Bone Marrow Mesenchymal Stem Cells Ameliorate Diabetes and Diabetic Renal Fibrosis by Modulating the Inflammatory Factor IL-11.

Casting Doubt on the Safety of “Off-the-shelf” Mesenchymal Stem Cells for Cell Therapy

Background: It is now established that having diabetes not only increases the chances of cancer also it complicates cancer treatment therapy. Doxorubicin (DOX) is a well known anticancer drug, however the clinical use of DOX was limited due to its cardiotoxic effects. One of the major concerns with DOX therapy has been its toxicity in patients who are less robust and more prone to toxic side effects, particularly patients with comorbid diseases such as diabetes mellitus. Several studies have demonstrated that mesenchymal stem cell (MSC) therapy has the potential to restore cardiac function following DOX induced cardiac injury. However, there is no study available on the effects of MSC therapy on DOX induced cardiac dysfunction in diabetics. Methods and Results: Diabetes was induced in male Wistar rats by streptozotcin injection (STZ, 65mg/kg body weight, i.p.). After 4 weeks of STZ injection, blood glucose levels in STZ group (301.58±23.97mg/dl) were significantly greater than control group (83.51±7.91mg/dl). These diabetic rats were treated with adriamycin (2.5mg/kg body weight, i.p) 3 times/week for two weeks (AD group); or with adriamycin+bone marrow MSCs (BM-MSC; 2x106 cells, via tail vein) or with adriamycin+adipose tissue derived MSCs (AD-MSC; 2x106 cells, via tail vein). Echocardiographic measurements showed a significant decline in cardiac function (%EF) following adriamycin treatment. Both BM-MSC and AT-MSC treatment improved %EF at 4 weeks. After 4 weeks of MSC injection, hearts from all the groups were excised and subjected to retrograde Langendorff perfusion and baseline levels of left ventricular developed pressure (LVDP), maximum rate of pressure rise dp/dt max and rate pressure product (RPP) were recorded. AD treatment caused a significant decrease in LVDP, dp/dt max and RPP levels. Both BM-MSCs and AD-MSCs injection significantly improved all these parameters. Conclusion: Both BM-MSC and AT-MSC were equally effective in preventing deterioration of cardiac function following doxorubicin therapy in diabetic rats. Furthermore, these findings should act as a stimulus for further research on the benefits of MSC therapy for diabetic subjects suffering from cancer.

This study was aimed to enhance the healing potential of rat bone marrow mesenchymal stem cells against chronic diabetic wounds through interleukin-7 (IL-7) transfection. IL-7 plays an important role in wound healing and acts as a survival factor in some cell types. This study involves isolation, propagation, and characterization of mesenchymal stem cells (MSCs) and their modification with IL-7 gene via retroviral transfection. Transfected MSCs were assessed for their effect on angiogenic genes by qPCR. Wound healing potential of transfected MSCs was analyzed by scratch assay in vitro and by transplanting these cells in rat diabetic wound models in vivo. Wound area was measured for a period of 15 days and subsequent histological analysis was performed. qPCR results showed increased expression of IL-7 gene (p ≤ 0.05) and also principal angiogenic genes, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), VEGF receptor 1 (FLT-1), and VEGF receptor 2 (FLK-1) (p ≤ 0.05). Neuropilin-1 (NRP-1) did not show any significant change. In vitro analysis of IL-7 MSCs showed intense cell-cell connections and tube formation as compared to the normal MSCs. Rate of wound closure was more (p ≤ 0.001) in case of diabetic group transplanted with IL-7 MSCs. Histological examination revealed enhanced vascular supply in skin tissues of diabetic animals transplanted with IL-7 transfected MSCs as compared to normal MSCs. Immunohistochemical results showed significantly higher expression of IL-7 (p ≤ 0.001) and α-smooth muscle actin(p ≤ 0.001) in the tissue sections of IL-7 transfected group as compared to normal MSCs and the diabetic control group; the latter indicates increase in the number of blood vessels. It is concluded from this study that IL-7 overexpression in MSCs can enhance the healing potential of MSCs and aid in wound closure in diabetic animals through the induction of angiogenic genes.

