Mesenchymal Stem Cells In Microenvironment Research Articles

TGF-β1 Induces Endoplasmic Reticulum Stress-Dependent Apoptosis in Human Placental Mesenchymal Stem Cells of Fetal Origin through PERK Signaling Pathway

Background: Mesenchymal Stem Cells (MSCs) are pivotal in immunomodulation, hematopoiesis, and tissue repair. The interplay between MSCs and the pathological microenvironment influences their proliferation and differentiation. Transforming Growth Factor-Beta 1 (TGF- β1) serves as a key cytokine in the MSC microenvironment. This study aimed to scrutinize the impact of TGF-β1 on human placenta-derived MSCs of fetal origin (fPMSCs) and elucidate its underlying mechanism. Methods: fPMSCs were isolated, and surface markers were identified by flow cytometry. Cell proliferation in fPMSCs was assessed using Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2’-Deoxy Uridine (EdU). Apoptosis was detected via Annexin V/PI staining, and apoptosis-related proteins were detected by western blot. Endoplasmic reticulum (ER) stress-related proteins were detected by western blot, and Flou-4 AM staining was utilized to assess intracellular Ca2+ levels under TGF-β1 exposure. The impact of 4-PBA treatment on ER stress and apoptosis was assessed by western blot and Annexin V/PI staining. Additionally, the PERK and p-PERK expressions were evaluated via Western blot. Results: CCK-8 and EdU assays revealed inhibited proliferation of fPMSCs under TGF-β1 exposure. Annexin V/PI staining demonstrated a significant induction of apoptosis in fPMSCs following TGF-β1 treatment. Furthermore, TGF-β1 treatment significantly elevated intracellular Ca2+ levels and the expressions of GRP78, p-eIF2α, and CHOP. Interruption of ER stress with 4-PBA mitigated TGF-β1-induced apoptosis in fPMSCs. Moreover, TGF-β1 increased p-PERK expression. Inhibition of PERK autophosphorylation with GSK2606414 suppressed TGF-β1-induced apoptosis and ER stress in fPMSCs. Conclusion: Our findings indicated that TGF-β1 induced ER stress-dependent apoptosis in fPMSCs through the PERK signaling pathway. These results offer insights into enhancing the therapeutic efficacy of fPMSCs by modulating TGF-β1-induced apoptosis.

Caffeine and naloxone treated mesenchymal stem cells improve symptoms and reduce inflammation in a mouse model of ulcerative colitis

Background and objectiveUlcerative colitis (UC) causes ulcers in the colon and rectum, leading to abdominal pain, diarrhea, and rectal bleeding, and if left untreated, can lead to serious complications. The therapeutic effects of mesenchymal stem cells (MSCs) on experimental models of UC have been proven. Since the microenvironment around these cells is crucial in maintaining cell proliferation, differentiation, metabolism, and overall function, this study aims to evaluation the role of caffeine and naloxone as a new microenvironment for MSCs in reducing inflammation and improving symptoms in an experimental model of UC. Material and methodA group of 40 outbred NMRI mice were studied and divided randomly into four equal groups (N = 10 each group). UC was induced in all groups using acetic acid. The first group (control) was treated with phosphate buffer saline (PBS), the second group with MSCs-Caffeine, the third with MSCs-Naloxone, and the fourth with Mesalazine. The disease activity index (DAI), tissue damage, myeloperoxidase (MPO) activity, nitric oxide (NO) levels, and the production of IL-1, IL-6, and TNF-α cytokines were evaluated. ResultOur research demonstrated that all treatments were effective in improving the symptoms and reducing inflammatory markers in mice with colitis. Among the two MSCs treatments, the MSCs-Caffeine was found to be the most potent in reducing the levels of NO, IL-1, IL-6, tissue damage (P < 0.001) and as well as TNF-α (P < 0.0001) in compared to the control group. ConclusionMSCs treated with caffeine and naloxone can enhance the immunoregulatory potential of these. As a result, treated MSCs can lead to improved clinical signs and reduced inflammatory parameters in mice with UC, making this approach a useful way for controlling and treating the disease. However, additional research is needed to access the mechanism behind the stronger immune system regulatory effects of treated MSCs in UC treatment.

