[Prerequisite and organisation of health-care pathways for Cell and Gene therapies, using Mesenchymal Stromal Cells (MSC) or Chimeric Antigen Receptor (CAR) T cells, in patients with autoimmune systemic diseases]. / Prérequis et organisation du parcours de soins en vue de l'utilisation d'une thérapie cellulaire ou génique par cellules stromales mésenchymateuses (CSM) ou par cellules T porteuses d'un récepteur antigénique chimérique (CAR-T cells) chez les patients avec maladies auto-immunes systémiques.

First-line treatments of autoimmune systemic diseases (ARD) are based on the use of various types of immunosuppressive or immunomodulatory drugs, either alone or in association, according to standardized reference protocols. Prolonged use of these drugs in severe or refractory ARD is associated with high morbidity and increased mortality. Innovative cell therapies represent a new promising approach for patients with ARDs, with the recent clinical use of: a) mesenchymal stromal cells (MSCs), based on their immunomodulatory, antifibrotic and pro-angiogenic properties and b) Chimeric Antigen Receptors (CAR) T cell therapies T lymphocytes, where genetically modified expression of a chimeric antigen receptor (CAR-T cells). Therapeutic use of MSC or CAR-T cells, remains indications of exception in patients with severe ARDs resistant to prior standard therapies with new prerequisite and organisation of health-care pathways as compared to traditional drugs, not only for the Cell and Gene Therapy (CGT) product definition and delivery process, but also for the patient clinical management before and after administration of the CGT product. The aim of this workshop under the auspices of the French Speaking Society of Bone Marrow and Cell transplantation (SFGM-TC) working group on autoimmune diseases (MATHEC) is to describe: a) the prerequisite for French hospitals to set-up the specific health-care pathways for MSC or CART therapy in ARDs patients, in accordance with regulatory and safety needs to perform academic or industry sponsored clinical trials, and b) the care-pathway for ARD patients treated with CGT, highlighting the importance of working in tandem between the ARD and the CAR-T cell specialist all along the indication, procedures and follow-up of ARDs. Patient safety considerations are central to guidance on patient selection to be validated collectively at the multidisciplinary team meeting (MDTM) based on recent (less than 3 months) thorough patient evaluation. MSC and CAR-T procedural aspects and follow-up are then carried out within appropriately experienced and SFGM-TC accredited centres in close collaboration with the ADs specialist.

Comparative study of systemic and local delivery of mesenchymal stromal cells for the treatment of chronic kidney disease.

Renal fibrosis, characterized by excessive extracellular matrix accumulation, leads to a progressive decline of renal function and is a common endpoint of chronic kidney disease (CKD). Current treatments primarily focus on managing underlying diseases, offering limited direct intervention for the fibrotic process. This study explores the anti-fibrotic potential of human adipose-derived mesenchymal stromal cells (MSCs) and their derived extracellular vesicles (EVs) in the context of CKD, emphasizing the effects of systemic versus local delivery methods. Preconditioned MSCs (Pr-MSCs) were treated with TNF-α and IFN-γ to enhance their immunomodulatory capabilities, and demonstrated significant anti-fibrotic effects in vitro, reducing mRNA expression of fibrosis markers in TGF-ß stimulated HKC-8 cells. Our in vivo findings from a murine unilateral ureteral obstruction (UUO) model of CKD showed that local deliveries of Pr-MSCs reduced collagen deposition and increased expression of the anti-inflammatory cytokine IL-10. Systemic administration of Pr-MSCs did not show any significant effect on UUO-induced injury. In addition, EVs did not replicate the anti-fibrotic effects observed with their parent cells, suggesting that soluble proteins or metabolites secreted by Pr-MSCs might be the primary mediators of the anti-fibrotic and immunomodulatory effects. This study provides critical insights into the therapeutic efficacy of MSCs, highlighting the importance of delivery methods and the potential of preconditioning strategies in enhancing MSC-based therapies for renal fibrosis.

Understanding and controlling the variables for stromal vascular fraction therapy.

