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.

Sustained release of stem cell secretome from nano-villi chitosan microspheres for effective treatment of atopic dermatitis.

Canine atopic dermatitis (AD) arises from hypersensitive immune reactions. AD symptoms entail severe pruritus and skin inflammation, with frequent relapses. Consequently, AD patients require continuous management, imposing financial burdens and mental fatigue on pet owners. In this study, we aimed to investigate the therapeutic relevance of secretome from canine adipose tissue-derived mesenchymal stem cells (MSCs), especially after encapsulation in nano-villi chitosan microspheres (CS-MS) to expect improved efficacy. Conditioned media (CM) from MSCs significantly inhibited the proliferation of splenocytes, induced the generation of regulatory T cells, and decreased mast cell degranulation. We found that beneficial soluble factors known to reduce AD symptoms, including transforming growth factor-beta 1, were detectable after sequential concentration and lyophilization of CM. The CS-MS, developed by a phase inversion regeneration method, showed high loading and sustained release of the secretome. Local injection of secretome-loaded CS-MS (ST/SC-MS) effectively reduced clinical severity compared to groups treated with secretome. Histological analysis revealed that ST/SC-MS potently suppressed epidermal hyperplasia, immunocyte infiltration and mast cell activation in the lesion. Taken together, this study presents a novel therapeutic approach exhibiting more potent and prolonged immunoregulatory efficacy of MSC secretome for canine AD treatment.

Impact of BMI and Cardiorespiratory Fitness on Oxidative Stress in Plasma and Circulating Exosomes Following Acute Exercise.

The impact of cardiorespiratory fitness (VO2max) and obesity on indices of oxidative stress in plasma and circulating exosome-like extracellular vesicles (ELVs) were examined following acute exercise. Indices of oxidative stress in plasma and isolated plasma ELVs were examined in aerobically trained (NW-Tr; n = 15) and untrained (NW-UTr; n = 18) normal-weight individuals and aerobically untrained individuals with obesity (Ob-Utr; n = 10) prior to and immediately following acute maximal treadmill running. Following exercise, ELV flotillin-1 expression (p = 0.008) and plasma total antioxidant capacity (TAC; p = 0.010) increased more in NW-UTr compared to NW-Tr and Ob-UTr participants, whereas plasma protein carbonyls (PC) decreased more in Ob-UTr compared to NW-Tr and NW-UTr groups. ELV glutathione (GSH) concentrations decreased more in NW-Tr compared to NW-UTr and Ob-UTr participants (p = 0.009), whereas lipid peroxidase (LPO) concentrations increased more in Ob-UTr compared to NW-Tr and NW-UTr participants (p = 0.003). Body mass index (BMI) was associated negatively with plasma TAC and PC (p < 0.05) and positively with ELV LPO concentration responses (p = 0.009). Finally, plasma-to-total (plasma + ELV) GSH ratios decreased in Ob-UTr compared to NW-Tr and NW-UTr participants (p = 0.006), PC ratios increased in NW-Tr and NW-UTr compared to Ob-UTr subjects (p = 0.008), and reactive oxygen/nitrogen species ratios increased in NW-UTr and decreased in Ob-UTr participants (p < 0.001). BMI, independently of VO2max, differentially regulates indices of oxidative stress within plasma and circulating ELVs prior to and immediately following acute maximal treadmill exercise.

Secretome analysis using Affinity Proteomics and Immunoassays: a focus on Tumor Biology.

The study of the cellular secretome using proteomic techniques continues to capture the attention of the research community across a broad range of topics in biomedical research. Due to their untargeted nature, independence from the model system employed, historically superior depth of analysis, as well as comparative affordability, mass spectrometry-based approaches traditionally dominate such analyses. More recently, however, affinity-based proteomic assays have massively gained in analytical depth, which together with their high sensitivity, dynamic range coverage as well as high throughput capabilities render them exquisitely suited to secretome analysis. In this review, we revisit the analytical challenges implied by secretomics and provide an overview of affinity-based proteomic platforms currently available for such analyses, using the study of the tumor secretome as an example for basic and translational research.

Cross-talks between perivascular adipose tissue and neighbors: multifaceted nature of nereids.