Creating an atlas of the bone microenvironment during oral inflammatory-related bone disease using single-cell profiling.

Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases.

The role of microenvironment in stem cell-based regeneration of intervertebral disc.

Regenerative medicine for intervertebral disc (IVD) disease, by utilizing chondrocytes, IVD cells, and stem cells, has progressed to clinical trials in the treatment of back pain, and has been studied in various animal models of disc degeneration in the past decade. Stem cells exist in their natural microenvironment, which provides vital dynamic physical and chemical signals for their survival, proliferation and function. Long-term survival, function and fate of mesenchymal stem cells (MSCs) depend on the microenvironment in which they are transplanted. However, the transplanted MSCs and the endogenous disc cells were influenced by the complicated microenvironment in the degenerating disc with the changes of biochemical and biophysical components. It is important to understand how the MSCs and endogenous disc cells survive and thrive in the harsh microenvironment of the degenerative disc. Furthermore, materials containing stem cells and their natural microenvironment have good clinical effects. However, the implantation of tissue engineering IVD (TE-IVD) cannot provide a complete and dynamic microenvironment for MSCs. IVD graft substitutes may need further improvement to provide the best engineered MSC microenvironment. Additionally, the IVD progenitor cells inside the stem cell niches have been regarded as popular graft cells for IVD regeneration. However, it is still unclear whether actual IVD progenitor cells exist in degenerative spinal conditions. Therefore, the purpose of this review is fourfold: to discuss the presence of endogenous stem cells; to review and summarize the effects of the microenvironment in biological characteristics of MSC, especially those from IVD; to explore the feasibility and prospects of IVD graft substitutes and to elaborate state of the art in the use of MSC transplantation for IVD degeneration in vivo as well as their clinical application.

TGFβ1-Pretreated Exosomes of Wharton Jelly Mesenchymal Stem Cell as a Therapeutic Strategy for Improving Liver Fibrosis

Background: Mesenchymal stem cells (MSCs) are the most promising tools for cell treatment and human tissue regeneration, e.g., in liver fibrosis. Mesenchymal stem cells repair tissue damage through paracrine mediators such as exosomes. Types and concentrations of inflammatory mediators, including transforming growth factor-beta (TGFβ1), in MSCs microenvironment can affect MSCs’ function and therapeutic potency. Objectives: This experimental study aimed to explore the effects of Wharton jelly MSCs (WJ-MSCs) exosomes on fibrotic gene expression and Smad2/3 phosphorylation (phospho-Smad2/3 (p-Smad2/3)). Moreover, we further investigated whether WJ-MSCs pretreatment with different concentrations of TGFβ1 changes the anti-fibrotic properties of their exosomes. Methods: After isolation from the umbilical cord, WJ-MSCs were characterized by observing differentiation and measuring surface biomarkers using flowcytometry. The WJ-MSC-derived exosomes were extracted and identified using transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting. Real-time PCR and western blot for extracellular matrix (ECM) and p-Smad2/3 expression detection were used to investigate the effect of exosomes from untreated and TGFβ1-pretreated WJ-MSCs on activated hepatic stellate cells (HSCs). Results: Phospho-Smad2/3, α-smooth muscle actin (α-SMA), and collagen1α1 levels were enhanced following treatment with TGFβ1, whereas E-cadherin was decreased. However, the outcomes were reversed after treatment with WJ-MSC-derived exosomes. Exosomes from TGFβ1-pretreated WJ-MSCs induced a significant decrease in p-Smad2/3 levels in activated HSCs, accompanied by the upregulation of E-cadherin gene expression and downregulation of α-SMA and collagen1α1 when compared to untreated WJ-MSC-derived exosomes. The p-Smad2/3 proteins were significantly decreased (fold change: 0.23, P-value &lt; 0.0001) after exposure to low-dose TGFβ1-pretreated WJ-MSC-derived exosomes (0.1 ng/mL), showing the best effect on activated HSCs. Conclusions: Exosomes derived from untreated WJ-MSCs could regress TGFβ-Smad2/3 signaling and the expression of fibrotic markers in activated LX-2 cells. However, these effects were significantly profound with applying exosomes derived from 0.1 ng/mL TGFβ-pretreated WJ-MSCs. We also observed the dose-response effects of TGFβ on WJ-MSCs-derived exosomes. Therefore, exosomes derived from TGFβ-pretreated WJ-MSCs may be critical in improving fibrosis and benefit liver fibrosis patients.

