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Mesenchymal stromal cells (MSCs) have recently emerged as an interesting therapeutic approach for patients with progressive systemic sclerosis (SSc), a rare and life-threatening orphan autoimmune disease. Whereas MSC immunomodulatory potential is considered as a central mechanism for their clinical benefit, very few data are available on the impact of MSCs on immune cell subsets in vivo. In the current extended study of a phase I/II clinical trial exploring the injection of a single dose of allogeneic bone marrow-MSCs (alloBM-MSCs) in patients with severe SSc (NCT02213705), we performed a longitudinal in-depth characterization of circulating immune cells in 19 MSC-treated patients, including 14 responders and 5 non-responders. By a combination of flow cytometry and transcriptomic analyses, we highlighted an increase in circulating CD24hiCD27posCD38lo/neg memory B cells, the main IL-10-producing regulatory B cell (Breg) subset, and an upregulation of IL10 expression in ex-vivo purified B cells, specifically in responder patients, early after the alloBM-MSC infusion. In addition, a deeper alteration of the B-cell compartment before alloBM-MSC treatment, including a higher expression of profibrotic cytokines IL6 and TGFβ by sorted B cells was associated with a non-responder clinical status. Finally, BM-MSCs were able to directly upregulate IL-10 production in activated B cells in vitro. These data suggest that cytokine-producing B cells, in particular Breg, are pivotal effectors of BM-MSC therapeutic activity in SSc. Their quantification as activity biomarkers in MSC potency assays and patient selection criteria may be considered to reach optimal clinical benefit when designing MSC-based clinical trials.

Calcium phosphates (CaP) are widely used synthetic bone graft substitutes, having bioactivity that is regulated by a set of intertwined physico-chemical and structural properties. While some CaPs have shown to be as effective in regenerating large bone defects as autologous bone, there is still the need to understand the role of individual material properties in CaP performance. Here, we aimed to decouple the effects of chemical composition and surface-microstructure of a beta-tricalcium phosphate (TCP) ceramic, with proven osteoinductive potential, on human mesenchymal stromal cells (hMSCs) differentiation. To this end, we replicated the surface structure of the TCP ceramic into polylactic acid without inorganic additives, or containing the chemical constituents of the ceramic, i.e., a calcium salt, a phosphate salt, or TCP powder. The microstructure of the different materials was characterized by confocal laser profilometry. hMSCs were cultured on the materials, and the expression of a set of osteogenic genes was determined. The cell culture medium was collected and the levels of calcium and phosphate ions were quantified by inductively-coupled plasma mass spectrometry. The results revealed that none of the tested combinations of properties in polymer/composite replicas was as potent in supporting the osteogenic differentiation of hMSCs as the original ceramic. Nevertheless, we observed some effects of the surface structure in the absence of inorganics, as well as combined effects of surface structure and the added salts, in particular calcium, on osteogenic differentiation. The approach presented here can be used to study the role of independent properties in other CaP-based biomaterials.

Advanced therapy medicinal products (ATMPs) offer revolutionary new prospects for the treatment of conditions of unmet medical need. Among them, Mesenchymal Stromal Cells (MSCs) have emerged as promising candidates for cell-based regenerative medical therapies. Currently, several early phase clinical trials have been completed and many are ongoing to explore MSC safety and efficacy in a wide range of nephropathologies. Kidney diseases are a current major health concern with an increasing global prevalence. The current standards of care focus on supportive treatments, emphasizing the need to develop novel and efficient therapies to delay or halt disease progression. However, before these therapies can be routinely used in patients, there are still hurdles to overcome. One of the current roadblocks relates to the lack of standardization of manufacturing protocols. Studies have shown that cell culture processing variables can have significant effects on MSC phenotype and functionality. In addition, MSCs can be isolated from various sources and there is now a growing body of literature highlighting unique and intrinsic properties according to the tissue origin and donor characteristics. Having an optimal, well-designed scale-up strategy for MSC manufacturing is critical to ensure product quality. In this thesis, we first sought to develop a protocol to expand the most clinically used MSC populations – adipose, bone marrow, and umbilical cord – in a multicentre study to compare the properties of the cells emanating from these three sources when cultured in a de-centralised manufacturing approach. By implementing a harmonised expansion workflow, we identified unique growth patterns and differentiation potential that followed comparable trends among centres. We harmonized our culture expansion conditions including the use of a single source of foetal bovine serum (FBS) in order to minimize culture condition-induced alterations in cell biology. Due to the inherent heterogeneity within the MSC-field, in terms of both culture conditions and source material, the development of strategies to select the optimal ‘source’ for a particular clinical condition would facilitate successful clinical translation. For this, the identification of features beyond basic cell characteristics are in need of further investigation. In this thesis, we focused on assessing the ability of MSCs from different tissue origin to support angiogenesis, essential during tissue regeneration. Bone marrow derived MSCs and their secretome showed significantly enhanced abilities to promote tubulogenesis and endothelial cell migration in vitro, partially due to a superior secretion of angiogenic cytokines such as vascular endothelial growth factor (VEGF). Additionally, the central metabolism is a critical driver of many cellular functions and has been shown to vary and alter the therapeutic benefit of MSCs undergoing large-expansion procedures. The comparison of metabolic characteristics and cellular fitness between MSC populations revealed a robust and stem cell-like phenotype in umbilical cord derived MSCs that conferred them higher abilities to withstand challenging microenvironments. Finally, we investigated the therapeutic efficacy of MSC-derived bioproducts, namely their secretome and extracellular vesicles, to restore metabolic disturbances in an in vitro model of renal ischaemia/reperfusion injury (IRI). Collectively, results to date emphasize the heterogeneity of MSCs in their nature and mechanism of action. The development of culture conditions appropriately designed to support cell expansion while enhancing therapeutic potential will allow for the elucidation of their unique regenerative properties, facilitating the development of tailored and efficient therapies for a given medical condition. 2025-05-08

Cartilage tissue presents low self-repair capability and lesions often undergo irreversible progression. Structures obtained by tissue engineering, such as those based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for research and therapeutic purposes. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. Herein, this work reports a reproducible bioprinting process followed by a successful post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs are bioprinted, ionically crosslinked, and post chondrogenically differentiated for 28 days. The expression of glycosaminoglycan, collagen II and IV are observed. After 56 days in culture, the bioprinted constructs are still stable and show satisfactory cell metabolic activity with profuse extracellular matrix production. These results show a promising procedure to obtain 3D models for cartilage research and ultimately, an in vitro proof-of-concept of their potential use as stable chondral tissue implants.

Inflammation-associated disorders are significant causes of morbidity in horses. Equine single-donor mesenchymal stromal cells (sdMSCs) hold promise as cell-therapy candidates due to their secretory nonprogenitor functions. This has been demonstrated by mononuclear cell suppression assays (MSAs) showing that sdMSCs are blood mononuclear cell (BMC) suppressive in vitro. sdMSCs derived from umbilical cord blood are of clinical interest due to their ease of procurement, multipotency, and immunomodulatory ability. Due to the inherent donor-to-donor heterogeneity of MSCs, the development of robust and easily deployable methods of potency assessment is critical for improving MSCs' predictability in treating inflammatory diseases. This study focuses on the development of robust in vitro potency assays and the assessment of potential sdMSC therapeutic end products generated from pooled sdMSCs (pMSCs). We hypothesized that, compared to MSA using only one donor, MSA using pooled BMCs (pBMCs) is a more robust sdMSC potency assay due to reduced donor BMC heterogeneity. pBMCs were generated by pooling equine BMCs isolated from peripheral blood of five donors in equal ratios. pBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stored in liquid nitrogen until use. Similarly, pooling sdMSCs from multiple equine donors in equal ratios generated pMSCs. sdMSC cultures were assessed with pBMCs in MSA using Bromodeoxyuridine ELISA and CFSE. Proliferation assessment of BMCs from individual donors revealed varied responses to concanavalin A (ConA) stimulation. MSA using BMCs from single donors further demonstrated BMC donor variability. Utilizing this assay, we have also found that the immunosuppressive potencies of pMSCs are at least equal, if not more, than the calculated mean of individual cultures. MSA based on pBMCs provides a consistent and reproducible equine sdMSC potency assay. This knowledge could be used in production monitoring of cellular potency and as release criteria before clinical use.

