MSC-conditioned Medium Research Articles

Property of Hemorrhoid Tissue Mesenchymal Stem Cell Tow-Dimensional and Tridimensional Cultures for the Treatment of Anal Incontinence

Abstract Background Autologous mesenchymal stem cells (MSC) from different origins, including adipose tissue, have been proposed for the treatment of anal incontinence with encouraging preclinical and clinical outcomes. Cell transplantation as tridimensional structures instead of individual cell suspension seems to increase cell viability and implantation. We previously developed a protocol for MSC isolation directly from haemorrhoidal tissue (he-MSC) in human and demonstrated that the isolated cells fulfil MSC criteria. Aims Evaluate the characteristics and secretome of he-MSC in both two-dimensional or tridimensional cultures. Methods Informed consent was obtained from all patients. Tissue samples were procured from haemorrhoidectomy specimens or liposuction waste product and processed and characterized according to previously reported methods. He-MSC were cultured as spheroids using the “hanging-drop technique” or agarose pits. Immunofluorescence was performed to assess the expression of mesenchymal proteins and cell viability. Conditioned media of tow-dimensional cultures or spheroids in suspension were generated using low serum culture medium and analysed with semi-quantitative cytokine arrays (120 cytokines) and ELISA assays. Results Cultured cells demonstrated expression of vimentin and good viability, even as spheroids (2.6±2% cell death). Cytokine profile of he-MSC secretome was similar to that of adipose tissue-derived MSC (AT-MSC), with shared core cytokines (FGF-9, OPG, CCL2, CCL11, CCL13, IGFBP-4, IGFBP-6). Concentration of HGF measured by ELISA was higher in he-MSC conditioned medium than in AT- MSC conditioned medium (19613±3528 versus 179±84 pg/ml p<0.001) whereas VEGF concentration followed an opposite trend (7627±947pg/ml versus 181±74 pg/ml p<0.001). Production of mesenchymal proteins and growth factors was not affected by the spheroid configuration of cells. Conclusion We demonstrated that he-MSC exhibit a similar secretome profile compared to AT-MSC. Generation of spheroids did not compromise cell viability or their ability to produce structural proteins and growth factors, validating he-MSC spheroids use for further in vivo studies prior to clinical validation.

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity.

Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.

Skin-derived precursor conditioned medium alleviated photoaging via early activation of TGF-β/Smad signaling pathway by thrombospondin1: In vitro and in vivo studies

Photoaging is one major exogenous factor of skin aging. Efficacy and safety of current anti-photoaging therapies remained to be improved. Our previous studies indicated that skin-derived precursors (SKPs) alleviated photodamage by early activation of TGF-β/Smad signaling pathway via thrombospondin1 (TSP1). However, the research concerning SKP conditioned medium (SKP-CM) has never been reported. In the current study, we aimed to explore the anti-photoaging effects of SKP-CM both in vitro and in vivo, and to elucidate the possible mechanisms. Mouse SKP-CM (mSKP-CM) collection was optimized by a comparative method. The concentration of protein and growth factors in mSKP-CM was detected using BCA protein assay kit and growth factor protein chip. The anti-photoaging effects of mSKP-CM and its regulation of key factors in the TGF-β/Smad signaling pathway were explored using UVA + UVB photoaged mouse fibroblasts (mFBs) and nude mice dorsal skin. The research revealed that mSKP-CM contained significantly higher-concentration of protein and growth factors than mouse mesenchymal stem cell conditioned medium (mDMSC-CM). mSKP-CM alleviated mFBs photoaging by restoring cell viability and relieving senescence and death. ELISA, qRT-PCR, and western blot results implied the potential mechanisms were associated with the early activation of TGF-β/Smad signaling pathway by TSP1. In vivo experiments demonstrated that compared with the topical intradermal mDMSC-CM injection and retinoic acid cream application, the photodamaged mice dorsal skin intradermally injected with mSKP-CM showed significantly better improvement. Consistent with the in vitro results, both western blot and immunohistochemistry results confirmed that protein expression of TSP1, smad2/3, p-smad2/3, TGF-β1, and collagen I increased, and matrix metalloproteinases decreased. In summary, both in vitro and in vivo experiments demonstrated that mSKP-CM alleviated photoaging through an early activation of TGF-β/Smad signaling pathway via TSP1. SKP-CM may serve as a novel and promising cell-free therapeutical approach for anti-photoaging treatment and regenerative medicine.

