Stem Cell Secretome Research Articles

Umbilical Cord Mesenchymal Stem Cell Secretome Improves Clinical Outcomes and Changes Biomarkers in Knee Osteoarthritis.

(1) Background: The current treatment for osteoarthritis is ineffective due to its focus on pain relief and lack of cartilage repair. Viscosupplementation such as hyaluronic acid improves symptoms but remains unnoticed for several months. Researchers are exploring cell-based therapies such as mesenchymal stem cells secretome and mesenchymal stem cells, which can repair cartilage damage. The objective of the research is to evaluate and compare the effectiveness of the secretome derived from umbilical cord mesenchymal stem cells (UC-MSCs) with hyaluronic acid (HA). (2) Methods: An open-label clinical trial involving 30 knee osteoarthritis patients divided into two groups received UC-MSC secretome and hyaluronic acid doses. The study assessed clinical outcomes using VAS and WOMAC and measured MMP-3 and TGF-β1 levels before and after treatment. (3) Results: A study of 30 subjects found that the UC-MSC secretome group showed a decrease in pain in the OA knee compared to the HA group. The therapy was most effective after the third injection, and the group showed a decrease in the MMP-3 ratio and an increase in TGF-β1 compared to the hyaluronic acid group. (4) Conclusions: UC-MSC secretome intra-articular injections showed superior clinical improvement, biomarker changes, and no side effects compared to hyaluronic acid over a 5-week interval.

ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4

BackgroundMesenchymal stem cells (MSCs) have immunomodulatory properties and therapeutic effects on autoimmune diseases through their secreted factors, referred to as the secretome. However, the specific key factors of the MSC secretome and their mechanisms of action in immune cells have not been fully determined. Most in vitro experiments are being performed using immune cells, but experiments using natural killer (NK) cells have been neglected, and a few studies using NK cells have shown discrepancies in results. NK cells are crucial elements of the immune system, and adjustment of their activity is essential for controlling various pathological conditions. The aim of this study was to elucidate the role of the adipose tissue-derived stem cell (ADSC) secretome on NK cell activity.MethodsTo obtain the ADSC secretome, we cultured ADSCs in medium and concentrated the culture medium using tangential flow filtration (TFF) capsules. We assessed NK cell viability and proliferation using CCK-8 and CFSE assays, respectively. We analyzed the effects of the ADSC secretome on NK cell activity and pathway-related proteins using a combination of flow cytometry, ELISA, cytotoxicity assay, CD107a assay, western blotting, and quantitative real-time PCR. To identify the composition of the ADSC secretome, we performed LC–MS/MS profiling and bioinformatics analysis. To elucidate the molecular mechanisms involved, we used mRNA sequencing to profile the transcriptional expression of human blood NK cells.ResultsThe ADSC secretome was found to restrict IL-2-mediated effector function of NK cells while maintaining proliferative potency. This effect was achieved through the upregulation of the inhibitory receptor CD96, as well as downregulation of activating receptors and IL-2 receptor subunits IL-2Rα and IL-2Rγ. These changes were associated with attenuated JAK-STAT and AKT pathways in NK cells, which were achieved through the upregulation of cytokine-inducible SH2-containing protein (CIS, encoded by Cish) and dual specificity protein phosphatase 4 (DUSP4). Furthermore, proteomic analysis revealed twelve novel candidates associated with the immunomodulatory effects of MSCs.ConclusionsOur findings reveal a detailed cellular outcome and regulatory mechanism of NK cell activity by the ADSC secretome and suggest a therapeutic tool for treating NK-mediated inflammatory and autoimmune diseases using the MSC secretome.

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. Based on the invitro and invivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR.