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds.

The physiological O2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O2, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O2. At 21% O2, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young’s modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O2 hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

Heterospheroid formation improves therapeutic efficacy of mesenchymal stem cells in murine colitis through immunomodulation and epithelial regeneration

Tissue repairing capacity and immunomodulatory effects of mesenchymal stem cells (MSCs) have been extensively utilized for treating various inflammatory disorders; however, inconsistent efficacy and therapeutic outcomes due to low survival rate after transplantation often restrain their clinical potential. To overcome these limitations, 3-dimensional culture (3D-culture) was established to augment stemness and paracrine functions of MSCs, although hypoxic stress at the core often leads to unexpected cell death. Thus, we designed a novel strategy to improve the microenvironment of MSCs by creating heterospheroids (HS) consisting of MSCs and quercetin (QUR)-loaded microspheres (MSCHS), to achieve local drug delivery to the cells. Notably, MSCHS exhibited resistance for senescence-associated phenotype and oxidative stress-induced apoptosis compared to 3D-cultured MSCs (MSC3D), as well as to 2D-cultured cells (MSC2D) in vitro. In a murine model of colitis, MSC3D and MSCHS exhibited enhanced anti-inflammatory impact than MSC2Dvia attenuating neutrophil infiltration and regulating helper T cell (Th) polarization into Th1 and Th17 cells. Interestingly, MSCHS provided better therapeutic outcomes compared to MSC3D, partially due to their enhanced survival capacity in vivo. Moreover, we found that MSC-derived paracrine factor, prostaglandin E2 (PGE2), can directly drive the epithelial regeneration process by inducing specialized tissue-repairing cell generation using the intestinal organoid culture. Importantly, MSC3D and MSCHS displayed an outstanding regeneration-inducing potency compared to MSC2D owing to their superior PGE2 secretion. Taken together, we suggest a convergent strategy of MSCHS formation with reactive oxygen species (ROS) scavenger, QUR, which can maximize the inflammation-attenuating and tissue-repairing capacity of MSCs, as well as the engraftment efficiency after transplantation.

Bioglass enhances the production of exosomes and improves their capability of promoting vascularization

Recently, exosomes have been extensively applied in tissue regeneration. However, their practical applications are severely restricted by the limited exosome secretion capability of cells. Therefore, developing strategies to enhance the production of exosomes and improve their biological function attracts great interest. Studies have shown that biomaterials can significantly enhance the paracrine effects of cells and exosomes are the main signal carriers of intercellular paracrine communication, thus biomaterials are considered to affect the exosome secretion of cells and their biological function. In this study, a widely recognized biomaterial, 45S5 Bioglass® (BG), is used to create a mild and cell-friendly microenvironment for mesenchymal stem cells (MSCs) with its ion products. Results showed that BG ion products can significantly improve exosome production of MSCs by upregulating the expression of neutral sphingomyelinase-2 (nSMase2) and Rab27a which enhanced the nSMases and Rab GTPases pathways, respectively. Besides, microRNA analysis indicates that BG ion products can modulate the cargoes of MSCs-derived exosomes by decreasing microRNA-342-5p level while increasing microRNA-1290 level. Subsequently, the function of exosomes is modified as their capabilities of promoting the vascularization of endothelial cells and facilitating the intradermal angiogenesis are enhanced. Taken together, BG ion products are confirmed to enhance exosome production and simultaneously improve exosome function, suggesting a feasible approach to improve the practical application of exosomes in regenerative medicine.