In regenerative medicine, the isolation of mesenchymal stromal cells (MSCs) from the adipose tissue's stromal vascular fraction (SVF) is a critical area of study. Our review meticulously examines the isolation process of MSCs, starting with the extraction of adipose tissue. The choice of liposuction technique, anatomical site, and immediate processing are essential to maintain cell functionality. We delve into the intricacies of enzymatic digestion, emphasizing the fine-tuning of enzyme concentrations to maximize cell yield while preventing harm. The review then outlines the filtration and centrifugation techniques necessary for isolating a purified SVF, alongside cell viability assessments like flow cytometry, which are vital for confirming the efficacy of the isolated MSCs. We discuss the advantages and drawbacks of using autologous vs allogeneic SVF sources, touching upon immunocompatibility and logistical considerations, as well as the variability inherent in donor-derived cells. Anesthesia choices, the selection between hypodermic needles vs liposuction cannulas, and the role of adipose tissue lysers in achieving cellular dissociation are evaluated for their impact on SVF isolation. Centrifugation protocols are also analyzed for their part in ensuring the integrity of the SVF. The necessity for standardized MSC isolation protocols is highlighted, promoting reproducibility and successful clinical application. We encourage ongoing research to deepen the understanding of MSC biology and therapeutic action, aiming to further the field of regenerative medicine. The review concludes with a call for rigorous research, interdisciplinary collaboration, and strict adherence to ethical and regulatory standards to safeguard patient safety and optimize treatment outcomes with MSCs.

Microvesicles derived from mesenchymal stem cells inhibit acute respiratory distress syndrome-related pulmonary fibrosis in mouse partly through hepatocyte growth factor.

BACKGROUND: Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome (ARDS) patients. Mesenchymal stromal cell-derived microvesicles (MSC-MVs) have been shown to exert antifibrotic effects in lung diseases. AIM: To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models. METHODS: MSC-MVs with low hepatocyte growth factor (HGF) expression (siHGF-MSC-MVs) were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model. Following intubation, respiratory mechanics-related indicators were measured via an experimental small animal lung function tester. Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging. Immunohistochemical, western blotting, ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators. RESULTS: The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice. Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores. However, low expression of HGF (siHGF-MSC-MVs) significantly inhibited the effects of MSC-MVs (P < 0.05). Compared with the ARDS pulmonary fibrosis group, the MSC-MVs group exhibited suppressed expression of type I collagen antigen, type III collagen antigen, and the proteins transforming growth factor-ß and α-smooth muscle actin, whereas the siHGF-MVs group exhibited significantly increased expression of these proteins. In addition, pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group, and the effects of the MSC-MVs were significantly inhibited by low HGF expression (all P < 0.05). CONCLUSION: MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.

Differential effects of TLR3 and TLR4 activation on MSC-mediated immune regulation.

Mesenchymal stromal cells (MSCs) have evolved as an invaluable therapeutic cell type due to their broad therapeutic properties. Bone marrow-derived MSCs are currently being applied in numerous clinical trials, and the initial results have been encouraging. However, heterogeneous responsiveness amongst patients is also being experienced; therefore, the efficacy of MSCs in vivo is still debatable. Host microenvironment plays an essential role in determining the fate of MSCs in vivo. Recent studies have indicated the role of toll-like receptors (TLR) in modulating the biological properties of MSCs. TLRs are expressed by MSCs, and activation of TLR3 and TLR4 can alter the functionality of MSCs. While MSCs can suppress the effector and memory T cell function by promoting regulatory T cells, the effect of TLR activation on MSC-mediated immune cell induction is still not well understood. This study was performed to understand the TLR licensing of MSCs and its impact on MSC-mediated immunomodulation. We found that TLR3 mediated activation of MSCs (TLR3-MSCs) increased the expression of G-CSF & IL-10 while TLR4-mediated activation of MSCs led to an increase in CXCL-1, CXCL-10, and CXCL-12. To study the immunological aspect, an in vitro co-culture model was established-to imitate the brief in vivo interaction of MSCs and immune cells. We found that TLR3-MSCs led to increase in CD4 and CD8 naive T (TNAI) cells and vice versa for effector (TEFF) and memory T (TMEM) cells, while TLR4-MSCs did not show any effect. Moreover, only TLR3-MSCs led to a non-significant increase in the regulatory T cells (TREGS) and Double negative regulatory cells. No change in B cell profile was evident while TLR3-MSCs depicted an increasing trend in regulatory B cells which was not statistically significant. TLR3 MSCs also inhibited the T cell proliferation in our setup. Our data indicate that TLR3 priming may regulate the function of MSCs through immunomodulation. Understanding the role of TLRs and other microenvironmental factors causing subdued responses of MSCs in vivo would allow the uninhibited use of MSCs for many diseased conditions.