Mesenchymal stem cell secretome ameliorates over-expression of soluble fms-like tyrosine kinase-1 (sFlt-1) and fetal growth restriction (FGR) in animal SLE model

Introduction In the near future, stem cell research may lead to several major therapeutic innovations in medical practice. Secretome, a “by-product” of stem cell line cultures, has many advantages. Its easiness of storage, usage, and fast direct effect are some of those to consider. Fetal growth restriction (FGR) remains one of the significant challenges in maternal-fetal and neonatal medicine. Placentation failure is one of the most profound causal and is often related to increasing sFlt-1 in early pregnancy. This study aimed to investigate hUC-MSC secretome in ameliorating sFlt-1 and how to improve outcomes in preventing FGR in an animal model. Materials and methods Pristane-induced systemic lupus erythematosus (SLE) in a mouse model was used to represent placentation failure and its consequences. Twenty-one mice were randomized into three groups: (I) normal pregnancy, (II) SLE, and (III) SLE with secretome treatment. Pristane was administered in all Groups four weeks prior mating period. Secretome was derived from human umbilical cord mesenchymal stem cells (hUC-MSC) conditioned medium on the 3rd and 4th passage, around day-21 until day-28 from the start of culturing process. Mesenchymal stem cell was characterized using flow cytometry for CD105+, CD90+, and CD73+ surface antigen markers. Immunohistochemistry anlysis by using Remmele’s Immunoreactive Score (IRS) was used to quantify the placental sFlt-1 expression in each group. Birth weight and length were analyzed as the secondary outcome. The number of fetuses obtained was also calculated for pregnancy loss comparison between Groups. Results The administration of secretome of hUC-MSC was found to lower the expression of the placental sFlt-1 significantly in the pristane SLE animal model (10.30 ± 1.40 vs. 4.98 ± 2.57; p < 0.001) to a level seen in normal mouse pregnancies in Group I (3.88 ± 0.49; p = 0.159). Secretome also had a significant effect on preventing fetal growth restriction in the pristane SLE mouse model (birth weight: 354.29 ± 80.76 mg vs. 550 ± 64.03 mg; p < 0.001 and birth length: 14.43 ± 1.27 mm vs. 19.00 ± 1.41 mm), comparable to the birth weight and length of the normal pregnancy in Group I (540.29 ± 75.47 mg and 18.14 ± 1.34 mm, p = 0.808 and = 0.719). Secretome administration also showed a potential action to prevent high number of pregnancy loss as the number of fetuses obtained could be similar to those of mice in the normal pregnant Group (7.71 ± 1.11 vs. 7.86 ± 1.06; p = 0.794). Conclusions Administration of secretome lowers sFlt-1 expression in placenta, improves fetal growth, and prevents pregnancy loss in a mouse SLE model.

Intranasal administration of induced pluripotent stem cell-derived cortical neural stem cell-secretome as a treatment option for Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is the most common neurodegenerative disorder in the elderly, resulting in gradual destruction of cognitive abilities. Research on the development of various AD treatments is underway; however, no definitive treatment has been developed yet. Herein, we present induced pluripotent stem cell (iPSC)-derived cortical neural stem cell secretome (CNSC-SE) as a new treatment candidate for AD and explore its efficacy.MethodsWe first assessed the effects of CNSC-SE treatment on neural maturation and electromagnetic signal during cortical nerve cell differentiation. Then to confirm the efficacy in vivo, CNSC-SE was administered to the 5×FAD mouse model through the nasal cavity (5 μg/g, once a week, 4 weeks). The cell-mediated effects on nerve recovery, amyloid beta (Aβ) plaque aggregation, microglial and astrocyte detection in the brain, and neuroinflammatory responses were investigated. Metabolomics analysis of iPSC-derived CNSC-SE revealed that it contained components that could exert neuro-protective effects or amplify cognitive restorative effects.ResultsHuman iPSC-derived CNSC-SE increased neuronal proliferation and dendritic structure formation in vitro. Furthermore, CNSC-SE-treated iPSC-derived cortical neurons acquired electrical network activity and action potential bursts. The 5×FAD mice treated with CNSC-SE showed memory restoration and reduced Aβ plaque accumulation.ConclusionsOur findings suggest that the iPSC-derived CNSC-SE may serve as a potential, non-invasive therapeutic option for AD in reducing amyloid infiltration and restoring memory.