Osteoarthritis (OA), a degenerative joint disorder, is a major reason of disability in adults. Accumulating evidences have proved that bone marrow mesenchymal stem cells (BMSCs)-carried exosomes play a significant therapeutic effect on OA. However, the precise regulatory network remains unknown. OA and normal cartilage samples were acquired from patients, and chondrocytes were exposed to IL-1β to conduct a cellular OA model. Exosomes prepared from BMSCs were identified using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Cell viability was determined with CCK-8 assay. Inflammatory injury was assessed by LDH and inflammatory factors (TNF-α and IL-6) using corresponding ELISA kits, respectively. Ferroptosis was evaluated by GSH, MDA and iron levels using corresponding kits, and ROS level with DCFH-DA. The expressions of genes/proteins were determined with RT-qPCR/western bolt. RNA immunoprecipitation and luciferase activity assay were conducted for testing the interactions of small nucleolar RNA host gene 7 (SNHG7)/ferroptosis suppressor protein 1 (FSP1) and miR-485-5p. The expressions of SNHG7 and FSP1 were both reduced in IL-1β-induced chondrocytes and OA cartilage tissues, and there was a positive correlation between them in clinical level. Moreover, SNHG7 was enriched in BMSCs-derived exosomes (BMSCs-Exos) and could be internalized by chondrocytes. Functional analysis illustrated that BMSCs-Exos administration repressed inflammatory injury, oxidative stress and ferroptosis in IL-1β-induced chondrocytes, while these changes were reinforced when SNHG7 was overexpressed in BMSCs-Exos. Notably, FSP1 silencing in chondrocytes abolished the beneficial effects mediated by exosomal SNHG7. Exosomal SNHG7 released from BMSCs inhibited inflammation and ferroptosis in IL-1β-induced chondrocytes through miR-485-5p/FSP1 axis. This work suggested that BMSCs-derived exosomal SNHG7 would be a prospective target for OA treatment.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD), which severely affects the quality of patients' lives. However, the current therapeutic approaches can only postpone its progression to ESRD. It is therefore imperative to develop a novel therapeutic strategy for renal injury in DKD, with the objective of restoring renal function and reversing the process of ESRD. In recent years, the potential of mesenchymal stem cell (MSC) therapy for DKD has garnered increasing attention within the scientific community. Preclinical research on MSC therapy has yielded promising results, and the safety of MSC treatment in vivo has been substantiated in clinical studies. An increasing body of evidence suggests that MSC therapy has significant potential for the treatment of DKD. This article reviews the existing research on MSCs and their derived exosomes in treating DKD and analyzes the underlying mechanism of MSC-based therapy for DKD. Additionally, we discuss the potential of combining MSC therapy with conventional pharmacological treatments, along with the constraints and prospects of MSC therapy for DKD. We hope this review can provide a precise and comprehensive understanding of MSCs for the treatment of DKD.

Neuronal plasticity of human Wharton's jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells

Interleukin-9 (IL-9) has been shown to be upregulated in rheumatoid arthritis (RA). The exact role of IL-9 has not yet been effectively studied. Mesenchymal stem cells (MSCs) have shown a promising immunomodulatory role towards repairing cartilage and restoring joint function. One of the key problems influencing the therapeutic efficacy of stem cell therapy is the poor cell survival following transplantation. This is attributed to oxidative and inflammatory stresses at the injured sites. Hesperidin (Hsd), a flavanone present in citrus fruits, has been studied as potential therapeutic agents that have anti-oxidant and anti-inflammatory activities. The objective of this study is to evaluate the therapeutic paracrine action of bone marrow MSCs on the IL-9 level in adjuvant-induced arthritis (AIA) and the enhancement effect of Hsd on transplanted MSCs. Articular tissue inflammation and cartilage damage were assessed by histological scoring. Antinuclear autoantibodies, tumour necrosis factor-alpha (TNF-α), IL-9, IL-4, interferon gamma (IFN-δ), and transforming growth factor-beta1 (TGF-β1), as well as malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels, were assessed in spleen tissue homogenates after treatment with MSCs either alone or combined with Hsd for 4 weeks in an AIA rat model. Results of this study confirmed that MSCs decreased IL-9 levels in AIA and provide novel insights into the application of Hsd on MSC-based treatments.HighlightsAdjuvant-induced arthritis (AIA) is one of the most widely used models that has a great similarity to rheumatoid arthritis (RA).Few studies in recent years have estimated IL-9 in rheumatic diseases and it remains an understudied cytokine.For the first time, bone marrow mesenchymal stem cells (MSCs) therapy has a vital role in splenocytes IL-9 level and further studies are required.Combined therapy of MSCs with antioxidants as hesperidin (Hsd) can alleviate oxidative stress and enhance stem cells immunomodulatory action.

AimTo compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair.MethodsHuman BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively.ResultsA greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source.ConclusionChondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.

What's in a Name?

Mesenchymal stem cell (MSC) therapy is a promising treatment for ischemic injury. However, the environmental regulatory mechanism is essentially unclear and thus greatly limits its application in clinical setting. Accumulating evidence suggests a vital role of aldehyde dehydrogenase-2 (ALDH2) in microenvironment homeostasis after ischemia. About 540 million people or 8% of population worldwide carry a loss-of-function allele of ALDH2. It is unknown whether ALDH2 functions as a host factor regulating the therapeutic potential of donor MSCs. Therefore, this study was designed to determine whether and how host ALDH2 regulates MSC retention and therapeutic efficacy after transplantation into ischemic tissues. Mice limb ischemia was performed by femoral artery ligation. A total of 10(6) MSCs were injected into the ischemic thigh muscles. One, 2, and 4 weeks after transplantation, MSC retention, blood perfusion recovery, limb necrosis, and fibrosis were analyzed. Compared with wild-type tissue, ALDH2 deficiency tissue significantly limited MSC retention and its perfusion recovery and limb salvage effects after ischemia. Importantly, local overexpression of ALDH2 optimized tissue microenvironment and significantly magnified all these MSC-induced improvement. Further study indicated that host ALDH2 regulated transplanted MSC survival and therapy as a microenvironment homeostasis mediator via local capillary density, energy supply, and oxidative stress regulating after ischemia. Our study establishes ALDH2 as a key mediator of host stem cell niche for optimal MSC therapy and suggests that ALDH2 deficiency present in the general population is a limiting host factor to be considered for MSC therapy.