Bone marrow mesenchymal stem cells in microenvironment transform into cancer-associated fibroblasts to promote the progression of B-cell acute lymphoblastic leukemia

Bone marrow microenvironment is essential for leukemia cells to survive and escape the killing effect of chemotherapeutics. Cancer-associated fibroblasts (CAFs) are the dominant stromal cells in tumor microenvironment (TME), but their role in B-cell acute lymphoblastic leukemia (B-ALL) remains unclear. Here, RT-PCR and Western blotting in bone marrow mononuclear cells revealed higher proportions of CAFs markers α-SMA and FAP in the newly diagnosed and relapsed B-ALL patients. In vitro experiments, bone marrow mesenchymal stem cells (BM-MSCs) acquired a CAFs phenotype after co-culture with leukemia cells, which produced high level of tumor-promoting growth factors and reduced the daunorubicin (DNR)-induced damage to B-ALL cells. As for its mechanism, CAFs activation was mediated by TGF-β up-regulation in the co-culture system, and TGF-β triggered MSCs conversion into CAFs relying on the SDF-1/CXCR4 pathway. Further LY2109761 and AMD3100 effectively decreased the activation of CAFs through inhibiting TGF-β receptor and CXCR4. Comparative experiments with MSCs and transformed CAFs prompted that CAFs had more obvious effect than MSCs on stimulating leukemia progression through accelerating leukemia cell migration and invasion. These results clarified the important role of CAFs in B-ALL progression and the possible mechanisms of CAFs activation in leukemia microenvironment, which might provide a theoretical basis for B-ALL patients to find more effective targeted therapies targeting the bone marrow microenvironment.

Assessment of the Effect of Infliximab on Immunomodulation Properties of Mesenchymal Stem Cells In Vitro.

Purpose: Mesenchymal stem cells (MSCs) have immunomodulatory traits making them a promising choice in the treatment of inflammatory diseases such as graft-versus-host disease (GVHD). Tumor necrosis factor-alpha (TNFα) is a major player of inflammatory disease which is blocked by infliximab to reduce the inflammation. The present study aims to assess the infliximab effects on the anti-inflammatory properties of MSCs. Methods: In this study, bone marrow mesenchymal stem cells (BMMSCs) were co-cultured with peripheral blood mononuclear cells (PBMCs) of GVHD patients in the presence of 10, 20 and 30 µg/mL of infliximab for 48 and 72 hours. The mRNA expression of indoleamine-2,3- dioxygenase (IDO) and inducible nitric oxide synthase (iNOS), as well as the secreted amount of prostaglandin E2 (PGE2) in the culture supernatant, were examined. Results: The results of this study show that the expression of IDO and iNOS genes, as well as the secretion amount of PGE2 in co-cultured groups raised dramatically, compared to the culture of BMMSCs or PBMCs alone. In co-culture groups containing infliximab, the expression of IDO and iNOS and also the amount of released PGE2 was significantly decreased compared to the control group without infliximab. However, no difference was found in the expression of assayed factors between 48 and 72 hours of treatments. Conclusion: As an anti-TNFα agent, infliximab can decrease the inflammation in the microenvironment of MSCs, which might mitigate the immunomodulatory effects of MSCs. These effects of anti-inflammatory agents on the immunomodulatory capacity of MSCs should be considered in MSC therapy.

Probucol enhances the therapeutic efficiency of mesenchymal stem cells in the treatment of erectile dysfunction in diabetic rats by prolonging their survival time via Nrf2 pathway