Treating Parkinson's Disease with Human Bone Marrow Mesenchymal Stem Cell Secretome: A Translational Investigation Using Human Brain Organoids and Different Routes of In Vivo Administration.

Parkinson's disease (PD) is the most common movement disorder, characterized by the progressive loss of dopaminergic neurons from the nigrostriatal system. Currently, there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Mesenchymal stem cells (MSCs) are one of the most extensively studied cell sources for regenerative medicine applications, particularly due to the release of soluble factors and vesicles, known as secretome. The main goal of this work was to address the therapeutic potential of the secretome collected from bone-marrow-derived MSCs (BM-MSCs) using different models of the disease. Firstly, we took advantage of an optimized human midbrain-specific organoid system to model PD in vitro using a neurotoxin-induced model through 6-hydroxydopamine (6-OHDA) exposure. In vivo, we evaluated the effects of BM-MSC secretome comparing two different routes of secretome administration: intracerebral injections (a two-site single administration) against multiple systemic administration. The secretome of BM-MSCs was able to protect from dopaminergic neuronal loss, these effects being more evident in vivo. The BM-MSC secretome led to motor function recovery and dopaminergic loss protection; however, multiple systemic administrations resulted in larger therapeutic effects, making this result extremely relevant for potential future clinical applications.

Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2 O2 -induced cytotoxicity.

Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 μg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.

Progress in the Development of Stem Cell-Derived Cell-Free Therapies for Skin Aging.

The skin is a vital organ as the body's largest barrier, but its function declines with aging. Therefore, research into effective regeneration treatments must continue to advance. Stem cell transplantation, a cell-based therapy, has become a popular skin-aging treatment, although it comes with drawbacks like host immune reactions. Stem cell-derived cell-free therapies have emerged as an alternative, backed by promising preclinical findings. Stem cell secretomes and extracellular vesicles (EVs) are the key components in cell-free therapy from stem cells. However, comprehensive reviews on the mechanisms of such treatments for skin aging are still limited. This review discusses stem cell-derived cell-free therapy's potential mechanisms of action related to skin aging prevention by identifying specific molecular targets suitable for the interventions. A search identified 27 relevant in vitro studies on stem cell-derived cell-free therapy interventions in skin aging model cells without restricting publication years using PubMed, Scopus, and Google Scholar. Stem cell-derived cell-free therapy can prevent skin aging through various mechanisms, such as (1) involvement of multiple regenerative pathways [NFkb, AP-1, MAPK, P-AKT, NRF2, SIRT-1]; (2) oxidative stress regulation [by reducing oxidants (HO-1, NQO1) and enhancing antioxidants (SOD1, CAT, GP, FRAP)]; (3) preventing ECM degradation [by increasing elastin, collagen, HA, TIMP, and reducing MMP]; (4) regulating cell activity [by reducing cell senescence (SA-β-gal), apoptosis, and cell cycle arrest (P53, P12, P16); and enhancing autophagy, cell migration, and cell proliferation (Ki67)] (5) Regulating the inflammatory pathway [by reducing IL-6, IL-1, TNF-⍺, and increasing TGF-β]. Several clinical trials have also revealed improvements in wrinkles, elasticity, hydration, pores, and pigmentation. Stem cell-derived cell-free therapy is a potential novel treatment for skin aging by cell rejuvenation through various molecular mechanisms.

Mesenchymal stem cell secretome and extracellular vesicles for neurodegenerative diseases: Risk-benefit profile and next steps for the market access.