Despite osteoarthritis (OA) representing a large burden for health and social care systems, affecting approximately 10 million people in the UK alone, there remains no effective intervention capable of regenerating the damaged joint cartilage in OA and current clinical management for end-stage disease remains joint replacement surgery. There is therefore a need for a change in healthcare strategies to manage early disease, a shift that is likely to include cell-based regenerative therapies. Regenerative medicine and cell therapies are poised to have a tremendous impact on the future of medicine by delivering more effective and personalised treatment than are currently available today. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which have immunomodulatory effects, and can differentiate into a variety of cell types, including cartilage progenitor cells, making MSCs a candidate for musculoskeletal cell therapy. However, there has been several challenges to translating basic science research into new therapies and two pivotal questions remain unanswered: • Where does the MSC therapy go after administration? • What is its precise mechanism of action? To address these questions, the effects of an intra-articular injection of human bone marrow derived MSCs into a mouse knee osteochondral injury model were investigated in young C57Bl/6 mice, aged C57Bl/6 mice and GNL3 heterozygote mice. MSC treatment was associated with improved osteochondral tissue repair in young C57Bl/6 mice but this was more limited in aged C57BL/6 mice and GNL3 heterozygote mice. Cage activity monitoring was used to assess mouse recovery following surgery, and the administration of human MSCs was associated with significantly improved recovery following injury when compared to untreated controls. To better understand the mechanisms of action of an MSC therapy, a novel gene reporter system which utilises the organic anion transporting protein, Oatp1a1, was used for cell tracking. Using a lentiviral vector system, transfected MSCs expressing this cell surface protein can transport several imageable small molecules across the cell membrane, including the MRI contrast agent, gadolinium-ethoxybenzyl-DTPA (Gd-EOB-DTPA, PrimovistTM) which is licensed for clinical use. Cells were successfully imaged in vitro and in vivo as a proof-of-concept for this approach in the musculoskeletal system. Whilst imaging provided circumstantial evidence of MSC therapy fate, single cell RNAseq of retrieved cells (following injection into the mouse after osteochondral injury) was performed to elucidate what transcriptomic changes were important to driving tissue repair over time. Here, the data demonstrated that the exogenous human MSC therapy can be retrieved from both the repair tissue (in the epiphysis) and synovial tissue of the damaged knee joint at both 1 and 4 weeks after intra-articular injection. The retrieved human MSC therapy from the mouse joint tissue showed spatial and temporal transcriptional heterogeneity which was pronounced between the two tissue sources. Supported by mass cytometry data, the paracrine role of MSC treatment was further emphasises, as MSC therapy was associated with the induction of regulatory T cells (Tregs) in the mouse knee joint tissues. As Tregs are involved in skeletal homeostasis, more work needs to be performed to investigate the crosstalk that exists between MSCs and Tregs. Overall, the presented work shows that exogenous human MSC therapy in the mouse may improve both histological and clinical outcomes following osteochondral injury. Following intra-articular injection of human MSCs, the transcriptomes of the retrieved human cells were studied for the first time and their heterogeneity described. Subpopulations with different functional roles may be implicated in the different phases of osteochondral repair. As tissue digestion and isolation protocols are refined, it is likely that rarer populations may be retrieved from the mouse osteochondral model, but the data presented here offers insights into the interaction between the MSC therapy and the host cells, opening new avenues for the role which MSCs can play as a cell therapy in osteochondral repair., Addenbrooke's Charitable Trust/Cambridge Biomedical Research Centre; Wellcome Trust

Despite a long history of investigation and sustained efforts in clinical testing with large numbers of clinical trials being initiated, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration (FDA). There are a multitude of barriers impeding the clinical progression of MSC therapies, to the forefront of these is the lack of standardised and consistent manufacturing protocols as well as the lack of biologically meaningful cell characterisation tools and standard release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, amongst other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. This work analysed the influence of five widely used culture media formulations on the isolation efficiency, growth parameters, trilineage differentiation potential and immunomodulatory potential of human bone marrow derived MSCs (BM-MSCs) and revealed significant differences in these parameters in response to the composition of the culture medium. However, analysis of surface immunophenotype showed that despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs; CD73, CD90 and CD105, a clear demonstration of the futility of the current standard. Further characterisation of the surface immunophenotype using a high-content screening panel of 243 antibodies revealed that the culture medium also influenced the surface proteome, with one third of markers assessed exhibiting variable expression profiles in response to culture medium and donor. Calculation of correlation coefficients between the aforementioned functional in vitro data and this phenotypic analysis identified a number of putative BM-MSC surface markers related to biological function for further investigation. Principal component analysis confirmed that BM-MSCs isolated and expanded in a proprietary serum-free medium previously developed in this laboratory, Purstem 2 (PS2), displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate (PL) and fetal bovine serum (FBS)-containing media. Given the safety and reproducibility issues surrounding the use of undefined media supplements such as PL and FBS, there is a need to move towards serum-free and chemically-defined culture media. PS2 medium supports the isolation and expansion of BM-MSCs from whole bone marrow but does not support isolation from the mononuclear cell (MNC) fraction of marrow only or of adipose derived stromal cells (ASCs). To address these shortcomings and develop a new iteration of the PS2 medium, growth factor and extracellular matrix molecule receptors expressed by BM-MSCs were identified and from these, candidate growth factors platelet derived growth factor (PDGF)-AA, PDGF-BB and epidermal growth factor (EGF) and an attachment factor fibronectin were selected for analysis. The three growth factors were added alone and in combinations to the existing PS2 and tested for their ability to isolate ASCs from the stromal vascular fraction (SVF) of adipose tissue with fibronectin coating of the tissue culture plates. Isolation and propagation of ASCs was successful in all media formulations containing PDGF-BB. Assessment of cell morphology, growth kinetics, trilineage differentiation propensity, immunomodulatory potential and surface immunophenotype demonstrated equivalent or greater performance in the serum/xeno-free media compared to 5% PL controls. However, these media did not support BM-MSC isolation from the MNC fraction of bone marrow only, indicating distinct biological differences between MSCs derived from different tissues. This highlights a deficit in our understanding of the biology of MSCs derived from different tissue sources and the need for bespoke culture media. 2025-02-28

Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with its 3′ UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2-targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in patients with osteoporosis—a condition characterized by increased marrow adiposity—demonstrating that its levels are negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis.

Применение мезенхимальных стромальных клеток (МСК) для научных исследований и клеточной терапии требует их выращивания в достаточных количествах in vitro. В качестве источника предшественников МСК в настоящее время наиболее часто используется костный мозг. Выделение фракции мононуклеаров из костного мозга возможно несколькими способами. Целью данного исследования является сравнение градиентного и седиментационного методов выделения мононуклеаров из костного мозга, предназначенных для дальнейшего получения МСК. В настоящей работе проведено сравнение результатов выделения мононуклеаров у 25 здоровых лиц и 45 больных множественной миеломой с применением градиента плотности Lympholite-H и 0,2%-ного раствора метилцеллюлозы. Последующий посев в питательную среду позволяет получить первичную культуру МСК. Проведены расчет эффективности выделения мононуклеаров, а также оценка функциональной активности выделенных клеток путем определения количества колониеобразующих единиц фибробластов. Морфологические признаки полученной культуры и иммунофенотипическая характеристика клеток соответствовали минимальным критериям отнесения их к МСК, которые установлены Международным обществом клеточной терапии. Эффективность фракционирования костного мозга с применением обоих методов сопоставима у здоровых лиц, тем не менее колониеобразующая способность клеток выше при использовании градиента плотности. У больных множественной миеломой предпочтителен метод выделения мононуклеаров с применением метилцеллюлозы., The use of mesenchymal stromal cells (MSCs) for research and cell therapy requires their cultivation in sufficient quantities in vitro. Bone marrow is currently the most common source of MSC precursors. There are several ways to isolate mononuclear cell fraction from the bone marrow. This study is aimed to compare two MSC separation techniques, density gradient separation and sedimentation methods for the isolation of mononuclear cells from the bone marrow. In this study we compare results of isolation of mononuclear cells in 25 healthy individuals and 45 patients with multiple myeloma using Lympholite-H density gradient and 0.2% methylcellulose solution. Subsequent seeding of the obtained cells allows to get a sufficient number of MSCs. We have evaluated the efficiency of mononuclear cell isolation and the functional activity of isolated cells by determining the number of colony-forming units of fibroblasts. The morphological features of the obtained culture and the immunophenotypic characteristics of the cells meet the minimum criteria defined by International Society for Cellular Therapy for classifying them as MSCs. Both methods of bone marrow fractionation show efficiency in healthy individuals, but colony-forming ability is higher with density gradient centrifugation. The isolation of mononuclear cells using methyl-cellulose is preferable in multiple myeloma patients.

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals., Biomaterials, 294

Reactive Oxygen Species are chemically unstable molecules generated during aerobic respiration, especially in the electron transport chain. ROS are involved in various biological functions; any imbalance in their standard level results in severe damage, for instance, oxidative damage, inflammation in a cellular system, and cancer. Oxidative damage activates signaling pathways, which result in cell proliferation, oncogenesis, and metastasis. Since the last few decades, mesenchymal stromal cells have been explored as therapeutic agents against various pathologies, such as cardiovascular diseases, acute and chronic kidney disease, neurodegenerative diseases, macular degeneration, and biliary diseases. Recently, the research community has begun developing several anti-tumor drugs, but these therapeutic drugs are ineffective. In this present review, we would like to emphasize MSCs-based targeted therapy against pathologies induced by ROS as cells possess regenerative potential, immunomodulation, and migratory capacity. We have also focused on how MSCs can be used as next-generation drugs with no side effects.