Exploration of altered miRNA expression and function in MSC-derived extracellular vesicles in response to hydatid antigen stimulation.

Hydatid disease is caused by Echinococcus parasites and can affect various tissues and organs in the body. The disease is characterized by the presence of hydatid cysts, which contain specific antigens that interact with the host's immune system. Mesenchymal stem cells (MSCs) are pluripotent stem cells that can regulate immunity through the secretion of extracellular vesicles (EVs) containing microRNAs (miRNAs). In this study, hydatid antigens were isolated from sheep livers and mice peritoneal cavities. MSCs derived from mouse bone marrow were treated with different hydatid antigens, and EVs were isolated and characterized from the conditioned medium of MSCs. Small RNA library construction, miRNA target prediction, and differential expression analysis were conducted to identify differentially expressed miRNAs. Functional enrichment and network construction were performed to explore the biological functions of the target genes. Real-time PCR and Western blotting were used for miRNA and gene expression verification, while ELISA assays quantified TNF, IL-1, IL-6, IL-4, and IL-10 levels in cell supernatants. The study successfully isolated hydatid antigens and characterized MSC-derived EVs, demonstrating the impact of antigen concentration on MSC viability. Key differentially expressed miRNAs, such as miR-146a and miR-9-5p, were identified, with functional analyses revealing significant pathways like Endocytosis and MAPK signaling associated with these miRNAs' target genes. The miRNA-HUB gene regulatory network identified crucial miRNAs and HUB genes, such as Traf1 and Tnf, indicating roles in immune modulation and osteogenic differentiation. Protein-protein interaction (PPI) network analysis highlighted central HUB genes like Akt1 and Bcl2. ALP activity assays confirmed the influence of antigens on osteogenic differentiation, with reduced ALP activity observed. Expression analysis validated altered miRNA and chemokine expression post-antigen stimulation, with ELISA analysis showing a significant reduction in CXCL1 expression in response to antigen exposure. This study provides insights into the role of MSC-derived EVs in regulating parasite immunity. The findings suggest that hydatid antigens can modulate the expression of miRNAs in MSC-derived EVs, leading to changes in chemokine expression and osteogenic capacity. These findings contribute to a better understanding of the immunomodulatory mechanisms involved in hydatid disease and provide potential therapeutic targets for the development of new treatment strategies.

Effect of Conditioned Medium from Human Adipose-Derived Mesenchymal Stem Cells on Human Sperm Quality During Cryopreservation.

The cryopreservation procedure decreases sperm quality, causing certain changes at structural and molecular levels affecting fertilizing ability. We aimed to investigate the impacts of human adipose-derived mesenchymal stem cells (HAd-MSCs) conditioned medium (CM) on the protection of human sperm from cryoinjury. Thirty normal semen specimens were evaluated in this study. Each specimen was separated into six groups and enhanced with varying concentrations of human Ad-MSCs-CM (0, 10, 30, 50, 70, and 100%). Sperm motility, viability, morphology, apoptosis, mitochondrial potential, and lipid peroxidation, and DNA fragmentation were evaluated before freezing and after thawing. The results showed that the total motility was preserved in 10% human Ad-MSCs-CM group. Also, DNA fragmentation was significantly lower in 10% compared to 0% human Ad-MSCs-CM (63.62 ± 17.72% vs.76.46 ± 4.87%, respectively, P < 0.004). Human Ad-MSCs-CM in groups of 10, 30, 50, and 70% reduced lipid peroxidation. The normal sperm morphology rate, mitochondrial membrane potential, and apoptosis showed no significant differences across various groups. It seems that human Ad-MSCs-CM can protect the sperm parameters during the cryopreservation by decreasing cryoinjury.

Therapeutic Potential of MSC-conditioned Medium: A Multifactorial Approach to Enhance Wound Healing

Abstract: Wound healing is a highly intricate process that involves a coordinated interplay of various cellular and molecular events. In recent years, mesenchymal stem cells (MSCs) have garnered significant attention as a promising therapeutic modality due to their remarkable regenerative properties. In addition to their capacity for direct tissue repair and differentiation, MSCs release a diverse repertoire of bioactive factors into the surrounding microenvironment, collectively termed an MSC-conditioned Medium (MSC-CM). This review aims to elucidate the therapeutic potential of MSC-CM as a multifactorial approach to enhance wound healing. MSCCM encompasses an extensive range of factors, including growth factors, cytokines, chemokines, extracellular matrix proteins, and non-coding RNAs, which collectively orchestrate and stimulate multiple cellular processes crucial for effective wound healing. Notably, these factors play pivotal roles in promoting angiogenesis, stimulating the proliferation and migration of various cell types involved in wound repair, modulating the immune response, and facilitating extracellular matrix remodeling. Moreover, compelling evidence from preclinical and clinical studies demonstrates that MSC-CM administration accelerates wound closure, enhances reepithelialization, and improves the quality of granulation tissue. By harnessing the therapeutic potential of MSC-CM, we can pave the way for significant advancements in wound care, ultimately leading to improved patient outcomes.