Neurodegenerative diseases represent a growing burden on healthcare systems worldwide. Mesenchymal stem cells (MSCs) have shown promise as a potential therapy due to their neuroregenerative, neuroprotective, and immunomodulatory properties, which are, however, linked to the bioactive substances they release, collectively known as secretome. This paper provides an overview of the most recent research on the safety and efficacy of MSC-derived secretome and extracellular vesicles (EVs) in clinical (if available) and preclinical models of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, acute ischemic stroke, and spinal cord injury. The article explores the biologically active substances within MSC-secretome/EVs, the mechanisms responsible for the observed therapeutic effects, and the strategies that may be used to optimize MSC-secretome/EVs production based on specific therapeutic needs. The review concludes with a critical discussion of current clinical trials and a perspective on potential future directions in translating MSC-secretome and EVs into the clinic, specifically regarding how to address the challenges associated with their pharmaceutical manufacturing, including scalability, batch-to-batch consistency, adherence to Good Manufacturing Practices (GMP) guidelines, formulation, and storage, along with quality controls, access to the market and relative costs, value for money and impact on total expenditure.

Photobiostimulation conjugated with stem cells or their secretome for temporomandibular joint arthritis in a rat model

BackgroundTemporomandibular joint (TMJ) arthritis is a debilitating, challenging condition and different methods have been implicated for its treatment. This study aimed to test the therapeutic potentials of low-level laser therapy (LLLT) associated with adipose derived stem cells (ADSC) or their derived secretome on a murine model induced arthritis.MethodsForty eight rats were divided into four groups where group I was the sham control, the rest of animals were subjected to arthritis induction using complete Freund’s adjuvant, then divided as follows: group II received phosphate buffered saline (PBS) intraarticular injection and irradiation of 0 j/cm2, group III received ADSCs derived secretome and irradiation of 38 j/cm2, and group IV received ADSCs and irradiation of 38 j/cm2 as well. One and three weeks after treatment, animals were euthanized, and paraffin blocks were processed for histological assessment by hematoxylin and eosin stain with histomorphometrical analysis. Histochemical evaluation of joint proteoglycan content was performed through toluidine blue stain, and immunohistochemical staining by the proinflammatory marker tumor necrosis factor-α (TNF-α) was performed followed by the relevant statistical tests.ResultsThe arthritis group showed histological signs of joint injury including cartilage atrophy, articular disc fibrosis, irregular osteochondral interface, and condylar bone resorption together with high inflammatory reaction and defective proteoglycan content. In contrast, the treated groups III and IV showed much restoration of the joint structure with normal cartilage and disc thickness. The inflammation process was significantly suppressed especially after three weeks as confirmed by the significant reduction in TNF-α positive immunostaining compared to the arthritic group, and the cartilage proteoglycan content also showed significant increase relative to the arthritic group. However, no significant difference between the results of the two treated groups was detected.ConclusionLLLT conjugated with ADSCs or ADSCs derived secretome can efficiently enhance the healing of arthritic TMJs. Stem cell secretome can be applied as a safe, potent therapy. However, further investigations are required to unravel its mechanism of action and pave its way as a safe, novel, cell free therapy.Graphical

A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models

BackgroundMesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation.MethodsWe used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays.ResultsOur results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells.ConclusionsThe MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.

The exosomal secretomes of mesenchymal stem cells extracted via 3D-printed lithium-doped calcium silicate scaffolds promote osteochondral regeneration.

The development of surface modification techniques has brought about a major paradigm shift in the clinical applications of bone tissue regeneration. Biofabrication strategies enable the creation of scaffolds with specific microstructural environments and biological components. Lithium (Li) has been reported to exhibit anti-inflammatory, osteogenic, and chondrogenic properties by promoting several intracellular signaling pathways. Currently, research focuses on fabricating scaffolds with simultaneous dual bioactivities to enhance osteochondral regeneration. In this study, we modified the surface of calcium silicate (CS) scaffolds with Li using a simple immersion technique and evaluated their capabilities for bone regeneration. The results showed that Li ions could be easily coated onto the surfaces of CS scaffolds without affecting the microstructural properties of CS itself. Furthermore, the modifications did not affect the printing capabilities of the CS, and porous scaffolds could be fabricated via extrusion. Moreover, the presence of Li improved the surface roughness and hydrophilicity, thus leading to enhanced secretion of osteochondral-related regeneration factors, such as alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen II (Col II) proteins. Subsequent in vivo studies, including histological and micro-CT analyses, confirmed that the Li-modified CS scaffolds promoted osteochondral regeneration. The transcriptome analysis suggested that the enhanced osteochondrogenic capabilities of our scaffolds were influenced by paracrine exosomes. We hope this study will inspire further research on osteochondral regeneration.