Perivascular adipose tissue (PVAT) is a unique fat depot surrounding blood vessels and plays a vital role in the progression of vascular remodeling and dysfunction. PVAT exhibits remarkable differences in structure, phenotype, origin, and secretome across anatomical locations. The proximity of PVAT to neighboring vascular beds favors a niche for bidirectional communication between adipocytes and vascular smooth muscle cells, endothelial cells, and immune cells. In this review, we update our understanding of PVAT's regional differences and provide a comprehensive exploration of how these differences impact cross-talks between PVAT and the vascular wall. Different PVAT depots show different degrees of vasoprotective function and resilience to pathological changes such as obesity and vasculopathies, shaping multifaceted interactions between PVAT depots and adjacent vasculatures. The depot-specific resilience may lead to innovative strategies to manage cardiometabolic disorders.

Topical application of a hyaluronic acid-based hydrogel integrated with secretome of human mesenchymal stromal cells for diabetic ulcer repair.

This preclinical proof-of-concept study aimed to evaluate the effectiveness of secretome therapy in diabetic mice with pressure ulcers. We utilized a custom-made hyaluronic acid (HA)-based porous sponge, which was rehydrated either with normal culture medium or secretome derived from human mesenchymal stromal cells (MSCs) to achieve a hydrogel consistency. Following application onto skin ulcers, both the hydrogel-only and the hydrogel + secretome combination accelerated wound closure compared to the vehicle group. Notably, the presence of secretome significantly enhanced the healing effect of the hydrogel, as evidenced by a thicker epidermis and increased revascularization of the healed area compared to the vehicle group. Notably, molecular analysis of healed skin revealed significant downregulation of genes involved in delayed wound healing and abnormal inflammatory response in ulcers treated with the hydrogel + secretome combination, compared to those treated with the hydrogel only. Additionally, we found no significant differences in therapeutic outcomes when comparing the use of secretome from fetal dermal MSCs to that from umbilical cord MSCs. This observation is supported by the proteomic profile of the two secretomes, which suggests a shared molecular signature responsible of the observed therapeutic effects.

Evaluation of the therapeutic potential of infrapatellar fat pad adipose-derived stem cells and their secretome for regenerating knee articular cartilage in a rat model of osteoarthritis.

Background: Mesenchymal stem cell (MSC) therapy has ameliorative effects for treating knee osteoarthritis (KOA) disease. Moreover, there is a growing interest in using MSCs-derived secretome (Sec) containing trophic factors secreted by MSCs for KOA treatment. Recently, some studies have suggested that the combination of MSCs and Sec has the potential to treat the diseases. Aims: This study aimed to evaluate the ameliorative effects of combined administration of infrapatellar fat pad (IPFP)-derived MSCs, a type of adipose-derived stem cells (ASCs), for treating degenerated cartilage in a rat model of KOA. Methods: IPFP-ASCs were isolated from the IPFP of male rats. Sec was obtained from IPFP-ASCs in the fourth passage. Eight weeks after the induction of KOA by collagenase II, the rats were divided into 5 groups (n=5), including a control group with no treatment, and four experimental groups that received sodium hyaluronate (Hyalgan®, Hya), ASCs, Sec, and IPFP-ASCs+Sec, respectively by an infrapatellar injection. To perform the pathological and radiological evaluations, the animals were sacrificed 8 weeks later. Results: Our findings indicated that combined administration of the IPFP-ASCs and Sec statistically (P<0.05) improved scores of medial tibial and femoral condyles and medial fabella osteophytes. Also, it statistically (P<0.05) enhances the cartilage surface, matrix, cell distribution and population viability, and subchondral bone indices. No statistical difference was observed between IPFP-ASCs+Sec and IPFP-ASCs. Conclusion: Administration of IPFP-ASCs+Sec has a therapeutic potential to treat KOA in rats. However, there is no difference in the combined administration of IPFP-ASCs and Sec with IPFP-ASCs alone.

Effect of bovine hydroxyapatite composite with secretome under normoxia and hypoxia conditions on inflammatory parameters in massive bone defect of rabbit radius bone.