Conditioned Medium Treatment for the Improvement of Functional Recovery after Spinal Cord Injury: A Meta-Analysis Study.

While there is no certain treatment for spinal cord injury (SCI), stem cellbased therapy may be an attractive alternative, but the survival and differentiation of cells in the host tissue are poor. Conditioned medium (CM) has several beneficial effects on cells. In this meta-analysis study, we examined the effect of CM on SCI treatment. After searching on MEDLINE, SCOPUS, EMBASE, and Web of Science, first and secondary screening were performed based on title, abstract, and full text. The data were extracted from the included studies, and meta-analysis was performed using STATA.14 software. A standardized mean difference (SMD) with a 95% confidence interval was used to report findings. Quality control and subgroup analysis were also performed. The results from 52 articles and 61 separate experiments showed that CM had a significantly strong effect on improving motor function after SCI (SMD = 2.58; 95% CI: 2.17 to 2.98; p < 0.001) and also analysis of data from 12 articles demonstrated that CM reduced the expression of GFAP marker (SMD = -4.16; p < 0.0001) compared to SCI group without any treatment. Subgroup analysis showed that treatment with CM of neural stem cells was better than CM of mesenchymal stem cells. It was more effective after a mild lesion than a moderate or severe one. The improvement was more pronounced with <4 weeks than >4 weeks follow-up. CM had a significant effect in improving motor function after SCI, especially in cases of mild lesions. It has been observed that if CM originates from the neural stem cells, it has a more significant effect than mesenchymal cells.

Novel therapeutic targets, including IGFBP3, of umbilical cord mesenchymal stem-cell-conditioned medium in intrauterine adhesion.

Mesenchymal stem cells play important roles in repairing injured endometrium. However, the molecular targets and potential mechanism of the endometrial recipient cells for stem cell therapy in intrauterine adhesion (IUA) are poorly understood. In this study, umbilical cord mesenchymal stem-cell-conditioned medium (UCMSCs-CM) produced positive effects on a Transforming growth factor beta (TGF-β) induced IUA cell model. RNA-sequencing was performed on clinical IUA tissues, and the top 40 upregulated and top 20 downregulated mRNAs were selected and verified using high-throughput (HT) qPCR in both tissues and cell models. Based on a bioinformatic analysis of RNA-sequencing and HT-qPCR results, 11 mRNAs were uncovered to be the intervention targets of UCMSCs-CM on IUA endometrium cell models. Among them, IGFBP3 was striking as a key pathogenic gene and a potential diagnostic marker of IUA, which exhibited the area under the curve (AUC), sensitivity, specificity were 0.924, 93.1% and 80.6%, respectively in 60 endometrial tissues. The silencing of IGFBP3 exerted positive effects on the IUA cell model through partially upregulating MMP1 and KLF2. In conclusion, RNA-sequencing combined with HT qPCR based on clinical tissues and IUA cell models were used in IUA research and our results may provide some scientific ideas for the diagnosis and treatment of IUA.

Cellular senescence is associated with osteonecrosis of the femoral head while mesenchymal stem cell conditioned medium inhibits bone collapse

Osteonecrosis of the femoral head (ONFH) is a type of ischemic osteonecrosis that causes pain, loss of function, and femoral head collapse. Here, we analyzed samples of femoral heads excised from patients with ONFH to clarify the relationship between ischemic osteonecrosis and cellular senescence. X-gal staining was strong and p16INK4a-positive cells were abundant in the transitional region of ONFH. The β-galactosidase-positive cells in the transitional region were also positive for nestin, periostin, or DMP-1. In contrast, no β-galactosidase-positive cells were detected in the healthy region. The senescence-associated p16INK4a, p21, and p53 were upregulated in ONFH tissue. We also examined and analyzed a mouse ischemic femoral osteonecrosis model in vivo to verify the association between ONFH and cellular senescence. Human mesenchymal stem cell-conditioned medium (MSC-CM) was administered to determine its therapeutic efficacy against cellular senescence and bone collapse. MSC-CM reduced the number of senescent cells and downregulated the aforementioned senescence-related genes. It also decreased the number of empty lacunae 4 weeks after ischemia induction and promoted bone formation. At 6 weeks post-surgery, MSC-CM increased the trabecular bone volume, thereby suppressing bone collapse. We conclude that cellular senescence is associated with ONFH and that MSC-CM suppresses bone collapse in this disorder.