Mechanoinduction of PTHrP/cAMP-signaling governs proteoglycan production in mesenchymal stromal cell-derived neocartilage.

Abnormal mechanical loading is one of the major risk factors for articular cartilage degeneration. Engineered mesenchymal stromal cell (MSC)-derived cartilage holds great promise for cell-based cartilage repair. However, physiological loading protocols were shown to reduce matrix synthesis of MSC-derived neocartilage in vitro and the regulators of this undesired mechanoresponse remain poorly understood. Parathyroid hormone-related protein (PTHrP) is involved in cartilage development and can affect extracellular matrix (ECM) production during MSC chondrogenesis opposingly, depending on a continuous or transient exposure. PTHrP is induced by various mechanical cues in multiple tissues and species; but whether PTHrP is regulated in response to loading of human engineered neocartilage and may affect matrix synthesis in a positive or negative manner is unknown. The aim of this study was to investigate whether dynamic loading adjusts PTHrP-signaling in human MSC-derived neocartilage and whether it regulates matrix synthesis and other factors involved in the MSC mechanoresponse. Interestingly, MSC-derived chondrocytes significantly upregulated PTHrP mRNA (PTHLH) expression along with its second messenger cAMP in response to loading in our custom-built bioreactor. Exogenous PTHrP(1-34) induced the expression of known mechanoresponse genes (FOS, FOSB, BMP6) and significantly decreased glycosaminoglycan (GAG) and collagen synthesis similar to loading. The adenylate-cyclase inhibitor MDL-12,330A rescued the load-mediated decrease in GAG synthesis, indicating a direct involvement of cAMP-signaling in the reduction of ECM production. According to COL2A1-corrected hypertrophy-associated marker expression, load and PTHrP treatment shared the ability to reduce expression of MEF2C and PTH1R. In conclusion, the data demonstrate a significant mechanoinduction of PTHLH and a negative contribution of the PTHrP-cAMP signaling axis to GAG synthesis in MSC-derived chondrocytes after loading. To improve ECM synthesis and the mechanocompetence of load-exposed neocartilage, inhibition of PTHrP activity should be considered for MSC-based cartilage regeneration strategies.

Quality Assessment of Long-Term Cryopreserved Human Bone-Derived Marrow Mesenchymal Stromal Cell Samples: Experience from the Texas Heart Institute Biorepository and Biospecimen Profiling Core.

In biomedical research, biorepositories are pivotal resources that safeguard and supply clinical samples for scientific investigators. Proper long-term cryopreservation conditions are essential to maintain biospecimen quality. In this study, we analyzed the efficacy of sample cryopreservation at the Texas Heart Institute Biorepository and Biospecimen Profiling Core (THI-BRC). Our assessments included a thorough review of internal processes, quality reports, and both internal and external audit outcomes. We examined the integrity of human bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) that were cryopreserved for over 5 years. These samples originated from randomly selected clinical trial participants or commercially sourced cell lines. Parameters such as cell viability, DNA and RNA integrity, population doubling time, sterility, and BM-MSC-specific attributes such as surface antigen expression and differentiation potential were studied. BM-MSC samples cryopreserved for ∼6 months served as our control. Our results demonstrated that the 5-year cryopreserved samples maintained their integrity compared with the shorter-term stored control samples. Moreover, THI-BRC has met accreditation agency standards and has not received any repeated deficiencies over 7 years. Collectively, our findings affirm that THI-BRC's biospecimen storage protocols align with accepted standards as confirmed by the quality assessment of long-term stored clinical samples.

