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.

The evolution of in vitro models of lung fibrosis: promising prospects for drug discovery.

Lung fibrosis is a complex process, with unknown underlying mechanisms, involving various triggers, diseases and stimuli. Different cell types (epithelial cells, endothelial cells, fibroblasts and macrophages) interact dynamically through multiple signalling pathways, including biochemical/molecular and mechanical signals, such as stiffness, affecting cell function and differentiation. Idiopathic pulmonary fibrosis (IPF) is the most common fibrosing interstitial lung disease (fILD), characterised by a notably high mortality. Unfortunately, effective treatments for advanced fILD, and especially IPF and non-IPF progressive fibrosing phenotype ILD, are still lacking. The development of pharmacological therapies faces challenges due to limited knowledge of fibrosis pathogenesis and the absence of pre-clinical models accurately representing the complex features of the disease. To address these challenges, new model systems have been developed to enhance the translatability of preclinical drug testing and bridge the gap to human clinical trials. The use of two- and three-dimensional in vitro cultures derived from healthy or diseased individuals allows for a better understanding of the underlying mechanisms responsible for lung fibrosis. Additionally, microfluidics systems, which replicate the respiratory system's physiology ex vivo, offer promising opportunities for the development of effective therapies, especially for IPF.

Amniotic MSC affect CD8 naive polarization toward SLEC/MPEC subsets by down-modulating IL-12Rß1 and IL-2Rα signaling pathways.

Mesenchymal stromal cells (MSCs) are known for their immunomodulatory activity. Here, we report that MSCs isolated from the amniotic membrane of human term placenta (hAMSCs) impact CD8 T cell fate through a multifaceted mechanism. We observed that hAMSCs are able to impact the metabolism of naive CD8 lymphocytes by downregulating the phosphorylation of mTOR and AKT, thus blocking cell differentiation. This effect is due to the ability of hAMSCs to reduce the expression of two receptors, IL-12Rß1 and IL-2RA, resulting in reduced phosphorylation of STAT4 and STAT5. In addition, hAMSCs reduce the expression of two transcriptional factors, Tbet and Eomes, directly involved in early effector cell commitment. Our results unravel an unknown feature of MSCs, offering alternative mechanistic insights into the effects of MSCs for the treatment of diseases characterized by an altered activation of memory subsets, such as autoimmune diseases and graft versus host disease.

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.

Systemic immune response in young and elderly patients after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. In addition to primary brain damage, systemic immune alterations occur, with evidence for dysregulated immune responses in aggravating TBI outcome and complications. However, immune dysfunction following TBI has been only partially understood, especially in the elderly who represent a substantial proportion of TBI patients and worst outcome. Therefore, we aimed to conduct an in-depth immunological characterization of TBI patients, by evaluating both adaptive (T and B lymphocytes) and innate (NK and monocytes) immune cells of peripheral blood mononuclear cells (PBMC) collected acutely (< 48 h) after TBI in young (18-45 yo) and elderly (> 65 yo) patients, compared to age-matched controls, and also the levels of inflammatory biomarkers. RESULTS: Our data show that young respond differently than elderly to TBI, highlighting the immune unfavourable status of elderly compared to young patients. While in young only CD4 T lymphocytes are activated by TBI, in elderly both CD4 and CD8 T cells are affected, and are induced to differentiate into subtypes with low cytotoxic activity, such as central memory CD4 T cells and memory precursor effector CD8 T cells. Moreover, TBI enhances the frequency of subsets that have not been previously investigated in TBI, namely the double negative CD27- IgD- and CD38-CD24- B lymphocytes, and CD56dim CD16- NK cells, both in young and elderly patients. TBI reduces the production of pro-inflammatory cytokines TNF-α and IL-6, and the expression of HLA-DM, HLA-DR, CD86/B7-2 in monocytes, suggesting a compromised ability to drive a pro-inflammatory response and to efficiently act as antigen presenting cells. CONCLUSIONS: We described the acute immunological response induced by TBI and its relation with injury severity, which could contribute to pathologic evolution and possibly outcome. The focus on age-related immunological differences could help design specific therapeutic interventions based on patients' characteristics.