Improvements in Diabetic Neuropathy with Topical Human Mesenchymal Stem Cell Conditioned Media

Improvements in Diabetic Neuropathy with Topical Human Mesenchymal Stem Cell Conditioned Media

Effects of adipose-derived mesenchymal stem cell conditioned medium on human tenocytes exposed to high glucose.

Diabetic tendinopathy is a common invalidating and challenging disease that may be treated using stem cells. However, the effects of adipose-derived mesenchymal stem cell conditioned medium (ASC-CM) in diabetic tendinopathy have never been explored. The present study evaluated the effects of ASC-CM on morphology, cell viability, structure, and scratch wound closure of human tenocytes (HTNC) exposed to high glucose (HG). Experimental study. HTNC were exposed to HG (25 mM) for 7, 14 and 21 days with or without ASC-CM for the last 24 h. CM was collected from 4 × 105 ASCs, centrifuged for 10 min at 200 g and sterilized with 0.22 μm syringe filter. At 7 days, HG-HTNC had decreased cell viability [72 ± 2%, p < 0.01 versus normal glucose (NG)] compared to NG-HTNC (90 ± 5%). A further decrement was detected after 14 and 21 days (60 ± 4% and 60 ± 5%, both, p < 0.01 versus NG and p < 0.01 versus HG7). While NG-HTNC evidenced a normal fibroblast cell-like elongated morphology, HG-HTNC showed increased cell roundness. In contrast, HG-HTNC exposed to ASC-CM showed a significant increase in cell viability, an improved cell morphology and higher scratch wound closure at all HG time points. Moreover, the exposure to ASC-CM significantly increased thrombospondin 1 and transforming growth factor beta 1 (TGF-β1) content in HG-HTNC. The TGF-β1 elevation was paralleled by higher Collagen I and Vascular Endothelial Growth Factor in HG-HTNC. ASC-CM may restore the natural morphology, cell viability and structure of HTNC, promoting their scratch wound closure through TGF-β1 increase.

Comparative analysis of the effects of limbal and adipose‐derived mesenchymal stem cell conditioned media on oxidative stress‐induced damage in retinal pigment epithelial cells

Aims/Purpose: The purpose of this investigation was to evaluate the impact of adipose and limbal tissue‐derived mesenchymal stem cell‐conditioned media on survival, and inflammation in H2O2‐induced retinal pigment epithelial cells.Methods: Following national regulations and with the approval of the Koç University Ethics and Investigation Committee, human limbal (L) and adipose‐derived (Ad) mesenchymal stem cells (MSCs) were isolated from cadaveric corneaoscleral rims from donors that originated from the cornea bank of Ministry of Health in Turkey and adipose tissue from discarded human liposuction surgery specimens, respectively. Conditioned medium (CM) that obtained by culturing MSCs in absence of serum was concentrated by filtration. Following a 24‐h pre‐treatment with H2O2, retinal pigment epithelial (RPE) cells were treated with MSC‐CM. Groups were defined as follows: Control, H2O2‐treatment (OS), OS‐Basal Medium, OS‐AdMSC‐CM and OS‐LMSC‐CM. Cell viability was assessed using MTT assay. Gene expression levels of pro‐ and anti‐inflammatory cytokines were analysed by qRT‐PCR.Results: Based on MTT assay, H2O2 inhibited the growth of RPE cells with IC50 values of 150 μM at 24 h. We found that MSC‐CM attenuated H2O2‐induced loss of cell viability. In comparison to the OS group, the LMSC‐CM and AdMSC‐CM treatments demonstrated significantly decreased levels of IL‐6, IL‐8, IL‐10 and TGF‐β1 gene expression.Conclusions: MSC‐CM ameliorated H2O2‐induced retinal pigment epithelial cell damage by alleviating inflammation.

Effects of bone marrow mesenchymal stem cell-conditioned medium on the proliferation and migration of liposarcoma cells.