Machine learning reveals the rules governing the efficacy of mesenchymal stromal cells in septic preclinical models.

BACKGROUND: Mesenchymal Stromal Cells (MSCs) are the preferred candidates for therapeutics as they possess multi-directional differentiation potential, exhibit potent immunomodulatory activity, are anti-inflammatory, and can function like antimicrobials. These capabilities have therefore encouraged scientists to undertake numerous preclinical as well as a few clinical trials to access the translational potential of MSCs in disease therapeutics. In spite of these efforts, the efficacy of MSCs has not been consistent-as is reflected in the large variation in the values of outcome measures like survival rates. Survival rate is a resultant of complex cascading interactions that not only depends upon upstream experimental factors like dosage, time of infusion, type of transplant, etc.; but is also dictated, post-infusion, by intrinsic host specific attributes like inflammatory microniche including proinflammatory cytokines and alarmins released by the damaged host cells. These complex interdependencies make a researcher's task of designing MSC transfusion experiments challenging. METHODS: In order to identify the rules and associated attributes that influence the final outcome (survival rates) of MSC transfusion experiments, we decided to apply machine learning techniques on manually curated data collected from available literature. As sepsis is a multi-faceted condition that involves highly dysregulated immune response, inflammatory environment and microbial invasion, sepsis can be an efficient model to verify the therapeutic effects of MSCs. We therefore decided to implement rule-based classification models on data obtained from studies involving interventions of MSCs in sepsis preclinical models. RESULTS: The rules from the generated graph models indicated that survival rates, post-MSC-infusion, are influenced by factors like source, dosage, time of infusion, pre-Interleukin-6 (IL-6)/ Tumour Necrosis Factor- alpha (TNF-α levels, etc. CONCLUSION: This approach provides important information for optimization of MSCs based treatment strategies that may help the researchers design their experiments.

Effectiveness of autologous emulsified stromal vascular fraction tissue injection for the treatment of complex perianal fistulas in inflammatory bowel diseases patients: a pilot study.

Complex fistulizing perianal disease is a disabling manifestation of inflammatory bowel disease (IBD), seriously compromising patients 'quality of life'. The success rate of available treatments is quite low, and nearly half of the patients will develop chronically active fistulas or experience fistula recurrence. Mesenchymal stem cell therapy has shown interesting results, but the complexity and the cost of production limit its widespread use. This study aims to report the results of the innovative use of autologous emulsified adipose-derived stromal vascular fraction tissue for treating complex fistulizing perianal disease. From March 2021 to March 2022, 10 patients underwent a two-step procedure: (1) examination under anaesthesia, with loose seton drainage and 4 weeks later and (2) curettage of the fistulous tract, internal fistula closure and an injection of autologous emulsified adipose-derived stromal vascular fraction tissue harvested from the subcutaneous layer of the patient's hip. Clinical and radiological (through magnetic resonance imaging) healing were assessed at 6 months. We included five patients affected by Crohn's disease, three by ulcerative colitis and two by indeterminate colitis. All patients were on concomitant biological therapy (50% on Infliximab). One patient required a re-treatment for a relapse and two different fistulas were separately treated in another one. Out of 12 total procedures performed, clinical healing was achieved in 10 cases (83%), while radiological healing in 6 patients (50%). No adverse events were recorded. Autologous emulsified adipose-derived stromal vascular fraction tissue can represent an effective, safe and cheap add-on therapy for patients with complex perianal fistulas in IBDs.

Morphological Changes in Tissue When Using Polypropylene Implants with Adsorbed Multipotent Stromal Cells in Experiment.

