A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

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.

Influence of the Surface Topography of Titanium Dental Implants on the Behavior of Human Amniotic Stem Cells.

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 ß1 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.

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

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

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

Stretch Causes cffDNA and HMGB1-Mediated Inflammation and Cellular Stress in Human Fetal Membranes.

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.

Pretreatment with Inflammatory Factors Altered the Secretome of Human Amniotic Epithelial Cells.

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.

A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery.

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.

Native characterization and QC profiling of human amniotic mesenchymal stromal cell vesicular fractions for secretome-based therapy.

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.

Osteogenic potential of adipose stem cells on hydroxyapatite-functionalized decellularized amniotic membrane.

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.

The Regenerative Capacity of Tissue-Engineered Amniotic Membranes.

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.

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

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

Group B streptococcus induces cellular senescence in human amnion epithelial cells through a partial interleukin-1-mediated mechanism.

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 ß-galactosidase activity, IL-6 and IL-8 release from the amnion epithelial cells. Cotreatment with IL1Ra reduced GBS-induced ß-galactosidase activity and IL-6 and IL-8 secretion. Direct treatment with IL-1α or IL-1ß 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.

Human Amniotic Membrane-Derived Mesenchymal Stem Cells Prevent Acute Graft-Versus-Host Disease in an Intestinal Microbiome-Dependent Manner.

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.

Hypoblast from human pluripotent stem cells regulates epiblast development.

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

An Exosome-Rich Conditioned Medium from Human Amniotic Membrane Stem Cells Facilitates Wound Healing via Increased Reepithelization, Collagen Synthesis, and Angiogenesis.

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% O2 and 5% CO2). The contents of GFs and NFs including keratinocyte growth factor, epidermal growth factor, fibroblast growth factor 1, transforming growth factor-ß, 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 H2O2 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 × 109 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.

The Human Placental Amniotic Membrane Mesenchymal-Stromal-Cell-Derived Conditioned Medium Inhibits Growth and Promotes Apoptosis of Human Cholangiocarcinoma Cells In Vitro and In Vivo by Suppressing IL-6/JAK2/STAT3 Signaling.

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.

Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

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.

Disclosing the molecular profile of the human amniotic mesenchymal stromal cell secretome by filter-aided sample preparation proteomic characterization.

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.

The effects of human amnion membrane-derived mesenchymal stem cells conditioned medium on ionizing radiation-induced premature ovarian failure and endoplasmic reticulum stress-related apoptosis mechanism.

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.

IL-33/ST2 axis of human amnion fibroblasts participates in inflammatory reactions at parturition.

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-1ß, 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-1ß, 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.

Canine amniotic membrane-derived mesenchymal stem cells ameliorate atopic dermatitis through regeneration and immunomodulation.

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-ß1, 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.