Liposarcoma constitutes a prevalent subtype of soft tissue sarcoma, represents approximately 20% of all sarcomas. However, conventional chemotherapeutic agents have shown restricted effectiveness in treating liposarcoma patients. Accumulating evidence indicates that mesenchymal stem cells (MSCs) have the characteristic of migration to tumor site, promote or suppress tumors. How human bone marrow mesenchymal stem cells (BMSCs) contribute to liposarcoma phenotype remains poorly understood. This study aims to investigate the effects of human bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on the proliferation and migration of liposarcoma cell lines 93T449 and SW872, as well as explore potential underlying mechanisms of BMSC-CM action on these cells. We transfected BMSCs with lentiviral constructs to knock down the transcriptional co-activator Yes-associated protein 1 (YAP1), conditioned medium (CM) obtained from BMSCs and shYAP1-BMSC, respectively. Liposarcoma cell lines 93T449 and SW872 were co-cultured with BMSC-CM or shYAP1-BMSC-CM. Cell proliferation ability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using flow cytometric assay. A wound healing assay was used to analyze cell migration. The expression levels of YAP1, Bcl-2, and matrix metalloproteinase-2 (MMP-2) were determined by western blot assay. Co-culturing liposarcoma cell lines 93T449 and SW872 with BMSC-CM promoted tumor cell proliferation, while shYAP1-BMSC-CM significantly inhibited cell viability and migration, induced apoptosis, and downregulated Bcl-2 and MMP-2 expression. These findings provide new insights into the impact of BMSC-CM on liposarcoma and suggest its possible involvement in liposarcoma cell growth.

Bovine umbilical mesenchymal stem cell-conditioned medium increased expression of GLUT-4 in pancreas of diabetic rats induced by nicotinamide-streptozotocin.

Diabetes is a degenerative disease associated with metabolic disorders. The majority of people have type 2 diabetes mellitus (DM) insulin resistance due to an unhealthy lifestyle. The development of DM treatment is also growing, one of which is using conditioned medium. This study aims to determine the effect of Bovine umbilical mesenchymal stem cell-conditioned medium (BUMSC-CM) on nicotinamide (NA) and streptozotocin (STZ) induced rats as an animal model of DM. The study began with the in vitro docking of Cholecalciferol with aldolase reductase and glucokinase. In the in vivo study, animal models were divided into five groups: group A (negative control), group B (diabetic rats), group C (NA+STZ+Metformin), group D (NA+STZ+ BUMSC-CM 0.2 ml/kg BW), and group E (NA+STZ+ BUMSC-CM 0.5 ml/kg BW). Blood sugar levels were checked, and BUMSC-CM was administered by intramuscular injection at four-day intervals for a duration of 16 days. Blood sugar levels were also sampled, and GLUT4 histochemical and immunohistochemical staining was performed. The results showed that Cholecalciferol can bind to aldolase reductase ASP43 and TYR48 and bind to glucokinase at TYR214 with hydrogen bonds. BUMSC-CM administration was able to reduce blood sugar well. In addition, BUMSC-CM also helped repair the tissue structure of the pancreas damaged by inflammation from STZ administration. This study can be concluded that the administration of BUMSC-CM can be an alternative cell-free therapy for patients with DM.

Secretome Therapy, Potential Treatment for Rotator Cuff Tear: A Case Study

Introduction: Rotator cuff tears (RCTs) are the most frequent pathologies within the shoulder girdle, and this injury is the most commonly diseased tendons in the human body. RCTs can cause pain and weakness in the shoulder. A 42-year-old woman had right shoulder pain and arm lift problems for almost a year. Ultrasonography revealed a full-thickness Supraspinatus tear. The patient underwent an Ultrasound-guided supraspinatus tendon injection of 2 ml umbilical cord Mesenchymal Stem Cell (MSC) secretome.&#x0D; Methods: This is a case report analyzing the role of secretome therapy in rotator cuff tear potential treatment. The study was conducted at RSCM Hospital.&#x0D; Results: At the one-week follow-up, the patient's Shoulder Pain and Disability Index (SPADI) score decreased from 56,9% to 48,5%, her pain scale reduced from 8 to 6, and her shoulder Active Range of Motion (AROM) improved. After a month, there was an improvement in her shoulder AROM. However, the ultrasound shows no significant improvement in the supraspinatus tear structure after the secretome injection.&#x0D; Conclusion: Secretome has a potential effect on reducing pain and improving functional outcomes in rotator cuff tears. These findings raise the prospect of secretome as a potential regenerative therapy for rotator cuff tears.&#x0D; Keywords: mesenchymal stem cells conditioned-medium, rotator cuff tear, secretome, supraspinatus tear.&#x0D;