The subcutaneous tissue of rats after implantation of polypropylene materials with adsorbed bone marrow-derived mesenchymal multipotent stromal cells (MMSCs) was studied using light microscopy. Inflammation in response to implantation was mild, and the foreign material was encapsulated into a thin strip of dense fibrous connective tissue with multinucleated macrophages. By 1 year after introduction of the monofilament and 6 and 12 months after implantation of the mesh product, some threads were deformed, broken, and had sharp edges. Small fragments of foreign material appeared in the adjacent tissues surrounded by their own relatively thick acellular capsule. As a result of preliminary adsorption of MMSCs on polypropylene, the thickness of the connective tissue capsule decreased, its vascularization increased, and the severity of inflammatory infiltration decreased. However, all effects of MMSCs adsorption in rats were transient and disappeared within 1 week.

3D-Bioprinted Co-Cultures of Glioblastoma Multiforme and Mesenchymal Stromal Cells Indicate a Role for Perivascular Niche Cells in Shaping Glioma Chemokine Microenvironment.

3D bioprinting has become a valuable tool for studying the biology of solid tumors, including glioblastoma multiforme (GBM). Our analysis of publicly available bulk RNA and single-cell sequencing data has allowed us to define the chemotactic profile of GBM tumors and identify the cell types that secrete particular chemokines in the GBM tumor microenvironment (TME). Our findings indicate that primary GBM tissues express multiple chemokines, whereas spherical monocultures of GBM cells significantly lose this diversity. Subsequently, the comparative analysis of GBM spherical monocultures vs. 3D-bioprinted multicultures of cells showed a restoration of chemokine profile diversity in 3D-bioprinted cultures. Furthermore, single-cell RNA-Seq analysis showed that cells of the perivascular niche (pericytes and endocytes) express multiple chemokines in the GBM TME. Next, we 3D-bioprinted cells from two glioblastoma cell lines, U-251 and DK-MG, alone and as co-cultures with mesenchymal stromal cells (representing cells of the perivascular niche) and assessed the chemokine secretome. The results clearly demonstrated that the interaction of tumors and mesenchymal cells leads to in a significant increase in the repertoire and levels of secreted chemokines under culture in 21% O2 and 1% O2. Our study indicates that cells of the perivascular niche may perform a substantial role in shaping the chemokine microenvironment in GBM tumors.

Acceptability of Allogeneic Mesenchymal Stromal Cell-Based Tissue Engineering for the Treatment of Periodontitis: A Qualitative Study in France.

INTRODUCTION AND AIMS: Periodontitis, the main cause of tooth loss in adults, is a public health concern; its incidence increases with age, and its prevalence increases with increasing life expectancy of the population. Innovative therapies such as cell therapy represent promising future solutions for guided tissue regeneration. However, these therapies may be associated with fears and mistrust from the general public. The aim of this study was to estimate the acceptability of an advanced therapy medicinal product combining allogeneic mesenchymal stromal cells from adipose tissue with a natural fibrin hydrogel in the treatment of periodontitis. METHODS: The methodology was based on a qualitative study conducted through semi-structured interviews with patients followed for periodontitis in the Oral Medicine Department of the Toulouse University Hospital, Toulouse, France. Qualitative studies are essential methodologies to understand the patterns of health behaviours, describe illness experiences, and design health interventions in a humanistic and person-centred way of discovering. RESULTS: Eleven interviews (with 4 men and 7 women) were required to reach thematic saturation. Analysis allowed 4 main themes to emerge: (1) perception of new treatments, science, and caregivers; (2) conditions that the treatment must meet; (3) patient perception of the disease; and (4) factors related to the content of the treatment. CONCLUSIONS: Patients find cell therapy for periodontitis to be acceptable. If they express a need to be informed about the benefit/risk ratio, they are not particularly worried about side effects of the treatment, for either allogeneic or blood-derived products. Periodontitis is a prototypical model of chronic inflammatory pathology and is multitissular, with hard- and soft-tissue lesions. In a patient-centred approach, the success of cell therapy will require a bilateral, informed decision, taking into account potential therapeutic effectiveness and patient expectations for regeneration.

Histone H3K9 Methylation Features under Hypoxic Conditions after the HIF1A Knockdown in Mesenchymal Stromal Cells In Vitro.