Amniotic Membrane-Derived Stromal Cells Release Extracellular Vesicles That Favor Regeneration of Dystrophic Skeletal Muscles.

Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing muscle stem cells (MuSCs) and an impairment of their activation and differentiation, which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study, we focused on an unexplored strategy to improve MuSC function and to preserve their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), that are multipotent cells recognized to have a role in tissue repair in different disease models. We demonstrate that the hAMSC secretome (CM hAMSC) and extracellular vesicles (EVs) isolated thereof directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Furthermore, we demonstrate that hAMSC secreted factors modulate the muscle stem cell niche in dystrophic-mdx-mice. Interestingly, local injection of EV hAMSC in mdx muscles correlated with an increase in the number of activated Pax7+/Ki67+ MuSCs and in new fiber formation. EV hAMSCs also significantly reduced muscle collagen deposition, thus counteracting fibrosis and MuSCs exhaustion, two hallmarks of DMD. Herein for the first time we demonstrate that CM hAMSC and EVs derived thereof promote muscle regeneration by supporting proliferation and differentiation of resident muscle stem cells. These results pave the way for the development of a novel treatment to counteract DMD progression by reducing fibrosis and enhancing myogenesis in dystrophic muscles.

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.

Comparison of EV-free fraction, EVs, and total secretome of amniotic mesenchymal stromal cells for their immunomodulatory potential: a translational perspective.

Amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties demonstrated in vitro and in vivo in various diseases in which the dysregulated immune system plays a major role. The immunomodulatory and pro-regenerative effects of MSCs, among which hAMSCs lie in the bioactive factors they secrete and in their paracrine activity, is well known. The mix of these factors (i.e., secretome) can be either freely secreted or conveyed by extracellular vesicles (EV), thus identifying two components in the cell secretome: EV-free and EV fractions. This study aimed to discern the relative impact of the individual components on the immunomodulatory action of the hAMSC secretome in order to obtain useful information for implementing future therapeutic approaches using immunomodulatory therapies based on the MSC secretome. To this aim, we isolated EVs from the hAMSC secretome (hAMSC-CM) by ultracentrifugation and validated the vesicular product according to the International Society for Extracellular Vesicles (ISEV) criteria. EVs were re-diluted in serum-free medium to maintain the EV concentration initially present in the original CM. We compared the effects of the EV-free and EV fractions with those exerted by hAMSC-CM in toto on the activation and differentiation of immune cell subpopulations belonging to both the innate and adaptive immune systems. We observed that the EV-free fraction, similar to hAMSC-CM in toto, a) decreases the proliferation of activated peripheral blood mononuclear cells (PBMC), b) reduces the polarization of T cells toward inflammatory Th subsets, and induces the induction of regulatory T cells; c) affects monocyte polarization to antigen-presenting cells fostering the acquisition of anti-inflammatory macrophage (M2) markers; and d) reduces the activation of B lymphocytes and their maturation to plasma cells. We observed instead that all investigated EV fractions, when used in the original concentrations, failed to exert any immunomodulatory effect, even though we show that EVs are internalized by various immune cells within PBMC. These findings suggest that the active component able to induce immune regulation, tested at original concentrations, of the hAMSC secretome resides in factors not conveyed in EVs. However, EVs isolated from hAMSC could exert actions on other cell types, as reported by others.

CM from intact hAM: an easily obtained product with relevant implications for translation in regenerative medicine.

BACKGROUND: It is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs. METHODS: Amniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM. RESULTS: We demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC. CONCLUSIONS: The results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. Moreover, the possibility of having a lyophilized secretome that maintains its original properties would allow for a ready-to-use product with easier handling, shipping and storage. The use of a lyophilized product will also facilitate clinicians by permitting customized reconstitution volumes and methods according to the most suitable formula required by the clinical application.

The Role of B Cells in PE Pathophysiology: A Potential Target for Perinatal Cell-Based Therapy?

