Stable Housekeeping Genes in Bone Marrow, Adipose Tissue, and Amniotic Membrane-Derived Mesenchymal Stromal Cells for Orthopedic Regenerative Medicine Approaches.

The therapeutic effect of mesenchymal stromal cells (MSCs) has been described for a variety of disorders, including those affecting musculoskeletal tissues. In this context, the literature reports several data about the regenerative effectiveness of MSCs derived from bone marrow, adipose tissue, and an amniotic membrane (BMSCs, ASCs, and hAMSCs, respectively), either when expanded or when acting as clinical-grade biologic pillars of products used at the point of care. To date, there is no evidence about the superiority of one source over the others from a clinical perspective. Therefore, a reliable characterization of the tissue-specific MSC types is mandatory to identify the most effective treatment, especially when tailored to the target disease. Because molecular characterization is a crucial parameter for cell definition, the need for reliable normalizers as housekeeping genes (HKGs) is essential. In this report, the stability levels of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) were sifted into BMSCs, ASCs, and hAMSCs. Adult and fetal/neonatal MSCs showed opposite HKG stability rankings. Moreover, by analyzing MSC types side-by-side, comparison-specific HKGs emerged. The effect of less performant HKG normalization was also demonstrated in genes coding for factors potentially involved in and predicting MSC therapeutic activity for osteoarthritis as a model musculoskeletal disorder, where the choice of the most appropriate normalizer had a higher impact on the donors rather than cell populations when compared side-by-side. In conclusion, this work confirms HKG source-specificity for MSCs and suggests the need for cell-type specific normalizers for cell source or condition-tailored gene expression studies.

AEC and AFMSC Transplantation Preserves Fertility of Experimentally Induced Rat Varicocele by Expressing Differential Regenerative Mechanisms.

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

The pathogenesis of cryptoglandular anal fistula: New insight into the immunological profile.

AIM: The aetiology of cryptoglandular anal fistula (AF) is poorly understood. Evidence suggests that persistence and/or recurrence of the disease is more related to inflammatory than infectious factors. The aim of this study was to investigate the immune profile of cryptoglandular AF and to perform a histopathological characterization. METHOD: Fistulectomy was performed in all patients; healthy ischioanal fat from the same patients was used as a control. Samples were evaluated by the Luminex xMAP system for the detection of 27 analytes. AF tissues were analysed using immunofluorescence. Staining was performed using primary antibodies to identify M1 inflammatory and M2 anti-inflammatory macrophages. Selective staining of total T lymphocytes and different T lymphocyte subsets was performed. RESULTS: Twenty patients with AF underwent a fistulectomy. Specific cytokine pathways differentiated AF from healthy tissue: pro-inflammatory cytokines interleukin (IL)-1ß, IL-4, IL-8 and IL-17 and the anti-inflammatory cytokine IL-10 were overexpressed in AF compared with controls. Chemokines involved in macrophage recruitment (CCL2, CCL3, CCL4) were higher in AF than in healthy fatty tissue. Moreover, we showed that Tc17 cells characterize AF patients, thus confirming the enzyme-linked immunosorbent assay data. Furthermore, elevated infiltration of CD68+ myeloid cells and a reduction of the M1/M2 ratio characterize AF patients. CONCLUSION: A combination of inflammatory cytokines, chemokines and growth factors reside in the wound microenvironment of AF patients. For the first time an important prevalence of Tc17 cells and a reduction in the M1/M2 ratio was observed, thus suggesting new insights into the immunological characterization of AF patients.

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.

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.

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.

Mesenchymal Stromal Cells: From Therapeutic Option to Therapeutic Target.

As our understanding of mesenchymal stromal cells (MSC) has evolved, they have come to be recognized as an integral part of the tumor tissue, and the exploitability of their intrinsic features in the field of oncology has reached a standstill [...].

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.

Recommendations from the COST action CA17116 (SPRINT) for the standardization of perinatal derivative preparation and in vitro testing.

Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to in vitro testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD in vitro. Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice.

Fight the Cancer, Hit the CAF!