BackgroundIntracavernous injection of mesenchymal stem cells (MSCs) is a promising method for diabetic mellitus-induced erectile dysfunction (DMED), but short survival time of MSCs in cavernous is a fatal defect for therapy. This study investigated therapeutic efficiency and potential mechanism of probucol combined with MSCs.MethodsIn vivo study, a total of forty-eight 10-week-old male Sprague-Dawley (SD) rats were used. Twelve rats received intraperitoneal injection of PBS as the sham group; the rest received intraperitoneal injection of 60 mg/kg streptozotocin to establish DM models. DM rats were randomly divided into three groups: received intracavernosal (IC) injection of either PBS (DM group), MSCs (M group), or administrated probucol after intracavernosal injection of MSCs (P + M group). Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated for histologic examination and Western blotting. In in vitro experiment, H2O2 was used to create oxidative stress environment to detect changes in cell viability. CCK8 was used to measure cell viability of MSCs treated with or without probucol. Intracellular ROS changes were detected by flow cytometry. Autophagy and apoptosis were detected by Western blotting and confocal microscopy.ResultsRecovery of erectile function was observed in the P + M group. The combination therapy decreased fibrosis and increased endothelial function compared with MSC therapy alone. Western blotting results confirmed the increased expression of Nrf2 and HO-1 in cavernous body. H2O2 induced high oxidative stress and reduced cell viability in vitro, which was gradually reversed with increased concentration of probucol. H2O2 reduced Nrf2 expression, which was reversed by probucol’s intervention. Furthermore, the expression of Bax, Caspase3, and Cleaved-Caspase3 decreased, and the expression of Bcl-2 increased in a dose-dependent manner because of probucol’s intervention. In addition, Beclin1 and LC3II both increased in a dose-dependent manner. Meanwhile, the expression of P62 decreased. In the study of autophagy flux, we found probucol did not block it.ConclusionProbucol enhanced therapeutic efficiency of MSCs in DMED by prolonging their survival time, which mediated through improving the transplanted microenvironment of MSCs, increasing self-antioxidant ability of MSCs, strengthening protective autophagy, and inhibiting apoptosis of MSCs via Nrf2 pathway.Graphical abstractSchematic model showing combined probucol and MSCs to improve DMED. Probucol increases self-antioxidant ability of MSCs, strengthening protective autophagy and inhibiting apoptosis via Nrf2/HO-1 and Nrf2/autophagy pathways.

Bone-Inspired Tube Filling Decellularized Matrix of Toad Cartilage Provided an Osteoinductive Microenvironment for Mesenchymal Stem Cells to Facilitate the Radius Defect Repair of Rabbit.

Toad bone not only contains the rich cartilage-like matrix but also presents low immunogenicity. It is inferred that decellularized toad bone matrix (dBECM) may provide the more profitable osteoinductive microenvironment for mesenchymal stem cells (MSCs) to promote the repair of bone defects. Herein, a hollow bone-inspired tube is first made from hydroxyapatite (HA) and poly (γ-glutamic acid) (PGA), and then MSCs/dBECM hydrogel is uniformly filled to its central cavity, constructing a biomimetic bone (dBECM + MSCs - PGA + HA). In vitro scratch and transwell experiments show that dBECM hydrogel not only effectively promotes migration and proliferation of MSCs but also induces their osteogenic differentiation. Moreover, the less inflammatory macrophages infiltrate at rat skin after subcutaneously injecting dBECM hydrogel, indicating its low potential for inflammatory attack. After implanting dBECM + MSCs - PGA + HA to critical radius defect of rabbit, X-ray and CT imaging shows that the cortex is effectively regenerated and the medullary cavity recanalization is completed at 20 weeks. Moreover, the expression of Collagen-II and OCN are obviously increased in the defect after implanting dBECM + MSCs - PGA + HA. The therapeutic mechanism of dBECM + MSCs - PGA + HA scaffold are highly associated with the enhanced angiogenesis. Collectively, the biomimetic dBECM + MSCs - PGA + HA scaffold may be a promising strategy to improve radius defect healing efficiency.

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration.

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

Directing hMSCs fate through geometrical cues and mimetics peptides.

The native microenvironment of mesenchymal stem cells (hMSCs)-the extracellular matrix (ECM), is a complex and heterogenous environment structured at different scales. The present study aims at mimicking the hierarchical microorganization of proteins or growth factors within the ECM using the photolithography technique. Polyethylene terephthalate substrates were used as a model material to geometrically defined regions of RGD + BMP-2 or RDG + OGP mimetic peptides. These ECM-derived ligands are under research for regulation of mesenchymal stem cells osteogenic differentiation in a synergic manner. The hMSCs osteogenic differentiation was significantly affected by the spatial distribution of dually grafted peptides on surfaces, and hMSCs cells reacted differently according to the shape and size of peptide micropatterns. Our study demonstrates the presence of a strong interplay between peptide geometric cues and stem cell differentiation toward the osteoblastic lineage. These tethered surfaces provide valuable tools to investigate stem cell fate mechanisms regulated by multiple ECM cues, thereby contributing to the design of new biomaterials and improving hMSCs differentiation cues.

Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds.Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed using a Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 h when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 h. Electrospun fibers seeded with MSCs were then applied to a rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA), and then analyzed by confocal microscopy. Immunostaining showed that expression of α-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation.MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models. Statement of significancePrevious studies have shown that the secretome of bone marrow-derived mesenchymal stem cells (MSC) is promotes corneal wound healing by facilitating improved wound closure rates and reduction of scarring and neovascularization. The present research is significant because it provides evidence for the modulation of the secretome as a function of the MSC culture environment. This leads to differential expression of therapeutic factors secreted, which can impact corneal epithelial and stromal healing after severe injury.In addition, this article shows that co-continuous delivery of the MSC secretome improves cell migration and proliferation over aliquoted delivery, and that MSCs grown on three-dimensional electrospun fiber constructs may provide a favorable microenvironment for cultured MSCs and as a carrier to deliver their secreted factors to the ocular surface.

Stem and progenitor cell microenvironment for bone regeneration and repair.

Stem cells reside in their native microenvironment, which provides dynamic physical and chemical cues essential to their survival, proliferation and function. A typical cell-based therapeutic approach requires the mesenchymal stem cells (MSC) to depart their native microenvironment, transplant to in-vivo environment, differentiate toward multiple lineages and participate in bone formation. The long-term survival, function and fate of MSC are dependent on the microenvironment in which they are transplanted. Transplantation of morselized autologous bone, which contains both stem cells and their native microenvironment, results in a good clinical outcome. However, implantation of bone graft substitutes does not provide the complete and dynamic microenvironment for MSC. Current bone graft therapeutics may need to be improved further to provide an optimal engineered MSC microenvironment.

Melatonin maximizes the therapeutic potential of non-preconditioned MSCs in a DEN-induced rat model of HCC

Pretreatment of mesenchymal stem cells (MSCs) with melatonin (Mel) improves their potential therapeutic effect on chronic diseases and cancers. However, this preconditioning strategy may direct the effect of Mel toward MSCs alone and deprive cancer cells of the oncostatic effect of Mel. Herein, we hypothesized that Mel given before transplantation of non-preconditioned MSCs may maximize the therapeutic outcome via the oncostatic effect of Mel by preparing a suitable tumor microenvironment for MSCs. Female rats (n = 60) were equally divided into 6 groups; normal control, diethylnitrosamine (DEN), DEN + Mel, DEN + MSCs, DEN + MSCs preconditioned with Mel, and DEN + MSCs + Mel. The obtained data revealed that administration of Mel before MSCs treatment without preconditioning yielded a better ameliorative effect against DEN-induced hepatocellular carcinoma (HCC) as evidenced by: 1) reduced serum levels of alpha fetoprotein and gamma-glutamyl transferase; 2) decreased number and area of glutathione S-transferase placental positive foci; 3) induced apoptosis (as indicated by increased cleaved caspase-3 activity, upregulated expression of proapoptotic genes Bax and caspase 3 and downregulated expression of anti-apoptotic genes Bcl2, survivin); 4) decreased malondialdehyde level and increased activities of superoxide dismutase, catalase, and glutathione peroxidase enzymes; and 5) reduced inflammation, angiogenesis and metastasis as indicated by downregulated expression of interleukin 1 beta, nuclear factor kappa B, vascular endothelial growth factor, and matrix metallopeptidase 9 genes and upregulated expression of metalloproteinase inhibitor 1 gene. Thus, administration of Mel before MSCs (without preconditioning) fostered the survival and therapeutic potential of MSCs in HCC, possibly through induction of apoptosis and inhibition of inflammation and oxidative stress. This new strategy showed better therapeutic outcomes and may improve MSC-based therapies for HCC.