Bone-marrow-mesenchymal-stromal-cells (BMSCs)- and platelet-rich-plasma (PRP)-based therapies have shown potential for treating osteoarthritis (OA). Recently, the combination of these two approaches was proposed, with results that overcame those observed with the separate treatments, indicating a possible role of PRP in ameliorating BMSCs’ regenerative properties. Since a molecular fingerprint of BMSCs cultivated in the presence of PRP is missing, the aim of this study was to characterize the secretome in terms of soluble factors and extracellular-vesicle (EV)-embedded miRNAs from the perspective of tissues, pathways, and molecules which frame OA pathology. One hundred and five soluble factors and one hundred eighty-four EV-miRNAs were identified in the PRP-treated BMSCs’ secretome, respectively. Several soluble factors were related to the migration of OA-related immune cells, suggesting the capacity of BMSCs to attract lympho-, mono-, and granulocytes and modulate their inflammatory status. Accordingly, several EV-miRNAs had an immunomodulating role at both the single-factor and cell level, together with the ability to target OA-characterizing extracellular-matrix-degrading enzymes and cartilage destruction pathways. Overall, anti-inflammatory and protective signals far exceeded inflammation and destruction cues for cartilage, macrophages, and T cells. This study demonstrates that BMSCs cultivated in the presence of PRP release therapeutic molecules and give molecular ground for the use of this combined and innovative therapy for OA treatment.

Mesenchymal stromal cells (MSCs) have gained special relevance in bone tissue regenerative applications. MSCs have been isolated from different depots, with adipose tissue being acknowledged as one of the most convenient sources, given the wide availability, high cellular yield, and obtainability. Recently, the falciform ligament (FL) has been regarded as a potential depot for adipose tissue-derived stromal cells (FL-ADSCs) isolation. Nonetheless, the osteogenic capability of FL-ADSCs has not been previously characterized. Thus, the present study aimed the detailed characterization of FL-ADSCs’ functionality upon osteogenic induction through a classic (dexamethasone-based-DEX) or an innovative strategy with retinoic acid (RA) in a comparative approach with ADSCs from a control visceral region. Cultures were characterized for cell proliferation, metabolic activity, cellular morphology, fluorescent cytoskeletal and mitochondrial organization, and osteogenic activity–gene expression analysis and cytochemical staining. FL-derived populations expressed significantly higher levels of osteogenic genes and cytochemical markers, particularly with DEX induction, as compared to control ADSCs that were more responsive to RA. FL-ADSCs were identified as a potential source for bone regenerative applications, given the heightened osteogenic functionality. Furthermore, data highlighted the importance of the selection of the most adequate osteogenic-inducing program concerning the specificities of the basal cell population.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized “self-developing” esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

Bone marrow-derived mesenchymal stromal cells (BMSCs)-based therapies show a great potential to manage inflammation and tissue degeneration in osteoarthritis (OA) patients. Clinical trials showed the ability to manage pain and activation of immune cells and allowed restoration of damaged cartilage. To date, a molecular fingerprint of BMSC-secreted molecules in OA joint conditions able to support clinical outcomes is missing; the lack of that molecular bridge between BMSC activity and clinical results hampers clinical awareness and translation into practice. In this study, BMSCs were cultured in synovial fluid (SF) obtained from OA patients and, for the first time, a thorough characterization of soluble factors and extracellular vesicles (EVs)-embedded miRNAs was performed in this condition. Molecular data were sifted through the sieve of molecules and pathways characterizing the OA phenotype in immune cells and joint tissues. One-hundred and twenty-five secreted factors and one-hundred and ninety-two miRNAs were identified. The combined action of both types of molecules was shown to, first, foster BMSCs interaction with the most important OA immune cells, such as macrophages and T cells, driving their switch towards an anti-inflammatory phenotype and, second, promote cartilage homeostasis assisting chondrocyte proliferation and attenuating the imbalance between destructive and protective extracellular matrix-related players. Overall, molecular data give an understanding of the clinical results observed in OA patients and can enable a faster translation of BMSC-based products into everyday clinical practice.

Mesenchymal stromal cells (MSCs) from both humans and horses, which represent a clinically relevant translation animal model for human cutaneous wound healing, were recently found to possess antimicrobial properties against planktonic bacteria, and in the case of equine MSCs, also against biofilms. This, together with previous findings that human and equine MSCs promote angiogenesis and wound healing, makes these cells an attractive approach to treat infected cutaneous wounds in both species. The anti‐biofilm activities of equine MSC, via secretion of cysteine proteases, have only been demonstrated in vitro, thus lacking information about in vivo relevance. Moreover, the effects of the equine MSC secretome on resident skin cells have not yet been explored. The goals of this study were to (a) test the efficacy of the MSC secretome in a physiologically relevant ex vivo equine skin biofilm explant model and (b) explore the impact of the MSC secretome on the antimicrobial defense mechanisms of resident skin cells. Our salient findings were that secreted factors from equine MSCs significantly decreased viability of methicillin‐resistant Staphylococcus aureus bacteria in mature biofilms in this novel skin biofilm explant model. Moreover, we demonstrated that equine MSCs secrete CCL2 that increases the antimicrobial activity of equine keratinocytes by stimulating expression of antimicrobial peptides. Collectively, these data contribute to our understanding of the MSC secretome's antimicrobial properties, both directly by killing bacteria and indirectly by stimulating immune responses of surrounding resident skin cells, thus further supporting the value of MSC secretome‐based treatments for infected wounds., CCL2 secreted by mesenchymal stromal cells promotes cutaneous antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes. The secretome of cultured equine mesenchymal stromal cells (MSCs) was collected as conditioned medium (CM) and applied to methicillin‐susceptible Staphylococcus aureus (MSSA) and methicillin‐resistant Staphylococcus aureus (MRSA) biofilms in an ex vivo cutaneous wound explant model, where it effectively reduced bacteria viability. Primary equine keratinocytes, in two‐dimensional cultures, significantly increased expression of the antimicrobial peptides (AMP) ß‐defensin and cathelicidin after stimulation with the MSC secretome. ß‐defensin expression was mediated by CCL2, a cytokine secreted by MSCs.

Buerger's disease or thromboangiitis obliterans is a type of obstructive vascular diseases categorized as vasculitis and usually present in 95% of young smoker men. The main pathogenetic mechanism is interplay between immune system and inflammation. Earlier our phase II study has shown that Stempeucel is safe when injected at 2 million cells/kg body weight by virtue of its anti‐inflammatory, immunomodulatory, and angiogenetic properties. The present study was conducted to further assess the safety and efficacy of Stempeucel in critical limb ischemia due to Buerger's disease after obtaining approval from Indian FDA based on the data generated in the phase II study. This is an open label, multicenteric phase IV PMS study conducted across India with experienced vascular surgeons. Fifty patients of critical limb ischemia due to Buerger's disease with Rutherford III‐5 or III‐6 were included in the study and each individual received a dose of 2 million cells/kg body weight of Stempeucel in the calf muscles and around the ulcer. These patients were evaluated over 12 months from drug administration. The present study showed the continued long term efficacy over a period of 12 months follow up in these patients corroborating the result obtained in the previous phase II studies. There was significant improvement in rest pain, ankle systolic pressure, and ankle brachial pressure index with accelerated ulcer healing. In conclusion, the present study shows that the intramuscular administration of Stempeucel continues to be safe, tolerable, and effective alternative treatment in patients with Buerger's disease., Stempeucel showed the continued long term efficacy over a period of 12 months follow up in patients with CLI due to Buerger's disease. Significant improvement in rest pain, ankle systolic pressure and ankle brachial pressure index with accelerated ulcer healing was observed. Stempeucel continues to be safe, tolerable and effective alternative treatment in patients with Buerger's disease.