Hydroxyapatite as a scaffold is capable of producing good bone regeneration formation. Incorporating secretome into scaffolds optimizes the bone healing process. The increase in proinflammatory, anti-inflammatory, and growth factors is one of the key factors in bone healing. In this study, we measured the levels of IL-6, IL-10, and FGF-2 to determine the effectiveness of bovine hydroxyapatite with secretome from normoxia and hypoxia on bone healing. This animal study employed a pure experimental research design, utilizing a post-test-only control group design. Bone marrow mesenchymal stem cells from rabbit thigh bones were used to derive secretomes under hypoxic and normoxic conditions. Bovine bone-derived hydroxyapatite (BHA) was treated with secretomes under both conditions. Rabbits' radius bones were implanted with BHA alone, BHA with normoxic secretome, and BHA with hypoxic secretome, then observed for 30 and 60 days. Levels of IL-6, IL-10, and FGF-2 were examined on days 30 and 60. On the 30th day, there was a significant increase in the levels of FGF-2, IL-6, and IL-10, with a dominance of strongly positive levels in BHA alone. However, on the 60th day, the levels of FGF-2, IL-6, and IL-10 started to decrease in all groups, with a dominance of moderately positive levels. Statistical tests showed significant results in all groups on days 30 and 60 (p < .05). Among the three groups, the best levels of growth factors and pro-inflammatory factors, and the lowest levels of anti-inflammatory factors were found in the BHA alone group on evaluation day 30.

Mesenchymal stem cell secretome: A promising therapeutic strategy for erectile dysfunction?

Objective: The secretome, comprising bioactive chemicals released by mesenchymal stem cells (MSCs), holds therapeutic promise in regenerative medicine. This review aimed to explore the therapeutic potential of the MSC secretome in regenerative urology, particularly for treating erectile dysfunction (ED), and to provide an overview of preclinical and clinical research on MSCs in ED treatment and subsequently to highlight the rationales, mechanisms, preclinical investigations, and therapeutic potential of the MSC secretome in this context. Methods: The review incorporated an analysis of preclinical and clinical research involving MSCs in the treatment of ED. Subsequently, it delved into the existing knowledge regarding the MSC secretome, exploring its therapeutic potential. The methods included a comprehensive examination of relevant literature to discern the processes underlying the therapeutic efficacy of the MSC secretome. Results: Preclinical research indicated the effectiveness of the MSC secretome in treating various models of ED. However, the precise mechanisms of its therapeutic efficacy remain unknown. The review provided insights into the anti-inflammatory, pro-angiogenic, and trophic properties of the MSC secretome. It also discussed potential advantages, such as avoiding issues related to cellular therapy, including immunogenicity, neoplastic transformation, and cost. Conclusion: This review underscores the significant therapeutic potential of the MSC secretome in regenerative urology, particularly for ED treatment. While preclinical studies demonstrate promising outcomes, further research is essential to elucidate the specific mechanisms underlying the therapeutic efficacy before clinical application. The review concludes by discussing future perspectives and highlighting the challenges associated with the clinical translation of the MSC secretome in regenerative urology.

The prospective role of mesenchymal stem cells in Parkinson's disease.

Parkinson's disease (PD) is a stressful neurodegenerative disorder affecting millions worldwide. PD leads to debilitating motor and cognitive symptoms such as tremors, rigidity, and difficulty walking. Current therapies for PD are symptomatic and don't address the root cause. Therefore, there is an urgent need for better management and intensive research into alternative therapies. Mesenchymal stem cell (MSC) therapy is among the leading contenders among these promising avenues. We examined preclinical and clinical evidence demonstrating the neuroprotective, anti-inflammatory, and regenerative properties of the MSCs. This review focuses on the complex pathophysiological mechanisms of PD, as well as the perspectives of MSCs and their derivatives, such as secretomes and exosomes, in the clinical management of PD. We also analyzed the challenges and limitations of each approach, including delivery methods, timing of administration, and long-term safety considerations.

Characterization of the Secretome from Spheroids of Adipose-Derived Stem Cells (SASCs) and Its Potential for Tissue Regeneration.