The tumor microenvironment (TME) is comprised of different cellular components, such as immune and stromal cells, which co-operate in unison to promote tumor progression and metastasis. In the last decade, there has been an increasing focus on one specific component of the TME, the stromal component, often referred to as Cancer-Associated Fibroblasts (CAF). CAF modulate the immune response and alter the composition of the extracellular matrix with a decisive impact on the response to immunotherapies and conventional chemotherapy. The most recent publications based on single-cell analysis have underlined CAF heterogeneity and the unique plasticity that strongly impact the TME. In this review, we focus not only on the characterization of CAF based on the most recent findings, but also on their impact on the immune system. We also discuss clinical trials and preclinical studies where targeting CAF revealed controversial results. Therefore, future efforts should focus on understanding the functional properties of individual subtypes of CAF, taking into consideration the peculiarities of each pathological context.

Perinatal derivatives: How to best validate their immunomodulatory functions.

Perinatal tissues, mainly the placenta and umbilical cord, contain a variety of different somatic stem and progenitor cell types, including those of the hematopoietic system, multipotent mesenchymal stromal cells (MSCs), epithelial cells and amnion epithelial cells. Several of these perinatal derivatives (PnDs), as well as their secreted products, have been reported to exert immunomodulatory therapeutic and regenerative functions in a variety of pre-clinical disease models. Following experience with MSCs and their extracellular vesicle (EV) products, successful clinical translation of PnDs will require robust functional assays that are predictive for the relevant therapeutic potency. Using the examples of T cell and monocyte/macrophage assays, we here discuss several assay relevant parameters for assessing the immunomodulatory activities of PnDs. Furthermore, we highlight the need to correlate the in vitro assay results with preclinical or clinical outcomes in order to ensure valid predictions about the in vivo potency of therapeutic PnD cells/products in individual disease settings.

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.

Amniotic membrane-mesenchymal stromal cells secreted factors and extracellular vesicle-miRNAs: Anti-inflammatory and regenerative features for musculoskeletal tissues.

Human amniotic membrane-derived mesenchymal stromal cells (hAMSCs) are easily obtained in large quantities and free from ethical concerns. Promising therapeutic results for both hAMSCs and their secreted factors (secretome) were described by several in vitro and preclinical studies, often for treatment of orthopedic disorders such as osteoarthritis (OA) and tendinopathy. For clinical translation of the hAMSC secretome as cell-free therapy, a detailed characterization of hAMSC-secreted factors is mandatory. Herein, we tested the presence of 200 secreted factors and 754 miRNAs in extracellular vesicles (EVs). Thirty-seven cytokines/chemokines were identified at varying abundance, some of which involved in both chemotaxis and homeostasis of inflammatory cells and in positive remodeling of extracellular matrix, often damaged in tendinopathy and OA. We also found 336 EV-miRNAs, 51 of which accounted for more than 95% of the genetic message. A focused analysis based on miRNAs related to OA and tendinopathy showed that most abundant EV-miRNAs are teno- and chondro-protective, able to induce M2 macrophage polarization, inhibit inflammatory T cells, and promote Treg. Functional analysis on IL-1ß treated tenocytes and chondrocytes resulted in downregulation of inflammation-associated genes. Overall, presence of key regulatory molecules and miRNAs explain the promising therapeutic results of hAMSCs and their secretome for treatment of musculoskeletal conditions and are a groundwork for similar studies in other pathologies. Furthermore, identified molecules will pave the way for future studies aimed at more sharply predicting disease-targeted clinical efficacy, as well as setting up potency and release assays to fingerprint clinical-grade batches of whole secretome or purified components.

Mesenchymal Stromal Cells and Their Secretome: New Therapeutic Perspectives for Skeletal Muscle Regeneration.