Administration of adipose-derived mesenchymal stem cell conditioned medium improves ovarian function in polycystic ovary syndrome rats: involvement of epigenetic modifiers system

BackgroundPolycystic ovary syndrome (PCOS) is a widespread heterogeneous disease that is in association with genetic, epigenetic, endocrine and environmental factors. Adipose-derived mesenchymal stem cell (ASC) and ASC-conditioned medium (ASC-CM) have shown promising abilities in tissue regeneration. In the present study, we aimed to investigate the effects of ASC and ASC-CM on epigenetic regulators, steroidal function and folliculogenesis in the letrozole-induced PCOS rats.ResultsBased on the measurement of the oral glucose tolerance test and physical parameters including body weight, estrus cycle pattern as well as ovary dimensions, PCOS-induced rats in sham and control (CTRL) groups showed signs of reproductive dysfunctions such as lack of regular estrus cyclicity, metabolic disorders such as increased ovary dimension, body weight and blood glucose level alteration which were improved especially by ASC-CM administration.

Mesenchymal stromal cells secretome restores bioenergetic and redox homeostasis in human proximal tubule cells after ischemic injury

BackgroundIschemia/reperfusion injury is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal stromal cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy; however, their full potential in treating AKI remains unknown.MethodsIn this study, we employed an in vitro model of chemically induced ischemia using antimycin A combined with 2-deoxy-d-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow, and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential), and overall cell metabolism by metabolomics.ResultsOur findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction, and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40–50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites.ConclusionOur in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.

Mesenchymal stem cell conditioned medium alleviates acute lung injury through KGF-mediated regulation of epithelial sodium channels

Acute lung injury (ALI) is a progressive inflammatory injury, and mesenchymal stem cells (MSCs) can be used to treat ALI. MSC-conditioned medium (MSC-CM) contains many cytokines, in which keratinocyte growth factor (KGF) is a soluble factor that plays a role in lung development. We aim to explore the protective effects of MSCs secreted KGF on ALI, and investigate the involvement of epithelial sodium channel (ENaC), which are important in alveolar fluid reabsorption. Both lipopolysaccharides (LPS)-induced mouse and alveolar organoid ALI models were established to confirm the potential therapeutic effect of MSCs secreted KGF. Meanwhile, the expression and regulation of ENaC were determined in alveolar type II epithelial (ATII) cells. The results demonstrated that MSC-CM and KGF could alleviate the extent of inflammation-related pulmonary edema in ALI mice, which was abrogated by a KGF neutralizing antibody. In an alveolar organoid ALI model, KGF in MSC-CM could improve the proliferation and decrease the differentiation of ATII cells. At the cellular level, the LPS-inhibited protein expression of ENaC could be reversed by KGF in MSC-CM. In addition, bioinformatics analysis and our experimental data provided the evidence that the NF-κB signaling pathway may be involved in the regulation of ENaC. Our research confirmed that the therapeutic effect of MSC-CM on edematous ALI was closely related to KGF, which may be involved in the proliferation and differentiation of ATII cells, as well as the upregulation of ENaC expression by the inhibition of NF-κB signaling pathway.

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.

Stem Cell-Based Therapy and Cell-Free Therapy as an Alternative Approach for Cardiac Regeneration.

The World Health Organization reports that cardiovascular diseases (CVDs) represent 32% of all global deaths. The ineffectiveness of conventional therapies in CVDs encourages the development of novel, minimally invasive therapeutic strategies for the healing and regeneration of damaged tissue. The self-renewal capacity, multilineage differentiation, lack of immunogenicity, and immunosuppressive properties of mesenchymal stem cells (MSCs) make them a promising option for CVDs. However, growing evidence suggests that myocardial regeneration occurs through paracrine factors and extracellular vesicle (EV) secretion, rather than through differentiation into cardiomyocytes. Research shows that stem cells secrete or surface-shed into their culture media various cytokines, chemokines, growth factors, anti-inflammatory factors, and EVs, which constitute an MSC-conditioned medium (MSC-CM) or the secretome. The use of MSC-CM enhances cardiac repair through resident heart cell differentiation, proliferation, scar mass reduction, a decrease in infarct wall thickness, and cardiac function improvement comparable to MSCs without their side effects. This review highlights the limitations and benefits of therapies based on stem cells and their secretome as an innovative treatment of CVDs.