INTRODUCTION: Spheroids are spherical aggregates of cells that mimic the three-dimensional (3D) architecture of tissues more closely than traditional two dimensional (2D) cultures. Spheroids of adipose stem cells (SASCs) show special features such as high multilineage differentiation potential and immunomodulatory activity. These properties have been attributed to their secreted factors, such as cytokines and growth factors. Moreover, a key role is played by the extracellular vesicles (EVs), which lead a heterogeneous cargo of proteins, mRNAs, and small RNAs that interfere with the pathways of the recipient cells. PURPOSE: The aim of this work was to characterize the composition of the secretome and exosome from SASCs and evaluate their regenerative potential. MATERIALS AND METHODS: SASCs were extracted from adipose samples of healthy individuals after signing informed consent. The exosomes were isolated and characterized by Dinamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western blotting analyses. The expression of mRNAs and miRNAs were evaluated through real-time PCR. Lastly, a wound-healing assay was performed to investigate their regenerative potential on different cell cultures. RESULTS: The SASCs' exosomes showed an up-regulation of NANOG and SOX2 mRNAs, typical of stemness maintenance, as well as miR126 and miR146a, related to angiogenic and osteogenic processes. Moreover, the exosomes showed a regenerative effect. CONCLUSIONS: The SASCs' secretome carried paracrine signals involved in stemness maintenance, pro-angiogenic and pro-osteogenic differentiation, immune system regulation, and regeneration.

Novel secreted regulators of glucose and lipid metabolism in the development of metabolic diseases.

The tight regulation of glucose and lipid metabolism is crucial for maintaining metabolic health. Dysregulation of these processes can lead to the development of metabolic diseases. Secreted factors, or hormones, play an essential role in the regulation of glucose and lipid metabolism, thus also playing an important role in the development of metabolic diseases such as type 2 diabetes and obesity. Given the important roles of secreted factors, there has been significant interest in identifying new secreted factors and new functions for existing secreted factors that control glucose and lipid metabolism. In this review, we evaluate novel secreted factors or novel functions of existing factors that regulate glucose and lipid metabolism discovered in the last decade, including secreted isoform of endoplasmic reticulum membrane complex subunit 10, vimentin, cartilage intermediate layer protein 2, isthmin-1, lipocalin-2, neuregulin-1 and neuregulin-4. We discuss their discovery, tissues of origin, mechanisms of action and sex differences, emphasising their potential to regulate metabolic processes central to diabetes. Additionally, we discuss the translational barriers, particularly the absence of identified receptors, that hamper their functional characterisation and further therapeutic development. Ultimately, the identification of new secreted factors may give insights into previously unidentified pathways of disease progression and mechanisms of glucose and lipid homeostasis.

Proteomic and secretomic comparison of young and aged dermal fibroblasts highlights cytoskeleton as a key component during aging.

Crucial for skin homeostasis, synthesis and degradation of extracellular matrix components are orchestrated by dermal fibroblasts. During aging, alterations of component expression, such as collagens and enzymes, lead to reduction of the mechanical cutaneous tension and defects of skin wound healing. The aim of this study was to better understand the molecular alterations underwent by fibroblasts during aging by comparing secretomic and proteomic signatures of fibroblasts from young (<35years) and aged (>55years) skin donors, in quiescence or TGF-stimulated conditions, using HLPC/MS. The comparison of the secretome from young and aged fibroblasts revealed that 16 proteins in resting condition, and 11 proteins after a 24h-lasting TGF-ß1-treatment, were expressed in significant different ways between the two cell groups (fold change>2, p-value <0.05), with a 77% decrease in the number of secreted proteins in aged cells. Proteome comparison between young and aged fibroblasts identified a significant change of 63 proteins in resting condition, and 73 proteins in TGF-ß1-stimulated condition, with a 67% increase in the number of proteins in aged fibroblasts. The majority of the differentially-expressed molecules belongs to the cytoskeleton-associated proteins and aging was characterized by an increase in Coronin 1C (CORO1C), and Filamin B (FLNB) expression in fibroblasts together with a decrease in Cofilin (CFL1), and Actin alpha cardiac muscle 1 (ACTC1) detection in aged cells, these proteins being involved in actin-filament polymerization and sharing co-activity in cell motility. Our present data reinforce knowledge about an age-related alteration in the synthesis of major proteins linked to the migratory and contractile functions of dermal human fibroblasts.

Secretome of brain microvascular endothelial cells promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage.

