes of drastic impairment of spermatogenesis and infertility. Cell-free-based approaches seem to be a promising strategy to prevent I/R injury, they have more stable biological properties, and they contain paracrine factors of mesenchymal stem cells. The purpose of this study was to evaluate the protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse sperm chromatin condensation and spermatogenesis improvement after I/R injury. hAMSCs were isolated and characterized by RT- PCR and flow cytometry, preparation of hAMSCs secreted factors was performed. Forty male mice were randomly divided into 4 groups: sham-operated, torsion detorsion, torsion detorsion+ intratesticular injection of DMEM/F-12, and torsion detorsion+ intratesticular injection of hAMSCs secreted factors. After one cycle of spermatogenesis, the mean number of germ cells, Sertoli, Leydig, myoid as well as tubular parameters, Johnson score, and spermatogenesis indexes were evaluated by H& E and PAS stainings. Sperm chromatin condensation and relative expression of c-kit and prm 1 genes were assessed by aniline blue staining and real-time PCR, respectively. The mean number of spermatogenic cells, Leydig, myoid, Sertoli, spermatogenesis parameters, Johnson score, as well as germinal epithelial height and diameters of seminiferous tubules decreased significantly after I/R injury. The thickness of basement membrane and percentage of sperm with excessive histone significantly increased, while the relative expression of c-kit and prm 1 significantly decreased in torsion detorsion group (p 0.001). hAMSCs secreted factors remarkably restored normal sperm chromatin condensation, spermatogenesis parameters and histomorphometric organization of seminiferous tubules via intratesticular injection (p 0.001). Thus, hAMSCs secreted factors may potentially salvage torsion-detorsion-induced infertility., Competing Interests: Conflict of Interests The authors declare no conflict of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Background: Inflammation of the fetal membranes is an indispensable event of labor onset at both term and preterm birth. Interleukin-33 (IL-33) is known to participate in inflammation via ST2 (suppression of tumorigenicity 2) receptor as an inflammatory cytokine. However, it remains unknown whether IL-33/ST2 axis exists in human fetal membranes to promote inflammatory reactions in parturition., Methods: The presence of IL-33 and ST2 and their changes at parturition were examined with transcriptomic sequencing, quantitative real-time polymerase chain reaction, Western blotting or immunohistochemistry in human amnion obtained from term and preterm birth with or without labor. Cultured primary human amnion fibroblasts were utilized to investigate the regulation and the role of IL-33/ST2 axis in the inflammation reactions. A mouse model was used to further study the role of IL-33 in parturition., Results: Although IL-33 and ST2 expression were detected in both epithelial and fibroblast cells of human amnion, they are more abundant in amnion fibroblasts. Their abundance increased significantly in the amnion at both term and preterm birth with labor. Lipopolysaccharide, serum amyloid A1 and IL-1β, the inflammatory mediators pertinent to labor onset, could all induce IL-33 expression through NF-κB activation in human amnion fibroblasts. In turn, via ST2 receptor, IL-33 induced the production of IL-1β, IL-6 and PGE2 in human amnion fibroblasts via the MAPKs-NF-κB pathway. Moreover, IL-33 administration induced preterm birth in mice., Conclusion: IL-33/ST2 axis is present in human amnion fibroblasts, which is activated in both term and preterm labor. Activation of this axis leads to increased production of inflammatory factors pertinent to parturition, and results in preterm birth. Targeting the IL-33/ST2 axis may have potential value in the treatment of preterm birth., (© 2023. The Author(s).)

Introduction: Disruption of fetal membranes before the onset of labor is referred to as premature rupture of membranes (PROM). Lack of maternal folic acid (FA) supplementation reportedly leads to PROM. However, there is a lack of information on the location of FA receptors in the amniotic tissue. Additionally, the regulatory role and potential molecular targets of FA in PROM in vitro have rarely been investigated., Methods: The three FA receptors (folate receptor α isoform [FRα], transporter of reduced folate [RFC], and proton-coupled folate transporter [PCFT]) in human amniotic epithelial stem cells (hAESCs) and amniotic tissue were localized using immunohistochemistry and immunocytochemistry staining. Effect and mechanism analyses of FA were performed in hAESCs and amniotic pore culture technique (APCT) models. An integrated pharmacological-bioinformatics approach was utilized to explore the potential targets of FA for the treatment of PROM., Results: The three FA receptors were widely expressed in human amniotic tissue, especially in the hAESC cytoplasm. FA stimulated the amnion regeneration in the in vitro APCT model. This mimics the PROM status, in which cystathionine-β-synthase, an FA metabolite enzyme, may play an important role. The top ten hub targets (STAT1, mTOR, PIK3R1, PTPN11, PDGFRB, ABL1, CXCR4, NFKB1, HDAC1, and HDAC2) of FA for preventing PROM were identified using an integrated pharmacological-bioinformatic approach., Discussion: FRα, RFC, and PCFT are widely expressed in human amniotic tissue and hAESCs. FA aids the healing of ruptured membrane., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