The pathophysiology of preeclampsia (PE) is poorly understood; however, there is a large body of evidence that suggests a role of immune cells in the development of PE. Amongst these, B cells are a dominant element in the pathogenesis of PE, and they have been shown to play an important role in various immune-mediated diseases, both as pro-inflammatory and regulatory cells. Perinatal cells are defined as cells from birth-associated tissues isolated from term placentas and fetal annexes and more specifically from the amniotic membrane, chorionic membrane, chorionic villi, umbilical cord (including Wharton's jelly), the basal plate, and the amniotic fluid. They have drawn particular attention in recent years due to their ability to modulate several aspects of immunity, making them promising candidates for the prevention and treatment of various immune-mediated diseases. In this review we describe main findings regarding the multifaceted in vitro and in vivo immunomodulatory properties of perinatal cells, with a focus on B lymphocytes. Indeed, we discuss evidence on the ability of perinatal cells to inhibit B cell proliferation, impair B cell differentiation, and promote regulatory B cell formation. Therefore, the findings discussed herein unveil the possibility to modulate B cell activation and function by exploiting perinatal immunomodulatory properties, thus possibly representing a novel therapeutic strategy in PE.

Extracellular Vesicles From Perinatal Cells for Anti-inflammatory Therapy.

Perinatal cells, including cells from placenta, fetal annexes (amniotic and chorionic membranes), umbilical cord, and amniotic fluid display intrinsic immunological properties which very likely contribute to the development and growth of a semiallogeneic fetus during pregnancy. Many studies have shown that perinatal cells can inhibit the activation and modulate the functions of various inflammatory cells of the innate and adaptive immune systems, including macrophages, neutrophils, natural killer cells, dendritic cells, and T and B lymphocytes. These immunological properties, along with their easy availability and lack of ethical concerns, make perinatal cells very useful/promising in regenerative medicine. In recent years, extracellular vesicles (EVs) have gained great interest as a new therapeutic tool in regenerative medicine being a cell-free product potentially capable, thanks to the growth factors, miRNA and other bioactive molecules they convey, of modulating the inflammatory microenvironment thus favoring tissue regeneration. The immunomodulatory actions of perinatal cells have been suggested to be mediated by still not fully identified factors (secretoma) secreted either as soluble proteins/cytokines or entrapped in EVs. In this review, we will discuss how perinatal derived EVs may contribute toward the modulation of the immune response in various inflammatory pathologies (acute and chronic) by directly targeting different elements of the inflammatory microenvironment, ultimately leading to the repair and regeneration of damaged tissues.

The Multifaceted Roles of MSCs in the Tumor Microenvironment: Interactions With Immune Cells and Exploitation for Therapy.

The tumor microenvironment (TME) plays a critical role in tumorigenesis and is composed of different cellular components, including immune cells and mesenchymal stromal cells (MSCs). In this review, we will discuss MSCs in the TME setting and more specifically their interactions with immune cells and how they can both inhibit (immunosurveillance) and favor (immunoediting) tumor growth. We will also discuss how MSCs are used as a therapeutic strategy in cancer. Due to their unique immunomodulatory properties, MSCs isolated from perinatal tissues are intensely explored as therapeutic interventions in various inflammatory-based disorders with promising results. However, their therapeutic applications in cancer remain for the most part controversial and, importantly, the interactions between administered perinatal MSC and immune cells in the TME remain to be clearly defined.

Amniotic MSCs reduce pulmonary fibrosis by hampering lung B-cell recruitment, retention, and maturation.

