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.

Methods and criteria for validating the multimodal functions of perinatal derivatives when used in oncological and antimicrobial applications.

Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo. Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, 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.

Perinatal derivatives: How to best characterize their multimodal functions in vitro. Part C: Inflammation, angiogenesis, and wound healing.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

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.

Shaping the Future of Perinatal Cells: Lessons From the Past and Interpretations of the Present.

Since their discovery and characterization, mesenchymal stromal cells (MSC) have been a topic of great interest in regenerative medicine. Over the last 10 years, detailed studies investigated the properties of MSC from perinatal tissues and have indicated that these cells may represent important tools for restoring tissue damage or promoting regeneration and repair of the tissue microenvironment. At first, perinatal tissue-derived MSC drew attention due to their potential differentiation capacities suggested by their early embryological origin. It is nowadays accepted that perinatal tissue-derived MSC are promising for a wide range of regenerative medicine applications because of their unique immune modulatory properties, rather than their differentiation ability. As a matter of fact, the activation and function of various cells of the innate and adaptive immune systems are suppressed and modulated by MSC from different perinatal tissues, such as human term placenta. However, the mechanisms by which they act on immune cells to facilitate tissue repair during pathological processes remain to be thoroughly elucidated to develop safe and efficient therapeutic approaches. In addition to immune modulatory ability, several other peculiar characteristics of placenta MSC, less explored and/or more debated, are being investigated. These include an understanding of the anti-microbial properties and the role of placental MSC in tumor progression. Moreover, a thorough investigation on preparation methods, bioactive factors, mechanisms of action of the cell secretome, and the development of potency assays to predict clinical efficacy of placenta MSC and their products, are necessary to provide a solid basis for their clinical application.

Conditioned medium from amniotic cells protects striatal degeneration and ameliorates motor deficits in the R6/2 mouse model of Huntington's disease.

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro-inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM-hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune-based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM-hAMSC display less severe signs of neurological dysfunction than saline-treated ones. CM-hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM-hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM-hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM-hAMSC could act by modulating inflammatory cells, and more specifically microglia.

Incorporating placental tissue in cord blood banking for stem cell transplantation.

INTRODUCTION: Human term placenta is comprised of various tissues from which different cells can be obtained, including hematopoietic stem cells and mesenchymal stem/stromal cells (MSCs). Areas covered: This review will discuss the possibility to incorporate placental tissue cells in cord blood banking. It will discuss general features of human placenta, with a brief review of the immune cells at the fetal-maternal interface and the different cell populations isolated from placenta, with a particular focus on MSCs. It will address the question as to why placenta-derived MSCs should be banked with their hematopoietic counterparts. It will discuss clinical trials which are studying safety and efficacy of placenta tissue-derived MSCs in selected diseases, and preclinical studies which have proven their therapeutic properties in other diseases. It will discuss banking of umbilical cord blood and raise several issues for improvement, and the applications of cord blood cells in non-malignant disorders. Expert commentary: Umbilical cord blood banking saves lives worldwide. The concomitant banking of non-hematopoietic cells from placenta, which could be applied therapeutically in the future, alone or in combination to their hematopoietic counterparts, could exploit current banking processes while laying the foundation for clinical trials exploring placenta-derived cell therapies in regenerative medicine.

Cardiac Restoration Stemming From the Placenta Tree: Insights From Fetal and Perinatal Cell Biology.

Efficient cardiac repair and ultimate regeneration still represents one of the main challenges of modern medicine. Indeed, cardiovascular disease can derive from independent conditions upsetting heart structure and performance: myocardial ischemia and infarction (MI), pharmacological cardiotoxicity, and congenital heart defects, just to name a few. All these disorders have profound consequences on cardiac tissue, inducing the onset of heart failure over time. Since the cure is currently represented by heart transplantation, which is extremely difficult due to the shortage of donors, much effort is being dedicated to developing innovative therapeutic strategies based on stem cell exploitation. Among the broad scenario of stem/progenitor cell subpopulations, fetal and perinatal sources, namely amniotic fluid and term placenta, have gained interest due to their peculiar regenerative capacity, high self-renewal capability, and ease of collection from clinical waste material. In this review, we will provide the state-of-the-art on fetal perinatal stem cells for cardiac repair and regeneration. We will discuss different pathological conditions and the main therapeutic strategies proposed, including cell transplantation, putative paracrine therapy, reprogramming, and tissue engineering approaches.

Immunological and Differentiation Properties of Amniotic Cells Are Retained After Immobilization in Pectin Gel.

Mesenchymal stromal cells from the human amniotic membrane (i.e., human amniotic mesenchymal stromal cells [hAMSCs]) of term placenta are increasingly attracting attention for their applications in regenerative medicine. Osteochondral defects represent a major clinical problem with lifelong chronic pain and compromised quality of life. Great promise for osteochondral regeneration is held in hydrogel-based constructs that have a flexible composition and mimic the physiological structure of cartilage. Cell loading within a hydrogel represents an advantage for regenerative purposes, but the encapsulation steps can modify cell properties. As pectin gels have also been explored as cell vehicles on 3D scaffolds, the aim of this study was to explore the possibility to include hAMSCs in pectin gel. Immobilization of hAMSCs into pectin gels could expand their application in cell-based bioengineering strategies. hAMSCs were analyzed for their viability and recovery from the pectin gel and for their ability to differentiate toward the osteogenic lineage and to maintain their immunological characteristics. When treated with a purposely designed pectin/hydroxyapatite gel biocomposite, hAMSCs retained their ability to differentiate toward the osteogenic lineage, did not induce an immune response, and retained their ability to reduce T cell proliferation. Taken together, these results suggest that hAMSCs could be used in combination to pectin gels for the study of novel osteochondral regeneration strategies.

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.

The Long Path of Human Placenta, and Its Derivatives, in Regenerative Medicine.

In the 1800s, a baby born with a caul, a remnant of the amniotic sack or fetal membranes, was thought to be lucky, special, or protected. Over time, fetal membranes lost their legendary power and were soon considered nothing more than biological waste after birth. However, placenta tissues have reclaimed their potential and since the early 1900s an increasing body of evidence has shown that these tissues have clinical benefits in a wide range of wound repair and surgical applications. Nowadays, there is a concerted effort to understand the mechanisms underlying the beneficial effects of placental tissues, and, more recently, cells derived thereof. This review will summarize the historical and current clinical applications of human placental tissues, and cells isolated from these tissues, and discuss some mechanisms thought to be responsible for the therapeutic effects observed after tissue and/or cell transplantation.