Background: Conditioned medium (CM) from human amnion-derived mesenchymal stem cells (hAMSCs) exhibits excellent pro-angiogenic capacity, and circ-100290 participates in this process. Autophagy is involved in the relevant mechanisms of angiogenesis, but it is unclear whether autophagy is related to the pro-angiogenesis effect of hAMSCs. This research sought to determine whether autophagy involved in the process of pro-angiogenesis induced by hAMSCs might be regulated by circ-100290., Methods: Upon treatment with CM from hAMSC or 3-methyladenine (3-MA), autophagosomes in human umbilical vein endothelial cells (HUVECs) were observed by transmission electron microscopy. HUVECs' angiogenic ability was evaluated by in vitro assays (transwell, wound healing, tube formation) and an in vivo Matrigel plug assay. Specific small interfering RNAs (siRNA) or inhibitors were used to regulate circ-100290 expression. Additionally, western blot and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were used to evaluate expression of the following indicators: Beclin-1, LC3-II, matrix metalloproteinase 2 (MMP2), MMP9, vascular endothelial growth factor (VEGF)-A, and endothelial nitric oxide synthase (eNOS)., Results: Incubation with hAMSC-CM increased autophagy, angiogenesis and the expressions of VEGF-A and eNOS in HUVECs, all of which were inhibited by 3-MA. Knocking down circ-100290 in hAMSC-CM-treated HUVECs reduced Beclin-1 expression and inhibited autophagy. This resulted in lower angiogenesis in the Matrigel plug assay showing that reduced angiogenesis occurred after circ-100290 silencing in hAMSC-CM-treated HUVECs., Conclusions: Circ-100290 promotes autophagy-mediated angiogenesis in hAMSC-CM-treated HUVECs., Competing Interests: All authors have completed the ICMJE uniform dis-closure form (available at: https://atm.amegroups.com/article/view/10.21037/atm-23-613/coif). The authors declare no conflict of interest., (© 2023 The Author(s). Published by Discovery Medicine.)

Glaucoma is one of the most devastating eye diseases, since the disease can develop into blindness and no effective therapeutics are available. Although the exact mechanisms and causes of glaucoma are unknown, increased intraocular pressure (IOP) has been demonstrated to be an important risk factor. Exosomes are lipid nanoparticles secreted from functional cells, including stem cells, and have been found to contain diverse functional molecules that control body function, inhibit inflammation, protect and regenerate cells, and restore damaged tissues. In the present study, exosome-rich conditioned media (ERCMs) were attained via hypoxic culture (2% O 2 ) of human amniotic membrane mesenchymal stem cells (AMMSCs) and amniotic membrane epithelial stem cells (AMESCs) containing 50 times more exosome particles than normoxic culture (20% O 2 ) medium (NCM). The exosome particles in ERCM were confirmed to be 77 nm in mean size and contain much greater amounts of growth factors (GFs) and neurotrophic factors (NFs) than those in NCM. The glaucoma-therapeutic effects of ERCMs were assessed in retinal cells and a hypertonic (1.8 M) saline-induced high-IOP animal model. CM-DiI-labeled AMMSC exosomes were found to readily penetrate the normal and H 2 O 2 -damaged retinal ganglion cells (RGCs), and AMMSC-ERCM not only facilitated retinal pigment epithelial cell (RPEC) proliferation but also protected against H 2 O 2 - and hypoxia-induced RPEC insults. The IOP of rats challenged with 1.8 M saline increased twice the normal IOP (12-17 mmHg) in a week. However, intravitreal injection of AMMSC-ERCM or AMESC-ERCM (3.9-4.5 × 10 8 exosomes in 10 μL/eye) markedly recovered the IOP to normal level in 2 weeks, similar to the effect achieved with platelet-derived growth factor-AB (PDGF-AB, 1.5 μg), a reference material. In addition, AMMSC-ERCM, AMESC-ERCM, and PDGF-AB significantly reversed the shrinkage of retinal layers, preserved RGCs, and prevented neural injury in the glaucoma eyes. It was confirmed that stem cell ERCMs containing large numbers of functional molecules such as GFs and NFs improved glaucoma by protecting retinal cells against oxidative and hypoxic injuries in vitro and by recovering IOP and retinal degeneration in vivo. Therefore, it is suggested that stem cell ERCMs could be a promising candidate for the therapy of glaucoma., Competing Interests: The authors declare no conflicts of interest.

Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease characterized by degeneration and necrosis of myofibers, chronic inflammation, and progressive muscle weakness resulting in premature mortality. Immunosuppressive multipotent mesenchymal stromal cell (MSC) therapy could be an option for DMD patients. We focused on amnion-derived mesenchymal stromal cells (AMSCs), a clinically viable cell source owing to their unique characteristics, such as non-invasive isolation, mitotic stability, ethical acceptability, and minimal risk of immune reaction and cancer. We aimed to identify novel immunomodulatory effects of AMSCs on macrophage polarization and their transplantation strategies for the functional recovery of skeletal and cardiac muscles., Methods: We used flow cytometry to analyze the expression of anti-inflammatory M2 macrophage markers on peripheral blood mononuclear cells (PBMCs) co-cultured with human AMSCs (hAMSCs). hAMSCs were intravenously injected into DMD model mice (mdx mice) to assess the safety and efficacy of therapeutic interventions. hAMSC-treated and untreated mdx mice were monitored using blood tests, histological examinations, spontaneous wheel-running activities, grip strength, and echocardiography., Results: hAMSCs induced M2 macrophage polarization in PBMCs via prostaglandin E 2 production. After repeated systemic hAMSC injections, mdx mice exhibited a transient downregulation of serum creatin kinase. Limited mononuclear cell infiltration and a decreased number of centrally nucleated fibers were indicative of regenerated myofibers following degeneration, suggesting an improved histological appearance of the skeletal muscle of hAMSC-treated mdx mice. Upregulated M2 macrophages and altered cytokine/chemokine expressions were observed in the muscles of hAMSC-treated mdx mice. During long-term experiments, a significant decrease in the grip strength in control mdx mice significantly improved in the hAMSC-treated mdx mice. hAMSC-treated mdx mice maintained running activity and enhanced daily running distance. Notably, the treated mice could run longer distances per minute, indicating high running endurance. Left ventricular function in DMD mice improved in hAMSC-treated mdx mice., Conclusions: Early systemic hAMSC administration in mdx mice ameliorated progressive phenotypes, including pathological inflammation and motor dysfunction, resulting in the long-term improvement of skeletal and cardiac muscle function. The therapeutic effects might be associated with the immunosuppressive properties of hAMSCs via M2 macrophage polarization. This treatment strategy could provide therapeutic benefits to DMD patients., (© 2023. The Author(s).)

Objectives: Sterile inflammation of fetal membranes is an indispensable event of normal parturition. However, triggers of sterile inflammation are not fully resolved. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver. Fetal membranes can also synthesize SAA1 but its functions are not well defined. Given the role of SAA1 in the acute phase response to inflammation, we postulated that SAA1 synthesized in the fetal membranes may be a trigger of local inflammation at parturition., Methods: The changes of SAA1 abundance in parturition were studied in the amnion of human fetal membranes. The role of SAA1 in chemokine expression and leukocyte chemotaxis was examined in cultured human amnion tissue explants as well as primary human amnion fibroblasts. The effects of SAA1 on monocytes, macrophages and dendritic cells were investigated in cells derived from a human leukemia monocytic cell line (THP-1)., Results: SAA1 synthesis increased significantly in human amnion at parturition. SAA1 evoked multiple chemotaxis pathways in human amnion fibroblasts along with upregulation of a series of chemokines via both toll-like receptor 4 (TLR4) and formyl peptide receptor 2 (FPR2). Moreover, SAA1-conditioned medium of cultured amnion fibroblasts was capable of chemoattracting virtually all types of mononuclear leukocytes, particularly monocytes and dendritic cells, which reconciled with the chemotactic activity of conditioned medium of cultured amnion tissue explants collected from spontaneous labor. Furthermore, SAA1 could induce the expression of genes associated with inflammation and extracellular matrix remodeling in monocytes, macrophages and dendritic cells derived from THP-1., Conclusions: SAA1 is a trigger of sterile inflammation of the fetal membranes at parturition., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objectives: This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies., Procedures: A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression., Results: Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins., Conclusions: This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis., (© 2022 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.)