316.1: Metabolomic and Lipidomic Profiling of the Amniotic Stromal Cell Secretome

316.1: Metabolomic and Lipidomic Profiling of the Amniotic Stromal Cell Secretome

Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro.

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro.

Managing Temporomandibular Joint Osteoarthritis by Dental Stem Cell Secretome

The potential therapeutic role of the Dental Pulp Stem Cells Secretome (SECR) in a rat model of experimentally induced Temporomandibular Joint (TMJ) Osteoarthritis (OA) was evaluated. Proteomic profiling of the human SECR under specific oxygen tension (5% O2) and stimulation with Tumor Necrosis Factor-alpha (TNF-α) was performed. SECR and respective cell lysates (CL) samples were collected and subjected to SDS-PAGE, followed by LC-MS/MS analysis. The identified proteins were analyzed with Bioinformatic tools. The anti-inflammatory properties of SECR were assessed via an in vitro murine macrophages model, and were further validated in vivo, in a rat model of chemically-induced TMJ-OA by weekly recording of the head withdrawal threshold, the food intake, and the weight change, and radiographically and histologically at 4- and 8-weeks post-treatment. SECR analysis revealed the presence of 50 proteins that were enriched and/or statistically significantly upregulated compared to CL, while many of those proteins were involved in pathways related to “extracellular matrix organization” and “immune system”. SECR application in vitro led to a significant downregulation on the expression of pro-inflammatory genes (MMP-13, MMP-9, MMP-3 and MCP-1), while maintaining an increased expression of IL-10 and IL-6. SECR application in vivo had a significant positive effect on all the clinical parameters, resulting in improved food intake, weight, and pain suppression. Radiographically, SECR application had a significant positive effect on trabecular bone thickness and bone density compared to the saline-treated group. Histological analysis indicated that SECR administration reduced inflammation, enhanced ECM and subchondral bone repair and regeneration, thus alleviating TMJ degeneration.Graphical

Conditioned medium from human dental pulp stem cells treats spinal cord injury by inhibiting microglial pyroptosis.

Human dental pulp stem cell transplantation has been shown to be an effective therapeutic strategy for spinal cord injury. However, whether the human dental pulp stem cell secretome can contribute to functional recovery after spinal cord injury remains unclear. In the present study, we established a rat model of spinal cord injury based on impact injury froma dropped weight and then intraperitoneally injected the rats with conditioned medium from human dental pulp stem cells. We found that the conditioned medium effectively promoted the recovery of sensory and motor functions in rats with spinal cord injury, decreased expression of the microglial pyroptosis markers NLRP3, GSDMD, caspase-1, and interleukin-1β, promoted axonal and myelin regeneration, and inhibited the formation of glial scars. In addition, in a lipopolysaccharide-induced BV2 microglia model, conditioned medium from human dental pulp stem cells protected cells from pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1β pathway. These results indicate that conditioned medium from human dental pulp stem cells can reduce microglial pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1β pathway, thereby promoting the recovery of neurological function after spinal cord injury. Therefore, conditioned medium from human dental pulp stem cells may become an alternative therapy for spinal cord injury.