The effects of HIF1A knockdown by RNA interference on the histone H3K9 methylation in human umbilical cord mesenchymal stromal cells in vitro under conditions of 24-h exposure to hypoxia (1% O2) were studied. Evaluation of transcriptional activity of genes involved in the regulation of H3K9 methylation (KDM3A, KDM4A, and EHMT2) and the cytofluorimetric analysis of the expression of the corresponding antigens and H3K9 methylation level demonstrated a pronounced stimulating effect of hypoxic exposure. Moreover, the expression of KDM4A and EHMT2 was regulated by HIF1A-mediated mechanism, unlike KDM3A; the level of the corresponding proteins depended on HIF1A. In addition, the HIF-1-dependent regulation of KDM3A, KDM4A, and EHMT2/G9a, and directly the H3K9 methylation level in mesenchymal stromal cells also took place under normoxia conditions.

A Nonenzymatic Procedure to Obtain Human Mesenchymal Stromal Cells from the Dermis.

Human mesenchymal stromal cells (MSCs) have gained significant interest as cell-based therapeutics for organ restoration in the field of regenerative medicine. More recently, substantial attention has been directed toward cell-free therapy, achieved through the utilization of soluble factors possessing trophic and immunomodulatory properties present in the MSC secretome. This collection of soluble factors can be found either freely in the secretome or packed within its vesicular fraction, known as extracellular vesicles (EVs). MSCs can be derived from various tissue sources, each involving different extraction methods and yielding varying cell amounts. In this study, we describe a nonenzymatic procedure for a straightforward isolation of MSCs from the fetal dermis and the adult dermis. The results demonstrate the isolation of a cell population with a uniform MSC immunophenotype from the earliest passages (approximately 90% positive for the classical MSC markers CD90, CD105, and CD73, while negative for the hematopoietic markers CD34 and CD45, as well as HLA-DR). Additionally, we describe the procedures for cell expansion, banking, and secretome collection.

The Matrix Protein Tropoelastin Prolongs Mesenchymal Stromal Cell Vitality and Delays Senescence During Replicative Aging.

Cellular senescence leads to the functional decline of regenerative cells such as mesenchymal stromal/stem cells (MSCs), which gives rise to chronic conditions and contributes to poor cell therapy outcomes. Aging tissues are associated with extracellular matrix (ECM) dysregulation, including loss of elastin. However, the role of the ECM in modulating senescence is underexplored. In this work, it is shown that tropoelastin, the soluble elastin precursor, is not only a marker of young MSCs but also actively preserves cell fitness and delays senescence during replicative aging. MSCs briefly exposed to tropoelastin exhibit upregulation of proliferative genes and concurrent downregulation of senescence genes. The seno-protective benefits of tropoelastin persist during continuous, long-term MSC culture, and significantly extend the MSC replicative lifespan. Tropoelastin-expanded MSCs further maintain youth-associated phenotype and function compared to age-matched controls, including preserved clonogenic potential, minimal senescence-associated beta-galactosidase activity, maintained cell sizes, reduced expression of senescence markers, suppressed secretion of senescence-associated factors, and increased production of youth-associated proteins. This work points to the utility of exogenously-supplemented tropoelastin for manufacturing MSCs that robustly maintain regenerative potential with age. It further reveals the active role of classical structural ECM proteins in driving cellular age-associated fitness, potentially leading to future interventions for aging-related pathologies.

Improving the future of clinical trials and translation of mesenchymal stromal cell therapies for neonatal disorders.

Despite recent advances in neonatal intensive care medicine, neonatal disorders such as (bronchopulmonary dysplasia [BPD], intraventricular hemorrhage [IVH], and hypoxic ischemic encephalopathy [HIE]) remain major causes of death and morbidity in survivors, with few effective treatments being available. Recent preclinical studies have demonstrated the pleiotropic host injury-responsive paracrine protective effects of cell therapy especially with mesenchymal stromal cells (MSCs) against BPD, IVH, and HIE. These findings suggest that MSCs therapy might emerge as a novel therapeutic modality for these currently devastating neonatal disorders with complex multifactorial etiologies. Although early-phase clinical trials suggest their safety and feasibility, their clinical therapeutic benefits have not yet been proven. Therefore, based on currently available preclinical research and clinical trial data, we focus on critical issues that need to be addressed for future successful clinical trials and eventual clinical translation such as selecting the right patient and optimal cell type, route, dose, and timing of MSCs therapy for neonatal disorders such as BPD, HIE, and IVH.