Ischemic heart disease, especially myocardial infarction, is an important cause of heart failure. However, therapeutic potential of mesenchymal stem cells (MSC) for myocardial or pulmonary ischemia reperfusion (IR) injury is not well studied. Interleukin-10 (IL-10) is an anti-inflammatory cytokine. In this study MSC and IL-10 engineered MSC were tested for their ability to prevent IR injury and promote tissue repair. Methods: Bone marrow cells from Lewis rat were cultured in plastic tissue culture flasks for 72 hours; cells in suspension were discarded and the adherent cells (MSC) were expanded. MSC were transduced with r vIL-10-retrovirus and selected on neomycin (1mg/ml for 7 days). Experimental design: following 120 minutes of left lung ischemia, Group A, rats received vIL10 transduced MSC (∼15 x 10 6 ; i.v.); Group B, rats received empty vector engineered MSC; Group C, received MSC; and Group D, received saline. Group E received no ischemia or MSC. Left lung ischemia was performed by clamping left pulmonary artery/vein/bronchus at end inspiration. At 24h, 72 h, & 7 days following ischemia, blood was collected from left and right pulmonary veins, separately. Results : Mean blood oxygenation (PaO2/FIO2 ratio, mmHg) at day 7 was significantly (P<0.05) reduced following IR injury in Group B (161± 77) and Group D (181± 64), compared to MSC-vIL10 (404± 32; Group A) & MSC (294±103;Group C) treated animals. In Group E, PaO2/FIO2 was 440± 42mm Hg. Significant improvement in lung oxygenation with MSC and vIL-10 therapy was observed as early as 3 days. MSC produced vIL-10 (∼5ng/ml) ex vivo . HP of lungs demonstrated reduced PMN cells with MSC therapy. Inflammatory mediators IL-1a, MCP-1, MIP-1a, and IL1-b protein levels were markedly increased in the injured lung; their analysis in MSC treated groups are underway. I.V. injected Tg GFP+MSC trafficked to the lung & other tissues. VEGF & HGF mRNA in MSC was 66% & 43% of β-actin, respectively. Ex vivo expanded MSC were CD34-, CD45+ (5%), CD29+ (92%), CD80-, CD86+ (10%), CD90+ (∼90%), MHC Class I low & MHC Class II-. Conclusions: Autologous MSC therapy prevented IR injury and served as an excellent vehicle to deliver cytokine. MSC therapy to prevent IR injury and enhance tissue regeneration in cardiopulmonary diseases seems promising.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6-12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2-5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier.

Objective To study the modification of bone marrow and human umbilical cord blood mesenchymal stem cells with hBMP-2 gene and compare the modification results. Methods Bone marrow and human umbilical cord blood mesenchymal stem cells were separated and cultured by density gradient centrifugation and adherent culture. The recombinant hBMP-2 plasmid was transfected into bone marrow and human umbilical cord blood mesenchymal stem cells by high-performance non-liposome reagent X-treme GENE. The fluorescence intensity was measured by inverted fluorescence microscopy and the transfection efficiency was calculated. The hBMP was detected by qPCR. The expression of-2 and chondronectin in two kinds of cells was detected by immunohistochemistry. Results Bone marrow and human umbilical cord blood mesenchymal stem cells were successfully isolated and cultured. The two cells had different growth characteristics. The recombinant hBMP-2 plasmid mediated by high-performance non-liposome reagent was successfully transfected into bone marrow and human umbilical cord blood mesenchymal stem cells. The efficiency of transfection of bone marrow mesenchymal stem cells (18.44±5.94)% was lower than that of human umbilical cord blood mesenchymal stem cells (27.74±7.59)%, and the difference was statistically significant (t=3.027, P<0.05). The expression of hBMP-2, cartilage connectin and collagen type II were detected in both transfected cells. Conclusion hBMP-2 gene can effectively modify bone marrow and human umbilical cord blood mesenchymal stem cells and promote chondrocyte differentiation. Key words: BMP2 protein, human; Mesenchymal stem cells; Liposomes

Bone marrow mesenchymal stem cells ameliorated kidney fibrosis by attenuating TLR4/NF-κB in diabetic rats