Effects of various xenogenic mesenchymal stem cell secretome fractions on the regenerative capacity of the liver in vitro

Liver diseases cause many deaths worldwide and wreak havoc on the economy. The main hepatoprotectors are plant substances and peptides. Regenerative medicine based on mesenchymal stem cells (MSCs) can offer a new set of biologically active substances for liver regeneration, which are part of their secretome. The work applied the methods of cell isolation and cultivation, ultrafiltration for fractionation of secretome components, and organotypic culture model. The study’s results indicate an increase in the regenerative capacity of the liver under the impact of the components of the MSC secretome; the fraction below 10 kDa shows the most significant activity. Keywords: organotypic liver culture; xenogenic stem cells; fractions of secretome; absorption spectrum, ultrafiltration

Secretome profile of TNF-α-induced human umbilical cord mesenchymal stem cells unveils biological processes relevant to skin wound healing.

Aim: To profile and study the proteins responsible for the beneficial effect of the TNF-α-induced human umbilical cord mesenchymal stem cells (hUCMSCs) secretome in wound healing. Methods: The hUCMSCs secretome was generated with (induced) or without (uninduced) TNF-α and was subsequently analyzed by liquid chromatography-mass spectrometry, immunoassay and in vitro scratch assay. Results: Proteomic analysis revealed approximately 260 proteins, including 51 and 55 unique proteins in the induced and uninduced secretomes, respectively. Gene ontology analysis disclosed that differential proteins in the induced secretome mainly involved inflammation-related terms. The induced secretome, consisting of higher levels of FGFb, VEGF, PDGF and IL-6, significantly accelerated wound closure and enhanced MMP-13 secretion in HaCaT keratinocytes. Conclusion: The secretome from induced hUCMSCs includes factors that promote wound closure.

Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair.

The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCsBMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects.

Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes.

Context: Osteoarthritis (OA) is an invalidating articular disease characterized by cartilage degradation and inflammatory events. In horses, OA is associated with up to 60% of lameness and leads to reduced animal welfare along with extensive economic losses; currently, there are no curative therapies to treat OA. The mesenchymal stromal cell (MSC) secretome exhibits anti-inflammatory properties, making it an attractive candidate for improving the management of OA. In this study, we determined the best storage conditions for conditioned media (CMs) and tested whether priming MSCs with cytokines can enhance the properties of the MSC secretome. Methods: First, properties of CMs collected from bone-marrow MSC cultures and stored at -80°C, -20°C, 4°C, 20°C or 37°C were assessed on 3D cultures of equine articular chondrocytes (eACs). Second, we primed MSCs with IL-1β, TNF-α or IFN-γ, and evaluated the MSC transcript levels of immunomodulatory effectors and growth factors. The primed CMs were also harvested for subsequent treatment of eACs, either cultured in monolayers or as 3D cell cultures. Finally, we evaluated the effect of CMs on the proliferation and the phenotype of eACs and the quality of the extracellular matrix of the neosynthesized cartilage. Results: CM storage at -80°C, -20°C, and 4°C improved collagen protein accumulation, cell proliferation and the downregulation of inflammation. The three cytokines chosen for the MSC priming influenced MSC immunomodulator gene expression, although each cytokine led to a different pattern of MSC immunomodulation. The cytokine-primed CM had no major effect on eAC proliferation, with IL-1β and TNF-α slightly increasing collagen (types IIB and I) accumulation in eAC 3D cultures (particularly with the CM derived from MSCs primed with IL-1β), and IFN-γ leading to a marked decrease. IL-1β-primed CMs resulted in increased eAC transcript levels of MMP1, MMP13 and HTRA1, whereas IFNγ-primed CMs decreased the levels of HTRA1 and MMP13. Conclusion: Although the three cytokines differentially affected the expression of immunomodulatory molecules, primed CMs induced a distinct effect on eACs according to the cytokine used for MSC priming. Different mechanisms seemed to be triggered by each priming cytokine, highlighting the need for further investigation. Nevertheless, this study demonstrates the potential of MSC-CMs for improving equine OA management.