The aim of this study was to compare the functional characteristics of mesenchymal stromal cells (MSCs) from a sheep model of traumatic temporomandibular joint (TMJ) fibrous and bony ankylosis. A sheep model of bilateral TMJ trauma-induced fibrous ankylosis on one side and bony ankylosis on the contralateral side was used. MSCs from fibrous ankylosed callus (FA-MSCs) or bony ankylosed callus (BA-MSCs) at weeks 1, 2, 4, and 8 after surgery were isolated and cultured. MSCs derived from the bone marrow of the mandibular condyle (BM-MSCs) were used as controls. The MSCs from the different sources were characterized morphologically, phenotypically, and functionally. Adherence and trilineage differentiation potential were presented in the ovine MSCs. These cell populations highly positively expressed MSC-associated specific markers, namely CD29, CD44, and CD166, but lacked CD31 and CD45 expressions. The BA-MSCs had higher clonogenic and proliferative potentials than the FA-MSCs. The BA-MSCs also showed higher osteogenic and chondrogenic potentials, but lower adipogenic capacity than the FA-MSCs. In addition, the BA-MSCs demonstrated higher chondrogenic, but lower osteogenic capacity than the BM-MSCs. Our study suggests that inhibition of the osteogenic and chondrogenic differentiations of MSCs might be a promising strategy for preventing bony ankylosis in the future.

Bone-marrow-derived mesenchymal stromal cells (BMSCs) showed therapeutic potential in the treatment of musculoskeletal diseases, including osteoarthritis (OA). Their soluble mediators and extracellular vesicles (EVs), which make up the secretome, suppress immune response, attenuate inflammation and promote cartilage repair. EVs, as well as the whole secretome, have been investigated as cell free approaches for OA although, to date, a disease-tailored molecular fingerprint is missing. In this study, soluble mediators and miRNAs were sifted in the BMSCs’ secretome and EVs, respectively, and analyzed in the frame of cell types and factors involved in OA. The majority of identified molecules repress the activation of immune cells and the production of OA-related inflammatory mediators, as well as promote cartilage protection by acting on both chondrocytes homeostasis and extracellular matrix-degrading enzymes. These data provide the molecular ground for the therapeutic potential of BMSCs for regenerative applications for OA and support the use of secretome or EVs as cell-free applications in joint diseases.

Background: In end-stage chronic liver disease, transplantation represents the only curative option. However, the shortage of donors results in the death of many patients. To overcome this gap, it is mandatory to develop new therapeutic options. In the present study, we decellularised pig livers and reseeded them with allogeneic porcine mesenchymal stromal cells (pMSCs) to understand whether extracellular matrix (ECM) can influence and/or promote differentiation into hepatocyte-like cells (HLCs). Methods: After decellularisation with SDS, the integrity of ECM-scaffolds was examined by histological staining, immunofluorescence and scanning electron microscope. DNA quantification was used to assess decellularisation. pMSCs were plated on scaffolds by static seeding and maintained in in vitro culture for 21 days. At 3, 7, 14 and 21 days, seeded ECM scaffolds were evaluated for cellular adhesion and growth. Moreover, the expression of specific hepatic genes was performed by RT-PCR. Results: The applied decellularisation/recellularisation protocol was effective. The number of seeded pMSCs increased over the culture time points. Gene expression analysis of seeded pMSCs displayed a weak induction due to ECM towards HLCs. Conclusions: These results suggest that ECM may address pMSCs to differentiate in hepatocyte-like cells. However, only contact with liver-ECM is not enough to induce complete differentiation.

It has been known that senescence-associated secretory phenotype (SASP) triggers senescence of the surrounding normal cells. However, SASP signaling regarding mesenchymal stromal cell aging remains to be fully elucidated. Therefore, the present study aimed to clarify the molecular mechanism of late (passage) MSC-induced paracrine SASP-mediated senescence of early (passage) MSCs during ex vivo expansion. Here, we conducted an extensive characterization of senescence features in bone-marrow (BM)-derived MSCs from healthy human donors. Late MSCs displayed an enlarged senescent-like morphology, induced SASP-related proinflammatory cytokines (IL-1α and IL-8), and reduced clonogenic capacity and osteogenic differentiation when compared to early MSCs. Of note, paracrine effects of SASP-related IL-1α and IL-8 from late MSCs induced cellular senescence of early MSCs via an NF-κB-dependent manner. Moreover, cellular senescence of early MSCs was promoted by the synergistic action of IL-1α and IL-8. However, inhibition of NF-κB by shRNA transfection or using inhibitors in early MSCs blocked early MSCs cellular senescence caused by paracrine SASP of late MSCs. In conclusion, these findings reveal that late MSCs display features of senescence and that, during ex vivo expansion, SASP-related proinflammatory cytokines contribute to activate a cellular senescence program in early MSCs that may ultimately impair their functionality.

Parathyroid hormone (PTH) is one of the key regulators of calcium and phosphate metabolism in the body, controlling bone metabolism and ion excretion by the kidneys. At present, attempts to use PTH as a therapeutic agent have been associated with side-effects, the nature of which is not always clear and predictable. In addition, it is known that in vivo impairment of PTH post-receptor signaling is associated with atypical differentiation behavior not only of bone cells, but also of connective tissues, including adipose tissue. In this work, we studied the functional responses of multipotent mesenchymal stromal cells (MSCs) to the action of PTH at the level of single cells. We used MSCs isolated from the periosteum and subcutaneous adipose tissue to compare characteristics of cell responses to PTH. We found that the hormone can activate three key responses via its receptor located on the surface of MSCs: single transients of calcium, calcium oscillations, and hormone-activated smooth increase in intracellular calcium. These types of calcium responses led to principally different cellular responses of MSCs. The cAMP-dependent smooth increase of intracellular calcium was associated with pro-osteogenic action of PTH, whereas phospholipase C dependent calcium oscillations led to a decrease in osteogenic differentiation intensity. Different variants of calcium responses are in dynamic equilibrium. Suppression of one type of response leads to increased activation of another type and, accordingly, to a change in the effect of PTH on cell differentiation.

Osteoarthritis (OA) is a debilitating joint disease affecting an increasing proportion of the elderly population worldwide annually. Current treatments for the disease are symptomatic with management of pain and side effects the only options. The field of osteoarthritis research has endeavored to understand the mechanisms of cartilage degeneration and disease progression. This has led to the development of several proposed cell and gene therapies that aim to reverse or slow the progression of OA. The underlying molecular mechanisms involved in OA are complex and remain to be elucidated in order to increase understanding of the disease to address the need to identify novel treatment options. One such novel protein is neuronal Interleukin-16 (nIL-16) previously identified as expressed in late osteoarthritic cartilage. nIL-16 is an IL-16 isoform previously reported in neural tissue only. The other known isoform of IL-16, peripheral IL-16 (pIL-16) has been associated with control of T-cell proliferation. nIl-16 has been reported to function as an anchoring protein via its’ PDZ domains in neural tissues leading to the hypothesis that its role in late-stage OA is to anchor ion channels to the chondrocyte membrane causing an influx of calcium during chondrocyte hypertrophy, eventually leading to inappropriate endochondral ossification. The main focus of this thesis was to determine if CRISPR/Cas9 gene editing applied to the PDZ domains of nIL-16 could contribute to elucidate their role in OA progression. CRISPR/Cas9 gene editing has begun to be widely used to understand the gene function in many different contexts. With the ability to target CRISPR/Cas9 to specific sites of interest, targeted double stranded breaks can create edits in the genomic sequence leading to insertions, deletions or frameshift mutations allowing investigation of the outcomes of these changes on gene and protein function. This thesis investigated the suitability of CRISPR/Cas9 technology as a means to investigate the role of nIL-16 in OA progression. The target sites of interest were the PDZ domains in the nIL-16 isoform. Different CRISPR/Cas formats and non-viral mechanisms of delivery of CRISPR/Cas9 to human MSCs were explored with the aim of identifying the most suitable method to investigate whether editing of these domains was possible in hMSCs. Peptide assisted CRISPR plasmid DNA and electroporation of CRISPR plasmid DNA and the CRISPR ribonucleoprotein were assessed as methods of CRISPR gene editing in hMSCs A comparison of the effects of both formats on hMSC survival, proliferation and growth post electroporation was analyzed through examination of gene expression changes in the NF-ΚB, PI3K/AKT/mTOR, MAPK/ERK signaling pathways, Autophagy, senescence, and the DNA damage repair response to double stranded breaks. Electroporation of both plasmid DNA and RNP successfully delivered CRISPR/Cas9 to hMSCs with the RNP having higher levels of viability and editing efficacy compared to the plasmid DNA format. Autophagy was activated in hMSCs regardless of the format of CRISPR used. However, the hMSCs treated with nIL-16 targeting plasmid DNA were visibly more stressed. This was reflected in the gene expression changes with metabolic stress evident through upregulated expression of signaling transducers such as mTOR, MAPK1 and SMAD4 as well as evidence of growth cycle arrest with upregulated p53 and downregulation of p16ink4a. When hMSCs were electroporated with the RNP complexes, there was an initial acute reaction to the electroporation of the RNP complexes but the hMSCs returned to a state of homeostasis within 24 hours of the electroporation with down regulation of NF-ΚB, PI3K/AKT/mTOR, MAPK/ERK pathways as well as rapid autophagic clearance of cell debris. The work in this thesis has demonstrated that the RNP format of CRISPR/Cas9 can efficiently perform edits of hMSCs. Using two RNP complexes targeting up and down stream of the nIL-16 PDZ domains of interest successfully edited the genomic sequence, and the cells were healthy and proliferating post electroporation. This method can therefore be used to further examine the role of nIL-16 in OA progression. Furthermore, this method can be applied to novel genes of interest in OA progression and beyond to uncover their functions in disease progression and to identify targets for drug or gene therapy development. 2024-11-24

Please see attached word document for systematic review and meta-analysis pre-registered protocol.