Mesenchymal stromal cells (MSCs) are multipotent cells found in different tissues: bone marrow, peripheral blood, adipose tissues, skeletal muscle, perinatal tissues, and dental pulp. MSCs are able to self-renew and to differentiate into multiple lineages, and they have been extensively used for cell therapy mostly owing to their anti-fibrotic and immunoregulatory properties that have been suggested to be at the basis for their regenerative capability. MSCs exert their effects by releasing a variety of biologically active molecules such as growth factors, chemokines, and cytokines, either as soluble proteins or enclosed in extracellular vesicles (EVs). Analyses of MSC-derived secretome and in particular studies on EVs are attracting great attention from a medical point of view due to their ability to mimic all the therapeutic effects produced by the MSCs (i.e., endogenous tissue repair and regulation of the immune system). MSC-EVs could be advantageous compared with the parental cells because of their specific cargo containing mRNAs, miRNAs, and proteins that can be biologically transferred to recipient cells. MSC-EV storage, transfer, and production are easier; and their administration is also safer than MSC therapy. The skeletal muscle is a very adaptive tissue, but its regenerative potential is altered during acute and chronic conditions. Recent works demonstrate that both MSCs and their secretome are able to help myofiber regeneration enhancing myogenesis and, interestingly, can be manipulated as a novel strategy for therapeutic interventions in muscular diseases like muscular dystrophies or atrophy. In particular, MSC-EVs represent promising candidates for cell free-based muscle regeneration. In this review, we aim to give a complete picture of the therapeutic properties and advantages of MSCs and their products (MSC-derived EVs and secreted factors) relevant for skeletal muscle regeneration in main muscular diseases.

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.

Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells.

Currently, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations have been performed for the treatment of hematological and nonhematological diseases using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered. One strategy to expand ex vivo CB-HSC is represented by the use of bone marrow mesenchymal stromal cells (BM-MSCs) as a feeder to enhance HSC proliferation while maintaining HSC stemness. Indeed, BM-MSCs have been recognized as one of the most relevant players in the HSC niche. Thus, it has been hypothesized that they can support the ex vivo expansion of HSC by mimicking the physiological microenvironment present in the hematopoietic niche. Due to the role of placenta in supporting fetal hematopoiesis, MSC derived from the amniotic membrane (hAMSC) of human term placenta could represent an interesting alternative to BM-MSC as a feeder layer to enhance the proliferation and maintain HSC stemness. Therefore, in this study we investigated if hAMSC could support the ex vivo expansion of HSC and progenitor cells. The capacity of hAMSCs to support the ex vivo expansion of CB-HSC was evaluated in comparison to the control condition represented by the CB-CD34+ cells without a feeder layer. The coculture was performed at two different CD34+ :MSC ratios (1:2 and 1:8) in both cell-to-cell contact and transwell setting. After 7 days, the cells were collected and analyzed for phenotype and functionality. Our results suggest that hAMSCs represent a valuable alternative to BM-MSC to support: (a) the ex vivo expansion of CB-HSC in both contact and transwell systems, (b) the colony forming unit ability, and (c) long-term culture initiating cells ability. Overall, these findings may contribute to address the unmet need of high HSC content in CB units available for transplantation.

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.

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.

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells.

Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and ΔCt method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields.

Mesenchymal Stromal Cells from Fetal and Maternal Placenta Possess Key Similarities and Differences: Potential Implications for Their Applications in Regenerative Medicine.

Placenta-derived mesenchymal stromal cells (MSC) have attracted more attention for their immune modulatory properties and poor immunogenicity, which makes them suitable for allogeneic transplantation. Although MSC isolated from different areas of the placenta share several features, they also present significant biological differences, which might point to distinct clinical applications. Hence, we compared cells from full term placenta distinguishing them on the basis of their origin, either maternal or fetal. We used cells developed by Pluristem LTD: PLacenta expanded mesenchymal-like adherent stromal cells (PLX), maternal-derived cells (PLX-PAD), fetal-derived cells (PLX-R18), and amniotic membrane-derived MSC (hAMSC). We compared immune modulatory properties evaluating effects on T-lymphocyte proliferation, expression of cytotoxicity markers, T-helper and T-regulatory cell polarization, and monocyte differentiation toward antigen presenting cells (APC). Furthermore, we investigated cell immunogenicity. We show that MSCs and MSC-like cells from both fetal and maternal sources present immune modulatory properties versus lymphoid (T cells) and myeloid (APC) cells, whereby fetal-derived cells (PLX-R18 and hAMSC) have a stronger capacity to modulate immune cell proliferation and differentiation. Our results emphasize the importance of understanding the cell origin and characteristics in order to obtain a desired result, such as modulation of the inflammatory response that is critical in fostering regenerative processes.