Ameliorative effects of mesenchymal stromal cells on senescence associated phenotypes in naturally aged rats.

BACKGROUND: Aging is a multifaceted process that affects all organ systems. With the increasing trend of population aging, aging-related diseases have resulted in significant medical challenges and socioeconomic burdens. Mesenchymal stromal cells (MSCs), due to their antioxidative stress, immunoregulatory, and tissue repair capabilities, hold promise as a potential anti-aging intervention. METHODS: In this study, we transplanted MSCs into naturally aged rats at 24 months, and subsequently examined levels of aging-related factors such as ß-galactosidase, superoxide dismutase, p16, p21 and malondialdehyde in multiple organs. Additionally, we assessed various aging-related phenotypes in these aged rats, including immune senescence, lipid deposition, myocardial fibrosis, and tissue damage. We also conducted a 16 S ribosomal ribonucleic acid (rRNA) analysis to study the composition of gut microbiota. RESULTS: The results indicated that MSCs significantly reduced the levels of aging-associated and oxidative stress-related factors in multiple organs such as the heart, liver, and lungs of naturally aging rats. Furthermore, they mitigated chronic tissue damage and inflammation caused by aging, reduced levels of liver lipid deposition and myocardial fibrosis, alleviated aging-associated immunodeficiency and immune cell apoptosis, and positively influenced the gut microbiota composition towards a more youthful state. This research underscores the diverse anti-aging effects of MSCs, including oxidative stress reduction, tissue repair, metabolic regulation, and improvement of immune functions, shedding light on the underlying anti-aging mechanisms associated with MSCs. CONCLUSIONS: The study confirms that MSCs hold great promise as a potential anti-aging approach, offering the possibility of extending lifespan and improving the quality of life in the elderly population.

Therapeutic efficacy of thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles on Escherichia coli-induced acute lung injury in mice.

BACKGROUND: Acute lung injury (ALI) following pneumonia involves uncontrolled inflammation and tissue injury, leading to high mortality. We previously confirmed the significantly increased cargo content and extracellular vesicle (EV) production in thrombin-preconditioned human mesenchymal stromal cells (thMSCs) compared to those in naïve and other preconditioning methods. This study aimed to investigate the therapeutic efficacy of EVs derived from thMSCs in protecting against inflammation and tissue injury in an Escherichia coli (E. coli)-induced ALI mouse model. METHODS: In vitro, RAW 264.7 cells were stimulated with 0.1 µg/mL liposaccharides (LPS) for 1 h, then were treated with either PBS (LPS Ctrl) or 5 × 107 particles of thMSC-EVs (LPS + thMSC-EVs) for 24 h. Cells and media were harvested for flow cytometry and ELISA. In vivo, ICR mice were anesthetized, intubated, administered 2 × 107 CFU/100 µl of E. coli. 50 min after, mice were then either administered 50 µL saline (ECS) or 1 × 109 particles/50 µL of thMSC-EVs (EME). Three days later, the therapeutic efficacy of thMSC-EVs was assessed using extracted lung tissue, bronchoalveolar lavage fluid (BALF), and in vivo computed tomography scans. One-way analysis of variance with post-hoc TUKEY test was used to compare the experimental groups statistically. RESULTS: In vitro, IL-1ß, CCL-2, and MMP-9 levels were significantly lower in the LPS + thMSC-EVs group than in the LPS Ctrl group. The percentages of M1 macrophages in the normal control, LPS Ctrl, and LPS + thMSC-EV groups were 12.5, 98.4, and 65.9%, respectively. In vivo, the EME group exhibited significantly lower histological scores for alveolar congestion, hemorrhage, wall thickening, and leukocyte infiltration than the ECS group. The wet-dry ratio for the lungs was significantly lower in the EME group than in the ECS group. The BALF levels of CCL2, TNF-a, and IL-6 were significantly lower in the EME group than in the ECS group. In vivo CT analysis revealed a significantly lower percentage of damaged lungs in the EME group than in the ECS group. CONCLUSION: Intratracheal thMSC-EVs administration significantly reduced E. coli-induced inflammation and lung tissue damage. Overall, these results suggest therapeutically enhanced thMSC-EVs as a novel promising therapeutic option for ARDS/ALI.