The incidence of inflammatory bowel diseases (IBD) is increasing worldwide. Mesenchymal stem/stromal cells (MSCs) have immunomodulatory functions and are a promising source for cell transplantation therapy for IBD. However, owing to their heterogeneous nature, their therapeutic efficacy in colitis is controversial and depends on the delivery route and form of transplanted cells. Cluster of differentiation (CD)73 is widely expressed in MSCs and used to obtain a homogeneous MSC population. Herein, we determined the optimal method for MSC transplantation using CD73+ cells in a colitis model. mRNA sequencing analysis showed that CD73+ cells exhibit downregulation of inflammatory genes and upregulation of extracellular matrix-related genes. Furthermore, three-dimensional CD73+ cell spheroids showed enhanced engraftment at the injured site through the enteral route, facilitated extracellular matrix remodeling, and downregulated inflammatory gene expression in fibroblasts, leading to attenuation of colonic atrophy. Therefore, the interaction between intestinal fibroblasts and exogenous MSCs via tissue remodeling is one mechanism that can be exploited for colitis prevention. Our results highlight that transplantation of homogeneous cell populations with well-characterized properties is beneficial for IBD treatment.

Foot ulceration is a major complication of diabetes mellitus, which results in significant human suffering and a major burden on healthcare systems. Mesenchymal stromal cells (MSCs) emerged as a promising therapeutic agent for treatment of diabetic wound healing because of their paracrine properties, including the secretion of angiogenic, immunomodulatory and anti-inflammatory factors. However, topical administration of MSCs via direct injection result in low cell viability and poor cell localization at the wound bed, which hinder the efficacy and clinical translation of a MSC therapy product. Here in, we used macromolecular crowding (MMC) concept to develop a MSC-scaffold bio-complex containing the native extracellular matrix (ECM) and investigated its therapeutic effect in a diabetic wound model. Firstly, we investigated the optimal MMC agent in human umbilical cord derived MSC (hUC-MSC) cultures. Seven types of carrageenan at five different concentrations were supplemented to MSC medium. 50 μg/ml l-MV carrageenan was identified to be the optimal cell culture condition, as it did not adversely affect (p > 0.05) cell viability, metabolic activity, proliferation, and the expression of MSC surface markers and immune phenotype markers, whereas it significantly increased (p < 0.05) the deposition of collagen types I, III and IV, fibronectin and laminin. The shortest cell culture time was 4 days. These data highlight the potential of l-MV carrageenan as crowding agents for developing ECM-rich cell therapy product and describes the optimal conditions for use. Secondly, as hUC-MSCs grown under the optimum MMC conditions, can only develop a thin cell layer, which was not suitable for transfer from the tissue culture dish directly to the wound. A collagen porous scaffold was utilised to deliver this ECM-rich MSC layer. Scaffolds were fabricated using either neutralised or non-neutralised collagen. Compared to neutralized scaffold, the non-neutralized scaffold is pliable, thicker and has significantly (p < 0.05) smaller pore size, which is suitable for pre-clinical wound healing studies. hUC-MSCs grown on the non-neutralized scaffold under MMC conditions maintained the cell viability and proliferation at three cell doses (150k, 300k, 600k) groups. hUC-MSCs deposited significantly higher (p < 0.05) fibronectin in the presence of 50 μg/ml l-MV carrageenan at 300k and 600k cell dose groups and no significant difference was observed between the two groups. These data suggest this MSC-scaffold bio-complex at 300k cell dose may have augmented therapeutic potential for the treatment of diabetic wounds. Thirdly, to evaluate the therapeutic effect of this MSC-scaffold bio-complex, we developed a diabetic model using immunodeficient mice, and reproduced the excisional splinting wound model to avoid the confounding effect of wound contraction. Diabetes was induced in animals using low-dose streptozotocin. Wounds were treated as per groups: Sham (no treatment control), SC (scaffold only), SC+MSC (MSC grown on scaffold), and SC+MSC+MMC (MSC grown on scaffold under MMC conditions). 60% of mice successfully developed diabetes and exhibited mild diabetic signs (polydipsia and polyuria) with no loss of body weight and fatality. Gross view of wounds on day 7 showed no significant difference in wound closure rate. Histology and immunohistochemistry analysis revealed no significant difference in re-epithelialization, granulation tissue area, blood vessel density and ECM deposition between the four groups. Donor comparison analysis revealed no significant difference in therapeutic effects between MSCs from 3 different donors. Correlation analysis revealed no correlation between wound gap and blood glucose level, and each group had similar blood glucose levels. Although current pre-clinical data did not support this MSC-scaffold bio-complex to improve diabetic wound healing, these results direct investigation of alternative scaffolds and cell doses as a next step. Collectively, this study provides a new method for the development of a novel MSC therapy product with augmented therapeutic potential. Further improvement of the wound model and optimization of this MSC therapy product are needed for future study. 2026-08-23

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.

Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self‐renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC‐specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol‐anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down‐regulated in bone marrow‐derived MSCs cultured on plastic, making it difficult to examine the self‐renewal and differentiation of Meflin‐positive cells at the single‐cell level. Here, we traced the lineage of Meflin‐positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin‐positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin‐positive cells or their lineage‐committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues., Contribution of Meflin‐positive cells to the development of multiple mesenchymal tissues. Meflin is a marker of MSCs/SSCs in the cartilage and bone, ADSCs in the adipose tissue, Pax7‐positive satellite cells and PDGFRα‐positive FAPs in the skeletal muscle.

Many cell types, including cancer cells, release tissue factor (TF)-exposing extracellular vesicles (EVs). It is unknown whether MSC-EVs pose a thromboembolism risk due to TF expression. Knowing that MSCs express TF and are procoagulant, we hypothesize that MSC-EVs also might. Here, we examined the expression of TF and the procoagulant activity of MSC-EVs and the impact of EV isolation methods and cell culture expansion on EV yield, characterization, and potential risk using a design of experiments methodology. MSC-EVs were found to express TF and have procoagulant activity. Thus, when MSC-derived EVs are employed as a therapeutic agent, one might consider TF, procoagulant activity, and thromboembolism risk and take steps to prevent them.

Mesenchymal stromal/stem cells (MSCs) and their secreted factors have been shown to have immunomodulatory and regenerative effects. In this study, we investigated human bone-marrow-derived MSC secretome (MSC-S) for the treatment of corneal epithelial wounds. Specifically, we evaluated the role of MSC extracellular vesicles (EV)/exosomes in mediating the wound-healing effects of the MSC-S. In vitro studies using human corneal epithelial cells showed that MSC-CM increased cell proliferation in HCEC and HCLE cells, while EV-depleted MSC-CM showed lower cell proliferation in both cell lines compared to the MSC-CM group. In vitro and in vivo experiments revealed that 1X MSC-S consistently promoted wound healing more effectively than 0.5X MSC-S, and MSC-CM promoted wound healing in a dose-dependent manner, while exosome deprivation delayed wound healing. We further evaluated the incubation period of MSC-CM on corneal wound healing and showed that MSC-S collected for 72 h is more effective than MSC-S collected for 48 h. Finally, we evaluated the stability of MSC-S under different storage conditions and found that after one cycle of freeze–thawing, MSC-S is stable at 4 °C for up to 4 weeks. Collectively, we identified the following: (i) MSC-EV/Exo as the active ingredient in MSC-S that mediates the wound-healing effects in the corneal epithelium, providing a measure to optimize its dosing for a potential clinical product; (ii) Treatment with EV/Exo-containing MSC-S resulted in an improved corneal barrier and decreased corneal haze/edema relative to EV/Exo-depleted MSC-S; (iii) The stability of MSC-CM for up to 4 weeks showed that the regular storage condition did not significantly impact its stability and therapeutic functions.