Cell-based therapeutic strategies have been proposed as an alternative for brain and blood vessels repair after stroke, but their clinical application is hampered by potential adverse effects. We therefore tested the hypothesis that secretome of these cells might be used instead to still focus on cell-based therapeutic strategies. We therefore characterized the composition and the effect of the secretome of brain microvascular endothelial cells (BMECs) on primary in vitro human models of angiogenesis and vascular barrier. Two different secretome batches produced in high scale (scHSP) were analysed by mass spectrometry. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used as well as in vitro models of EC monolayer (CMECs) and blood-brain barrier (BBB). Cells were also exposed to oxygen-glucose deprivation (OGD) conditions and treated with scHSP during reoxygenation. Protein yield and composition of scHSP batches showed good reproducibility. scHSP increased CD34+-EC proliferation, tubulogenesis, and migration. Proteomic analysis of scHSP revealed the presence of growth factors and proteins modulating cell metabolism and inflammatory pathways. scHSP improved the integrity of CMECs, and upregulated the expression of junctional proteins. Such effects were mediated through the activation of the interferon pathway and downregulation of Wnt signalling. Furthermore, OGD altered the permeability of both CMECs and BBB, while scHSP prevented the OGD-induced vascular leakage in both models. These effects were mediated through upregulation of junctional proteins and regulation of MAPK/VEGFR2. Finally, our results highlight the possibility of using secretome from BMECs as a therapeutic alternative to promote brain angiogenesis and to protect from ischemia-induced vascular leakage.

Augmenting osteoporotic bone regeneration through a hydrogel-based rejuvenating microenvironment.

Osteoporotic bone defects pose a significant challenge for bone regeneration as they exhibit impaired healing capacity and delayed healing period. To address this issue, this study introduces a hydrogel that creates a rejuvenating microenvironment, thereby facilitating efficient bone repair during the initial two weeks following bone defect surgery. The hydrogel, named GelHFS, was created through host-guest polymerization of gelatin and acrylated ß-cyclodextrin. Incorporation of the human fetal mesenchymal stem cell secretome (HFS) formed GelHFS hydrogel aimed at mimicking a rejuvenated stem cell niche. Our results demonstrated that GelHFS hydrogel promotes cell stellate spreading and osteogenic differentiation via integrin ß1-induced focal adhesion pathway. Implantation of GelHFS hydrogel in an osteoporotic bone defect rat model recruited endogenous integrin ß1-expressing cells and enhanced new bone formation and bone strength. Our findings reveal that GelHFS hydrogel provides a rejuvenating niche for endogenous MSCs and enhances bone regeneration in osteoporotic bone defect. These findings highlight the potential of GelHFS hydrogel as an effective therapeutic strategy for addressing challenging bone healing such as osteoporotic bone regeneration.

Conditioned medium of human mesenchymal stromal/stem cells cultured on decellularized extracellular matrix promotes murine skeletal muscle repair after acute injury.

Mesenchymal stromal/stem cells (MSCs) and their secretome are known to exert beneficial effects in many pathological states. However, MSCs therapeutic properties can be reduced due to unsuitable in vitro maintenance conditions. Standard culture protocols neglect the fact that MSCs exist in vivo in the closest connection with the extracellular matrix (ECM), the complex protein network providing an instructive microenvironment. We found recently that conditioned medium from human endometrial MSCs cultured on cell-derived decellularized extracellular matrix (CM-dECM) is dramatically enriched in a number of paracrine factors such as GM-CSF, FGF-2, HGF, MMP-1, MCP-1, IL-6, IL-8, CXCL-1, -2, -5, -6 (Ushakov et al., 2024). Given that several upregulated molecules belong to myokines that are known to participate in skeletal muscle regeneration, we hypothesized that CM-dECM may promote restoration of damaged muscle tissue. Here, we found that CM-dECM injections into barium chloride-injured murine m. tibialis anterior caused myofiber hypertrophy and promoted angiogenesis. Besides, CM-dECM significantly contributed to progression of murine C2C12 myoblasts cell cycle suggesting that muscle repair in vivo may be connected with stimulation of resident myoblasts proliferation. In this study, a role for secretome of endometrial MSCs cultured on dECM in injured murine skeletal muscle regeneration was outlined first. Our findings demonstrate that culture on dECM may be considered as a novel preconditioning approach enhancing MSCs therapeutic potential.