Effects of palmitate and astaxanthin on cell viability and proinflammatory characteristics of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have broad immunomodulatory activities. These cells are a stable source of cytokine production such as interleukin-6 (IL6), monocyte chemoattractant protein-1 (MCP-1/CCL2) and vascular endothelial growth factor (VEGF). Fatty acid elevation in chronic metabolic diseases alters the microenvironment of MSCs and thereby, might affect their survival and cytokine production. In the present study, we investigated the effects of palmitate, the most abundant saturated free fatty acid (FFA) in plasma, and astaxanthin, a potent antioxidant, on cell viability and apoptosis in human bone marrow-driven mesenchymal stem cells. We also elucidated how palmitate and astaxanthin influence the inflammation in MSCs. Human mesenchymal stem cells were collected from an aspirate of the femurs and tibias marrow compartment. The effect of palmitate on cell viability, caspase activity and pro-inflammatory cytokines expression and secretion were evaluated. In addition, activation of the MAP kinases and NF-kB signaling pathways were investigated. The results showed that astaxanthin protected MSCs from palmitate-induced cell death. We found that palmitate significantly enhanced IL-6, VEGF and MCP-1 expression, and secretion in MSC cells. Increased cytokine expression was parallel to the enhanced phosphorylation of P38, ERK and IKKα-IKKβ. In addition, pretreatment with JNK, ERK, P38, and NF-kB inhibitors could correspondingly attenuate palmitate-induced expression of VEGF, IL-6, and MCP-1. Our results demonstrated that fatty acid exposure causes inflammatory responses in MSCs that can be alleviated favorably by astaxanthin treatment.

Characterizing the Impact of 2D and 3D Culture Conditions on the Therapeutic Effects of Mesenchymal Stem Cell Secretome on Corneal Wound Healing &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;ex vivo&lt;/i&gt;

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds.Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed by Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 hr when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 hr. Electrospun fibers seeded with MSCs were then applied to a wounded rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA) and keratan sulfate (KS), and then analyzed by confocal microscopy. Immunostaining showed that expression of alpha-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation. KS expression was higher in corneas treated with MSCs seeded on fibers, suggesting that keratocytic phenotype was preserved.MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models.

MiR-21/STAT3 Signal Is Involved in Odontoblast Differentiation of Human Dental Pulp Stem Cells Mediated by TNF-α.

Dental pulp stem cells (DPSCs), as one type of mesenchymal stem cells (MSCs), have the capability of self-renewal and multipotency to differentiate into several cell lineages, including osteogenesis, odontoblasts, chondrogenesis, neurogenesis, and adipogenesis. It has found that tumor necrosis factor-α (TNF-α) can promote osteogenic differentiation of human DPSCs in our previous studies. Other experimentation revealed that signal transducer and activator of transcription 3 (STAT3) underwent a rapid activation both in osteogenesis and inflammation microenvironment of MSCs in vitro. MicroRNAs (miRNAs or miRs) have been proved in previous studies to regulate MSCs differentiation in vitro. In this study, we identified miR-21 as a key miRNA contributed the functional axis of odontoblast differentiation induced by STAT3. It is observed that the expression of miR-21 and STAT3 increased gradually in low concentration (1-10 ng/mL) of TNF-α, while they were suppressed in high concentration (50-100 ng/mL). The upregulation of miR-21 may facilitate the odontoblast differentiation of DPSCs coordinating with STAT3. SiSTAT3 or treated by the inhibitor of STAT3, cucurbitacin I (Cuc I), significantly increased primary miR-21 expression along with decreased mature miR-21 expression. Meanwhile, the inhibition of miR-21 (anti-miR-21) decreased the activation of STAT3 as well as suppressed the marker proteins of odontoblast differentiation. The results revealed a new function of miR-21, suggesting that miR-21/STAT3 signal may act as a modulator within a complex network of factors to regulate odontoblast differentiation of human DPSCs. It may provide a novel therapeutic strategy to regulate the odontoblast differentiation of DPSCs.