Cell-based therapies using periodontal ligament stromal cells (PDLSC) for periodontal regeneration may represent an alternative source for mesenchymal stromal cells (MSC) to MSC derived from bone marrow (MSC(M)) and adipose tissue (MSC(AT)). We aimed to characterize the osteogenic/periodontal potential of PDLSC in comparison to MSC(M) and MSC(AT). PDLSC were obtained from surgically extracted healthy human third molars, while MSC(M) and MSC(AT) were obtained from a previously established cell bank. Flow cytometry, immunocytochemistry, and cell proliferation analyses provided cellular characteristics from each group. Cells from the three groups presented MSC-like morphology, MSC-related marker expression, and multilineage differentiation capacity (adipogenic, chondrogenic, and osteogenic). In this study, PDLSC expressed osteopontin, osteocalcin, and asporin, while MSC(M) and MSC(AT) did not. Of note, only PDLSC expressed CD146, a marker previously applied to identify PDLSC, and presented higher proliferative potential compared to MSC(M) and MSC(AT). Upon osteogenic induction, PDLSC exhibited higher calcium content and enhanced upregulation of osteogenic/periodontal genes compared to MSC(M) and MSC(AT), such as Runx2, Col1A1 and CEMP-1. However, the alkaline phosphatase activity of PDLSC did not increase. Our findings suggest that PDLSC might be a promising cell source for periodontal regeneration, presenting enhanced proliferative and osteogenic potential compared to MSC(M) and MSC(AT).

Background & Aim: Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g.,2–8°C) could be a feasible alternative. In this study, we aim to determine the ability of alginate encapsulation to maintain cell viability, identity, and function in the context of MSC-based therapy manufacturing. Methods, Results & Conclusion: Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads (BeadReady™ kits kindly provided by Atelerix) for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity and functional immunophenotype, MSC tri-lineage differentiation potential, metabolic activity, and hematopoietic support capacity were determined and compared between timepoints. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor. info:eu-repo/semantics/publishedVersion

Treatments for acute respiratory distress syndrome are still unavailable, and the prevalence of the disease has only increased due to the COVID-19 pandemic. Mechanical ventilation regimens are still utilized to support declining lung function but also contribute to lung damage and increase the risk for bacterial infection. The anti-inflammatory and pro-regenerative abilities of mesenchymal stromal cells (MSCs) have shown to be a promising therapy for ARDS. We propose to utilize the regenerative effects of MSCs and the extracellular matrix (ECM) in a nanoparticle. Our mouse MSC (MMSC) ECM nanoparticles were characterized using size, zeta potential, and mass spectrometry to evaluate their potential as pro-regenerative and antimicrobial treatments. The nanoparticles had an average size of 273.4 nm (±25.6) and possessed a negative zeta potential, allowing them to surpass defenses and reach the distal regions of the lung. It was found that the MMSC ECM nanoparticles are biocompatible with mouse lung epithelial cells and MMSCs, increasing the wound healing rate of human lung fibroblasts while also inhibiting the growth of Pseudomonas aeruginosa, a common lung pathogen. Our MMSC ECM nanoparticles display characteristics of healing injured lungs while preventing bacterial infection, which can increase recovery time.

Amniotic fluid mesenchymal stromal cells (AF-MSCs) represent an autologous cell source to ameliorate congenital heart defects (CHDs) in children. The AF-MSCs, having cardiomyogenic potential and being of fetal origin, may reflect the physiological and pathological changes in the fetal heart during embryogenesis. Hence, the study of defects in the functional properties of these stem cells during fetal heart development will help obtain a better understanding of the cause of neonatal CHDs.Therefore, in the present study, we compared the proliferative and cardiomyogenic potential of AF-MSCs derived from ICHD fetuses (ICHD AF-MSCs) with AF-MSCs from structurally normal fetuses (normal AF-MSCs). Compared to normal AF-MSCs, the ICHD AF-MSCs showed comparable immunophenotypic MSC marker expression and adipogenic and chondrogenic differentiation potential, with decreased proliferation, higher senescence, increased expression of DNA-damaged genes, and osteogenic differentiation potential. Furthermore, the expression of cardiac progenitor markers (PDGFR-α, VEGFR-2, and SSEA-1), cardiac transcription factors (GATA-4, NKx 2-5, ISL-1, TBX-5, TBX-18, and MeF-2C), and cardiovascular markers (cTNT, CD31, and α-SMA) were significantly reduced in ICHD AF-MSCs. Overall, these results suggest that the AF-MSCs of ICHD fetuses have proliferation defects with significantly decreased cardiomyogenic differentiation potential. Thus, these defects in ICHD AF-MSCs highlight that the impaired heart development in ICHD fetuses may be due to defects in the stem cells associated with heart development during embryogenesis.

Inflammatory bowel disease (IBD) belongs to the group of diseases characterized by idiopathic inflammation of the digestive tract organs. Two basic IBD types are distinguished: ulcerative colitis and Crohns disease. The IBD symptoms including vomiting and diarrhea, abdominal pain, rectal bleeding, anemia have a significant negative impact on the general patients state of health. Besides, IBD patients are susceptible to the risk of a number of serious diseases such as colorectal cancer, thrombosis and primary sclerosing cholangitis. More than 4 million people in the USA and Europe suffer from IBD, with 70000 new cases diagnosed yearly in the USA only. In some cases, a surgical removal of the damaged digestive tract fragments is required to treat severe IBD forms. However, drug therapy of IBD has mainly been used in the last decades. The rate of remission with application of traditional IBD therapy is estimated as 20-30%, and is still no higher than 50% with the combined therapy. Cell therapy has been proven to be a very promising approach in the IBD treatment. In our review, we discuss mesenchymal stromal cells (MSC) and the most important preclinical and clinical results of their application for the IBD therapy.

Background and objectives Despite significant improvement in the treatment of tendon injuries, the full tissue recovery is often not possible because of its limited ability to auto-repair. The transplantation of mesenchymal stromal cells (MSCs) is considered as a novel approach in the treatment of tendinopathies. The question about the optimal culture conditions remains open. In this study we aimed to investigate if serum reduction, L-ascorbic acid supplementation or a combination of both factors can induce tenogenic differentiation of human adipose-derived MSCs (ASCs). Methods and results Human ASCs from 3 healthy donors were used in the study. The tested conditions were: 0.5 mM of ascorbic acid 2-phosphate (AA-2P), reduced serum content (2% FBS) or combination of these two factors. The combination of AA-2P and 2% FBS was the only experimental condition that caused a significant increase of the expression of all analyzed genes related to tenogenesis (SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3, DECORIN) in comparison to the untreated control (evaluated by RT-PCR, 5th day of experiment). Moreover, this treatment significantly increased the synthesis of SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3 proteins at the same time point (evaluated by Western blot method). Double immunocytochemical staining revealed that AA-2P significantly increased the extracellular deposition of both types of collagens. Semi-quantitative Electron Spin Resonance analysis of ascorbyl free radical revealed that AA-2P do not induce harmful transition metals-driven redox reactions in cell culture media. Conclusions Obtained results justify the use of reduced content of serum with the addition of 0.5 mM of AA-2P in tenogenic inducing media.

Cell‐based therapies hold promise to substantially curb complications from extreme preterm birth, the main cause of death in children below the age of 5 years. Exciting preclinical studies in experimental neonatal lung injury have provided the impetus for the initiation of early phase clinical trials in extreme preterm infants at risk of developing bronchopulmonary dysplasia. Clinical translation of promising therapies, however, is slow and often fails. In the adult population, results of clinical trials so far have not matched the enticing preclinical data. The neonatal field has experienced many hard‐earned lessons with the implementation of oxygen therapy or postnatal steroids. Here we briefly summarize the preclinical data that have permitted the initiation of early phase clinical trials of cell‐based therapies in extreme preterm infants and describe the INCuBAToR concept (Innovative Neonatal Cellular Therapy for Bronchopulmonary Dysplasia: Accelerating Translation of Research), an evidence‐based approach to mitigate the risk of translating advanced therapies into this vulnerable patient population. The INCuBAToR addresses several of the shortcomings at the preclinical and the clinical stage that usually contribute to the failure of clinical translation through (a) systematic reviews of preclinical and clinical studies, (b) integrated knowledge transfer through engaging important stakeholders early on, (c) early economic evaluation to determine if a novel therapy is viable, and (d) retrospective and prospective studies to define and test ideal eligibility criteria to optimize clinical trial design. The INCuBAToR concept can be applied to any novel therapy in order to enhance the likelihood of success of clinical translation in a timely, transparent, rigorous, and evidence‐based fashion., The INCuBAToR is a framework built into the classical clinical translation pathway from laboratory discovery to human trials. It is designed to mitigate the risk of translating cell therapies into the clinic by providing an evidence‐based approach. Systematic reviews, integrated knowledge translation, early economic evaluation, and retro‐ and prospective observational cohort studies can substantially enhance success of the clinical translation.

Background aim Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation. Methods The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers. Results As of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32–100%) at 12 weeks and early relief of pain. Conclusions The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.