Secretome from iPSC-derived MSCs exerts proangiogenic and immunosuppressive effects to alleviate radiation-induced vascular endothelial cell damage.

BACKGROUND: Radiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass, irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications, neurodegeneration, and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity, homeostasis, and repair. Here, we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines. METHODS: We generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome, their modulation, and the molecular pathways they elicit. RESULTS: Increasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ≤ 5 Gy) were treated with iMSC CM, endothelial cell viability, adherence, spheroid compactness, and proangiogenic sprout formation were significantly ameliorated, and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently, iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore, iMSC CM suppressed IR-induced NFκB activation, TNF-α release, and ROS production in THP1 cells. Additionally, iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3, iMSCs, or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2, IL6, IL8/CXCL8, ANG (Angiogenin), GROα/CXCL1, and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors, the PI3K/AKT/mTOR modulator PRAS40 and ß-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism, translation, mitochondrial respiration, DNA damage repair, and neurodevelopment. CONCLUSIONS: The iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.

Mesenchymal stromal cells and their secretory products reduce the inflammatory crosstalk between islets and endothelial cells.

PURPOSE: Preculturing isolated islets with Mesenchymal Stromal Cells (MSCs) improves their functional survival in vitro and subsequent transplantation outcomes in vivo. The MSC secretory product Annexin A1 (ANXA1) is a key modulator of MSC-mediated improvements in islet function. The current study aims to determine the influence of MSCs and defined MSC secretory products, including ANXA1, on the inflammatory crosstalk between isolated islets and Endothelial Cells (ECs), using in vitro models of the clinically-preferred intraportal islet transplantation niche. METHODS: Islets were cultured alone, with MSCs, or with MSC secretory products and exposed to pro-inflammatory cytokines. Islet gene expression of C-C Motif Chemokine Ligand 2 (CCL2), C-X-C Motif Chemokine Ligand (CXCL)-10 (CXCL10) and CXCL1 were assessed by RT-qPCR. EC activation was induced with 100 U/ml TNF for 24 h. Islet-EC co-cultures were used to determine the influence of MSCs, or MSC secretory products on the inflammatory crosstalk between isolated islets and ECs. VCAM-1 and ICAM-1 expression were assessed at the mRNA and protein level in ECs, using RT-qPCR and immunofluorescence. RESULTS: MSCs reduce pro-inflammatory cytokine-induced islet CCL2, CXCL10, and CXCL1 gene expression, which is partially mimicked by ANXA1. MSCs and ANXA1 have a similar capacity to reduce TNF-induced EC activation. Isolated islets exacerbate TNF-induced EC activation. Preculturing islets with MSCs reduces islet-exacerbated EC activation. ANXA1 reduces islet-exacerbated EC activation, when present during the islet preculture and islet-EC co-culture period. CONCLUSION: MSC-derived secretory factors, including ANXA1, may be used in islet transplantation protocols to target donor islet and host EC inflammation at the intraportal niche.

Compact bone mesenchymal stem cells-derived paracrine mediators for cell-free therapy in sepsis.

Sepsis, a life-threatening condition resulting in multiple organ dysfunction, is characterized by a dysregulated immune response to infection. Current treatment options are limited, leading to unsatisfactory outcomes for septic patients. Here, we present a series of studies utilizing compact bone mesenchymal stem cells (CB-MSCs) and their derived paracrine mediators, especially exosome (CB-MSCs-Exo), to treat mice with cecal ligation and puncture-induced sepsis. Our results demonstrate that CB-MSCs treatment significantly improves the survival rate of septic mice by mitigating excessive inflammatory response and attenuating sepsis-induced organ injuries. Furthermore, CB-MSCs-conditioned medium, CB-MSCs secretome (CB-MSCs-Sec), and CB-MSCs-Exo exhibit potent anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated murine macrophage (RAW264.7). Intriguingly, intravenous administration of CB-MSCs-Exo confers superior protection against inflammation and organ damage in septic mice compared to CB-MSCs in certain aspects. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomic analysis, we identify a range of characterized proteins derived from the paracrine activity of CB-MSCs, involved in critical biological processes such as immunomodulation and apoptosis. Our findings highlight that the paracrine products of CB-MSCs could serve as a promising cell-free therapeutic agent for sepsis.

Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.