Growing evidence suggests a mechanistic link between inflammation and the development and progression of fibrotic processes. Mesenchymal stromal cells derived from the human amniotic membrane (hAMSCs), which display marked immunomodulatory properties, have been shown to reduce bleomycin-induced lung fibrosis in mice, possibly by creating a microenvironment able to limit the evolution of chronic inflammation to fibrosis. However, the ability of hAMSCs to modulate immune cells involved in bleomycin-induced pulmonary inflammation has yet to be elucidated. Herein, we conducted a longitudinal study of the effects of hAMSCs on alveolar and lung immune cell populations upon bleomycin challenge. Immune cells collected through bronchoalveolar lavage were examined by flow cytometry, and lung tissues were used to study gene expression of markers associated with different immune cell types. We observed that hAMSCs increased lung expression of T regulatory cell marker Foxp3, increased macrophage polarization toward an anti-inflammatory phenotype (M2), and reduced the antigen-presentation potential of macrophages and dendritic cells. For the first time, we demonstrate that hAMSCs markedly reduce pulmonary B-cell recruitment, retention, and maturation, and counteract the formation and expansion of intrapulmonary lymphoid aggregates. Thus, hAMSCs may hamper the self-maintaining inflammatory condition promoted by B cells that continuously act as antigen presenting cells for proximal T lymphocytes in injured lungs. By modulating B-cell response, hAMSCs may contribute to blunting of the chronicization of lung inflammatory processes with a consequent reduction of the progression of the fibrotic lesion.

Perinatal Cells: A Promising COVID-19 Therapy?

The COVID-19 pandemic has become a priority in the health systems of all nations worldwide. In fact, there are currently no specific drugs or preventive treatments such as vaccines. The numerous therapies available today aim to counteract the symptoms caused by the viral infection that in some subjects can evolve causing acute respiratory distress syndromes (ARDS) with consequent admission to intensive care unit. The exacerbated response of the immune system, through cytokine storm, causes extensive damage to the lung tissue, with the formation of edema, fibrotic tissues and susceptibility to opportunistic infections. The inflammatory picture is also aggravated by disseminated intravascular coagulation which worsens the damage not only to the respiratory system, but also to other organs. In this context, perinatal cells represent a valid strategy thanks to their strong immunomodulatory potential, their safety profile, the ability to reduce fibrosis and stimulate reparative processes. Furthermore, perinatal cells exert antibacterial and antiviral actions. This review therefore provides an overview of the characteristics of perinatal cells with a particular focus on the beneficial effects that they could have in patients with COVID-19, and more specifically for their potential use in the treatment of ARDS and sepsis.

Autophagy: a potential key contributor to the therapeutic action of mesenchymal stem cells.

Macroautophagy/autophagy occurs at basal levels in all eukaryotic cells and plays an important role in maintaining bio-energetic homeostasis through the control of molecule degradation and organelle turnover. It can be induced by environmental conditions such as starvation, and is deregulated in many diseases including autoimmune diseases, neurodegenerative disorders, and cancer. Interestingly, the modulation of autophagy in mesenchymal stem cells (MSCs) represents a possible mechanism which, affecting MSC properties, may have an impact on their regenerative, therapeutic potential. Furthermore, the ability of MSCs to modulate autophagy of cells in injured tissues/organs has been recently proposed to be involved in the regeneration of damaged tissues and organs. In particular, MSCs can affect autophagy in immune cells involved in injury-induced inflammation reducing their survival, proliferation, and function and favoring the resolution of inflammation. In addition, MSCs can affect autophagy in endogenous adult or progenitor cells, promoting their survival, proliferation and differentiation supporting the restoration of functional tissue. This review provides, for the first time, an overview of the studies which highlight a possible link between the therapeutic properties of MSCs and their ability to modulate autophagy, and it summarizes examples of disorders where these therapeutic properties have been correlated with such modulation. A better elucidation of the mechanism(s) through which MSCs can modulate the autophagy of target cells and how autophagy can affect MSCs therapeutic properties, can provide a wider perspective for the clinical application of MSCs in the treatment of many diseases.Abbreviations: 3-MA: 3-methyladenine; AD: Alzheimer disease; ATG: autophagy-related; BECN1: beclin 1; BM: bone marrow; CD: cluster of differentiation; EAE: experimental autoimmune encephalomyelitis; IL: interleukin; INF: interferon; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; PD: Parkinson disease; PtdIns3K: class III phosphatidylinositol 3-kinase; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; SQSTM1: sequestosome 1; TBI: traumatic brain injury; TGF: transforming growth factor; TNF: tumor necrosis factor.

Perinatal Mesenchymal Stromal Cells and Their Possible Contribution to Fetal-Maternal Tolerance.