Kidney diseases are an important medical problem. Kidney injuries are accompanied by oxidative stress, cell death, capillary destruction, inflammation and fibrosis. Mesenchymal stromal cells (MSCs) have a complex effect on the regeneration by producing various regulatory molecules, including those inside extracellular vesicles, and therefore are considered as a promising therapeutic resource for cell therapy of kidney diseases. Their renoprotective effect has been shown in different experimental models, but the results of the clinical trials are ambiguous. Clinical use of MSCs is complicated by their low survival rate in the injured kidney, potential immunogenicity, tumorogenicity and fibrogenicity. Cell-free therapy with the secretory products of MSCs such as conditioned environments or extracellular vesicles is a promising direction for using their regenerative potential. However, introduction of MSCs and their secretory products into medical practice requires further research into the mechanisms of their proregenerative action, improvement of cultivation protocols, and more clinical trials.

Summary Cellular therapy is a promising tool for improving the outcome of organ transplantation. Various cell types with different immunoregulatory and regenerative properties may find application for specific transplant rejection or injury‐related indications. The current era is crucial for the development of cellular therapies. Preclinical models have demonstrated the feasibility of efficacious cell therapy in transplantation, early clinical trials have shown safety of several of these therapies, and the first steps towards efficacy studies in humans have been made. In this review, we address the current state of the art of cellular therapies in clinical transplantation and discuss monitoring tools and endpoints for these studies.

Organ preservation is a prerequisite for an urgent increase in the availability of organs for solid organ transplantation (SOT). An increasing amount of expanded criteria donor (ECD) organs are used clinically. Currently, the paradigm of organ preservation is shifting from simple reduction of cellular metabolic activity to maximal simulation of an ex vivo physiological microenvironment. An ideal organ preservation technique should not only preserve isolated organs but also offer the possibility of rehabilitation and evaluation of organ function prior to transplantation. Based on the fact that mesenchymal stromal cells (MSCs) possess strong regeneration properties, the combination of MSCs with machine perfusion (MP) is expected to be superior to conventional preservation methods. In recent years, several studies have attempted to use this strategy for SOT showing promising outcomes. With better organ function during ex vivo preservation and the potential of utilization of organs previously deemed untransplantable, this strategy is meaningful for patients with organ failure to help overcome organ shortage in the field of SOT.

High mobility group (HMG)A proteins are nonhistone chromatin proteins that bind to the minor groove of DNA, interact with transcriptional machinery, and facilitate DNA‐directed nuclear processes. HMGA1 has been shown to regulate genes involved with systemic inflammatory processes. We hypothesized that HMGA1 is important in the function of mesenchymal stromal cells (MSCs), which are known to modulate inflammatory responses due to sepsis. To study this process, we harvested MSCs from transgenic (Tg) mice expressing a dominant‐negative (dn) form of HMGA1 in mesenchymal cells. MSCs harvested from Tg mice contained the dnHMGA1 transgene, and transgene expression did not change endogenous HMGA1 levels. Immunophenotyping of the cells, along with trilineage differentiation revealed no striking differences between Tg and wild‐type (WT) MSCs. However, Tg MSCs growth was decreased compared with WT MSCs, although Tg MSCs were more resistant to oxidative stress‐induced death and expressed less IL‐6. Tg MSCs administered after the onset of Escherichia coli‐induced sepsis maintained their ability to improve survival when given in a single dose, in contrast with WT MSCs. This survival benefit of Tg MSCs was associated with less tissue cell death, and also a reduction in tissue neutrophil infiltration and expression of neutrophil chemokines. Finally, Tg MSCs promoted bacterial clearance and enhanced neutrophil phagocytosis, in part through their increased expression of stromal cell‐derived factor‐1 compared with WT MSCs. Taken together, these data demonstrate that expression of dnHMGA1 in MSCs provides a functional advantage of the cells when administered during bacterial sepsis., Graphical Abstract Administration of adipose‐derived MSCs expressing a dominant‐negative HMGA1 transgene results in less tissue injury and inflammation during a systemic infection, while improving neutrophil phagocytosis.

Colorectal cancer (CRC) is the third most commonly occurring cancer. While there have been improvements in treatment options for patients, it remains the second leading cause of cancer-related deaths worldwide. CRC develops in a complex multicellular microenvironment, with patients diagnosed with mesenchymal-rich tumours having the lowest disease-free survival. However, the role mesenchymal stromal cells (MSCs) play in tumour promotion and immune invasion has yet to be fully elucidated. Macrophages are the most abundant immune cell type found in CRC, and following the promising results from T cell targeting immunotherapies, macrophages are now under investigation for their potential to be target therapeutically in CRC. The focus of my PhD project was to investigate the role of the inflammatory tumour microenvironment on stromal cell-mediated modulation of macrophage phenotype and function. Inflammatory tumour conditioned media (TCM) generated from CRC cells treated with TNF-α increased the mRNA expression of immunomodulatory factors and pathways related to the regulation of immune cell function in Balb/c MSCs. Inflammatory tumour conditioned MSCdisplayed increased surface expression of the immunomodulatory markers PD-L1 and CD47. CD47 expression was found to be higher on MSC than on cancer cells, while PD-L1 levels were comparable. TCM generated from treating CRC cells with low dose cyclophosphamide (CTX) significantly increased the expression of MSC PD-L1 and CD47. These induced changes in the MSCs were mediated by changes in the cancer cell secretome. Bioinformatic and immunohistochemical analysis of CRC tumours revealed an association between MSCs and macrophages in the tumour microenvironment. Macrophages that were co-cultured with control, TCM or inflammatory TCM treated MSCs showed reduced expression of SIRPα, MHC II, arginase1 and IL-10 while increasing CD206 expression and a suppression of macrophage phagocytic capacity. Targeting PD-1 and SIRPα with receptor blocking antibodies was used to assess if MSC PD-L1 or CD47 were modulating the changes in macrophage phenotype and function. Neither targeting PD-1 nor CD47 altered MSC mediated changes in macrophage expression profile. However, blocking both PD-1 and SIRPα showed a strong trend towards the restoration of macrophage phagocytosis. Furthermore, blocking SIRP-α led to reduced TNF-α secretion following co-culture of macrophages with inflammatory conditioned MSCs. Following from this, a viable 3D hydrogel system containing CRC cells, MSCs and monocytes was developed to better study the complex tumour microenvironment in a more physiologically relevant model. The addition of monocytes and MSCs to the culture system alters the mRNA expression of ECM remodelling proteins fibronectin 1 and MMP2 and altered cancer cell expression of PD-L1, CD47 and EGFR. The presence of MSCs in the 3D model increased the expression of tumour promoting molecules such as serpin E1, CXCL12 and macrophage inhibitory factor. The secretome was further altered by treatment with TNF-α, resulting in the induction of different cytokines and chemokines including IL-6 and GM-CSF. The treatment of the 3D CRC model with PD153035, an EGFR inhibitor, revealed that hydrogels containing MSCs responded differently to this treatment. Taken together, this data demonstrates the role of inflammation on the ability of CRC cells to modulate the mesenchymal stromal compartment of the tumour and how stromal cells, in particular, MSCs, can alter macrophage phenotype and function. We have demonstrated that targeting the PD-L1/PD-1 or CD47/SIRPα signalling axis in MSC-rich inflammatory CRC tumours may aid the restoration of macrophage phagocytosis. Finally, we have a developed a 3D model to study the complex interactions that occur in CRC and that can be used to identify novel targets and test novel therapies. Overall, this project demonstrates that stromal cells in the inflammatory tumour microenvironment can influence macrophage function through the expression of PD-L1 and CD47. We propose that these factors could represent novel therapeutic targets in stromal rich CRC. 2026-06-27

Producción Científica, Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)–based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti–human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti–CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p, Spanish Government (RTI2018-096320-B-C22, FPU16-04015, PID2019-110709RB-I00, PID2020-118669RA-I00), Interreg V España Portugal POCTEP (0624_2IQBIONEURO_6_E), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Consejería de Educación de Castilla y León (CAS079P17), Instituto de Salud Carlos III (ISCIII) (PI19/01630), Programs of ISCIII- European Regional Development Fund (RD16/0011/0015, RD21/ 0017/0006)

Background Maternal blood pressure levels reflect cardiovascular adaptation to pregnancy and proper maternal-fetal exchanges through the placenta and are very sensitive to numerous environmental stressors. Maternal hypertension during pregnancy has been associated with impaired placental functions and with an increased risk for children to suffer from cardiovascular and respiratory diseases later on. Investigating changes in placental DNA methylation levels and cell-type composition in association with maternal blood pressure could help elucidate its relationships with placental and fetal development. Methods Taking advantage of a large cohort of 666 participants, we investigated the association between epigenome-wide DNA methylation patterns in the placenta, measured using the Infinium HumanMethylation450 BeadChip, placental cell-type composition, estimated in silico, and repeated measurements of maternal steady and pulsatile blood pressure indicators during pregnancy. Results At the site-specific level, no significant association was found between maternal blood pressure and DNA methylation levels after correction for multiple testing (false discovery rate < 0.05), but 5 out of 24 previously found CpG associations were replicated (p-value < 0.05). At the regional level, our analyses highlighted 64 differentially methylated regions significantly associated with at least one blood pressure component, including 35 regions associated with mean arterial pressure levels during late pregnancy. These regions were found enriched for genes implicated in lung development and diseases. Further mediation analyses show that a significant part of the association between steady blood pressure—but not pulsatile pressure—and placental methylation can be explained by alterations in placental cell-type composition. In particular, elevated blood pressure levels are associated with a decrease in the ratio between mesenchymal stromal cells and syncytiotrophoblasts, even in the absence of preeclampsia. Conclusions This study provides the first evidence that the association between maternal steady blood pressure during pregnancy and placental DNA methylation is both direct and partly explained by changes in cell-type composition. These results could hint at molecular mechanisms linking maternal hypertension to lung development and early origins of childhood respiratory problems and at the importance of controlling maternal blood pressure during pregnancy.