Role of the mesenchymal stromal cells in bone marrow failure of Fanconi Anemia patients.

Introduction: Fanconi anemia (FA) is an inherited disorder characterized by bone marrow failure, congenital malformations, and predisposition to malignancies. Alterations in hematopoietic stem cells (HSC) have been reported, but little is known regarding the bone marrow (BM) stroma. Thus, the characterization of Mesenchymal Stromal Cells (MSC) would help to elucidate their involvement in the BM failure. Methods: We characterized MSCs of 28 FA patients (FA-MSC) before and after treatment (hematopoietic stem cell transplantation, HSCT; or gene therapy, GT). Phenotypic and functional properties were analyzed and compared with MSCs expanded from 26 healthy donors (HD-MSCs). FA-MSCs were genetically characterized through, mitomycin C-test and chimerism analysis. Furthermore, RNA-seq profiling was used to identify dysregulated metabolic pathways. Results: Overall, FA-MSC had the same phenotypic and functional characteristics as HD-MSC. Of note, MSC-GT had a lower clonogenic efficiency. These findings were not confirmed in the whole FA patients' cohort. Transcriptomic profiling identified dysregulation in HSC self-maintenance pathways in FA-MSC (HOX), and was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). Discussion: Our study provides a comprehensive characterization of FA-MSCs, including for the first time MSC-GT and constitutes the largest series published to date. Interestingly, transcript profiling revealed dysregulation of metabolic pathways related to HSC self-maintenance. Taken together, our results or findings provide new insights into the pathophysiology of the disease, although whether these niche defects are involved in the hematopoietic defects seen of FA deserves further investigation.

Role of Mesenchymal Stem/Stromal Cells in Head and Neck Cancer-Regulatory Mechanisms of Tumorigenic and Immune Activity, Chemotherapy Resistance, and Therapeutic Benefits of Stromal Cell-Based Pharmacological Strategies.

Head and neck cancer (HNC) entails a heterogenous neoplastic disease that arises from the mucosal epithelium of the upper respiratory system and the gastrointestinal tract. It is characterized by high morbidity and mortality, being the eighth most common cancer worldwide. It is believed that the mesenchymal/stem stromal cells (MSCs) present in the tumour milieu play a key role in the modulation of tumour initiation, development and patient outcomes; they also influence the resistance to cisplatin-based chemotherapy, the gold standard for advanced HNC. MSCs are multipotent, heterogeneous and mobile cells. Although no MSC-specific markers exist, they can be recognized based on several others, such as CD73, CD90 and CD105, while lacking the presence of CD45, CD34, CD14 or CD11b, CD79α, or CD19 and HLA-DR antigens; they share phenotypic similarity with stromal cells and their capacity to differentiate into other cell types. In the tumour niche, MSC populations are characterized by cell quiescence, self-renewal capacity, low reactive oxygen species production and the acquisition of epithelial-to-mesenchymal transition properties. They may play a key role in the process of acquiring drug resistance and thus in treatment failure. The present narrative review examines the links between MSCs and HNC, as well as the different mechanisms involved in the development of resistance to current chemo-radiotherapies in HNC. It also examines the possibilities of pharmacological targeting of stemness-related chemoresistance in HNSCC. It describes promising new strategies to optimize chemoradiotherapy, with the potential to personalize patient treatment approaches, and highlights future therapeutic perspectives in HNC.