During pregnancy, a successful coexistence between the mother and the semi-allogenic fetus occurs which requires a dynamic immune system to guarantee an efficient immune protection against possible infections and tolerance toward fetal antigens. The mechanism of fetal-maternal tolerance is still an open question. There is growing in vitro and in vivo evidence that mesenchymal stromal cells (MSC) which are present in perinatal tissues have a prominent role in generating a functional microenvironment critical to a successful pregnancy. This review highlights the immunomodulatory properties of perinatal MSC and their impact on the major immune cell subsets present in the uterus during pregnancy, such as natural killer cells, antigen-presenting cells (macrophages and dendritic cells), and T cells. Here, we discuss the current understanding and the possible contribution of perinatal MSC in the establishment of fetal-maternal tolerance, providing a new perspective on the physiology of gestation.

The Immunomodulatory Properties of Amniotic Cells: The Two Sides of the Coin.

Among the many cell types useful in developing therapeutic treatments, human amniotic cells from placenta have been proposed as valid candidates. Both human amniotic epithelial and mesenchymal stromal cells, and the conditioned medium generated from their culture, exert multiple immunosuppressive activities. Indeed, they inhibit T and B cell proliferation, suppress inflammatory properties of monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells, while promoting induction of cells with regulatory functions such as regulatory T cells and anti-inflammatory M2 macrophages. These properties have laid the foundation for their use for the treatment of inflammatory-based diseases, and encouraging results have been obtained in different preclinical disease models where exacerbated inflammation is present. Moreover, an immune-privileged status of amniotic cells has been often highlighted. However, even if long-term engraftment of amniotic cells has been reported into immunocompetent animals, only few cells survive after infusion. Furthermore, amniotic cells have been shown to be able to induce immune responses in vivo and, under specific culture conditions, they can stimulate T cell proliferation in vitro. Although immunosuppressive properties are a widely recognized characteristic of amniotic cells, immunogenic and stimulatory activities appear to be less reported, sporadic events. In order to improve therapeutic outcome, the mechanisms responsible for the suppressive versus stimulatory activity need to be carefully addressed. In this review, both the immunosuppressive and immunostimulatory activity of amniotic cells will be discussed.

Effect of human amniotic epithelial cells on pro-fibrogenic resident hepatic cells in a rat model of liver fibrosis.

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial-mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF-ß, the main fibrogenic growth factor, through αvß6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvß6 integrin expression, with a consequent decrease in TGF-ß activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury-induced and fibrosis-promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

The dichotomy of placenta-derived cells in cancer growth.

Placenta-derived mesenchymal stromal cells (MSC) have often been considered to linger behind their equivalents from other tissues, such as MSC from bone marrow, in many aspects including their therapeutic potential in regenerative medicine. Nowadays however, it is clear that certain aspects make placental MSC attractive as a cellular therapy, such as their lack of ethical concerns and ease of isolation from human term placenta, a material long regarded as biological waste. Moreover, placental MSC virtually lack expression of human leukocyte antigens and co-stimulatory molecules, making them very attractive for transplantation in allogeneic settings. In the context of cancer, cell therapy remains an area of intense investigation whereby MSC have been shown to play opposing roles, and placental MSC are no exception. In this review, we will discuss dichotomy of placental MSC that underscores the challenges in understanding their therapeutic potential in oncology.

Is Immune Modulation the Mechanism Underlying the Beneficial Effects of Amniotic Cells and Their Derivatives in Regenerative Medicine?

Regenerative medicine aims to repair and regenerate damaged cells, tissues, and organs in order to restore function. Regeneration can be obtained either by cell replacement or by stimulating the body's own repair mechanisms. Importantly, a favorable environment is required before any regenerative signal can stimulate resident stem/stromal cells, and regeneration is possible only after the resolution of injury-induced inflammation. An exacerbated immune response is often present in cases of degenerative, inflammatory-based diseases. Here we discuss how amniotic membrane cells, and their derivatives, can contribute to the resolution of many diseases with altered immune response by acting on different inflammatory mediators.