Background Mesenchymal stromal cells (MSCs) may be of benefit in ARDS due to immunomodulatory and reparative properties. This trial investigates a novel CD362 enriched umbilical cord derived MSC product (REALIST ORBCEL-C), produced to Good Manufacturing Practice standards, in patients with moderate to severe ARDS due to COVID-19 and ARDS due to other causes. Methods Phase 1 is a multicentre open-label dose-escalation pilot trial. Patients will receive a single infusion of REALIST ORBCEL-C (100 × 106 cells, 200 × 106 cells or 400 × 106 cells) in a 3 + 3 design. Phase 2 is a multicentre randomised, triple blind, allocation concealed placebo-controlled trial. Two cohorts of patients, with ARDS due to COVID-19 or ARDS due to other causes, will be recruited and randomised 1:1 to receive either a single infusion of REALIST ORBCEL-C (400 × 106 cells or maximal tolerated dose in phase 1) or placebo. Planned recruitment to each cohort is 60 patients. The primary safety outcome is the incidence of serious adverse events. The primary efficacy outcome is oxygenation index at day 7. The trial will be reported according to the Consolidated Standards for Reporting Trials (CONSORT 2010) statement. Discussion The development and manufacture of an advanced therapy medicinal product to Good Manufacturing Practice standards within NHS infrastructure are discussed, including challenges encountered during the early stages of trial set up. The rationale to include a separate cohort of patients with ARDS due to COVID-19 in phase 2 of the trial is outlined. Trial registration ClinicalTrials.gov NCT03042143. Registered on 3 February 2017. EudraCT Number 2017-000584-33

In regenerative medicine related to orthopedic conditions, mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) have been proposed as innovative clinical options. The definition of EV-shuttled signals and their modulation under orthopedic settings, such as osteoarthritis (OA), is crucial for MSC-related research, both for basic science and for use in clinical settings, either as therapeutics or as producers of cell-free products such as EVs or secretome. The objective of this work is to compare the literature available on high-throughput EV-miRNA data obtained from adipose-derived MSCs (ASCs) in standard conditions or cultured in high levels of IFNγ, low-level inflammatory conditions mimicking OA synovial fluid (SF), and OA-SF. The first result was that both IFNγ and low-level inflammatory treatment led to an increase, whereas SF led to a reduction in EV release. Second, more than 200 EV-miRNAs were found to be shared across the different conditions. After a bioinformatics search through experimentally validated and OA-related targets, pathways and tissues, several miRNAs resulted in the restoration of cartilage and synovium stability and the homeostasis of inflammatory cells, including macrophages, promoting their switch towards an M2 anti-inflammatory phenotype. Third, IFNγ and especially SF culturing were able to modulate the overall EV-miRNA fingerprint, although the main molecular messages related to OA resulted conserved between treatments with the majority of modulations within 2-fold range. In conclusion, ASC EV-miRNAs may be modulated in their overall landscape by OA-related culturing conditions albeit resulted largely stable in their specific OA-protective signals allowing for a faster clinical translation of these new cell-free therapies for joint diseases.

The clinical translation of mesenchymal stromal cell (MSC)-based therapies remains challenging due to rapid cell death and poor control over cell behavior. Compared to monodisperse cells, the aggregation of MSCs into spheroids increases their tissue-forming potential by promoting cell-cell interactions. However, MSCs initially lack engagement with an endogenous extracellular matrix (ECM) when formed into spheroids. Previously the instructive nature of an engineered, cell-secreted ECM is demonstrated to promote survival and differentiation of adherent MSCs. Herein, it is hypothesized that the incorporation of this cell-secreted ECM during spheroid aggregation would enhance MSC osteogenic potential by promoting cell-matrix and cell-cell interactions. ECM-loaded spheroids contained higher collagen and glycosaminoglycan content, and MSCs exhibited increased mechanosensitivity to ECM through Yes-associated protein (YAP) activation via integrin α2β1 binding. ECM-loaded spheroids sustained greater MSC viability and proliferation and are more responsive to soluble cues for lineage-specific differentiation than spheroids without ECM or loaded with collagen. The encapsulation of ECM-loaded spheroids in instructive alginate gels resulted in spheroid fusion and enhanced osteogenic differentiation. These results highlight the clinical potential of ECM-loaded spheroids as building blocks for the repair of musculoskeletal tissues.

Despite the advent of promising technologies in tissue engineering, finding a biomimetic 3D bio-construct capable of enhancing cell attachment, maintenance, and function is still a challenge in producing tailorable scaffolds for bone regeneration. Here, osteostimulatory effects of the butterfly wings as a naturally porous and non-toxic chitinous scaffold on mesenchymal stromal cells are assessed. The topographical characterization of the butterfly wings implied their ability to mimic bone tissue microenvironment, whereas their regenerative potential was validated after a 14-day cell culture. In vivo analysis showed that the scaffold induced no major inflammatory response in Wistar rats. Topographical features of the bioconstruct upregulated the osteogenic genes, including COL1A1, ALP, BGLAP, SPP1, SP7, and AML3 in differentiated cells compared to the cells cultured in the culture plate. However, butterfly wings were shown to provide a biomimetic microstructure and proper bone regenerative capacity through a unique combination of various structural and material properties. Therefore, this novel platform can be confidently recommended for bone tissue engineering applications.

The mesenchymal stromal cells (MSCs) residing within the stromal component of visceral adipose tissue appear to be greatly affected by obesity, with impairment of their functions and presence of senescence. To gain further insight into these phenomena, we analyzed the changes in total proteome content and secretome of mouse MSCs after a high-fat diet (HFD) treatment compared to a normal diet (ND). In healthy conditions, MSCs are endowed with functions mainly devoted to vesicle trafficking. These cells have an immunoregulatory role, affecting leukocyte activation and migration, acute inflammation phase response, chemokine signaling, and platelet activities. They also present a robust response to stress. We identified four signaling pathways (TGF-β, VEGFR2, HMGB1, and Leptin) that appear to govern the cells’ functions. In the obese mice, MSCs showed a change in their functions. The immunoregulation shifted toward pro-inflammatory tasks with the activation of interleukin-1 pathway and of Granzyme A signaling. Moreover, the methionine degradation pathway and the processing of capped intronless pre-mRNAs may be related to the inflammation process. The signaling pathways we identified in ND MSCs were replaced by MET, WNT, and FGFR2 signal transduction, which may play a role in promoting inflammation, cancer, and aging © 2020. Acar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. All Rights Reserved. The mesenchymal stromal cells (MSCs) residing within the stromal component of visceral adipose tissue appear to be greatly affected by obesity, with impairment of their functions and presence of senescence. To gain further insight into these phenomena, we analyzed the changes in total proteome content and secretome of mouse MSCs after a high-fat diet (HFD) treatment compared to a normal diet (ND). In healthy conditions, MSCs are endowed with functions mainly devoted to vesicle trafficking. These cells have an immunoregulatory role, affecting leukocyte activation and migration, acute inflammation phase response, chemokine signaling, and platelet activities. They also present a robust response to stress. We identified four signaling pathways (TGF-β, VEGFR2, HMGB1, and Leptin) that appear to govern the cells’ functions. In the obese mice, MSCs showed a change in their functions. The immunoregulation shifted toward pro-inflammatory tasks with the activation of interleukin-1 pathway and of Granzyme A signaling. Moreover, the methionine degradation pathway and the processing of capped intronless pre-mRNAs may be related to the inflammation process. The signaling pathways we identified in ND MSCs were replaced by MET, WNT, and FGFR2 signal transduction, which may play a role in promoting inflammation, cancer, and aging © 2020. Acar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. All Rights Reserved. Erciyes University Research Project Fund BAP-FYL-2017-7399 Università degli Studi della Campania Luigi Vanvitelli CUP B23D18000250007