Cytotoxicity In Mesenchymal Stem Cells Research Articles

Optimizing the Delivery of mRNA to Mesenchymal Stem Cells for Tissue Engineering Applications.

Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

Biocompatibility of graphene oxide nanosheets functionalized with various amino acids towards mesenchymal stem cells

Graphene and its derivatives have gained popularity due to their numerous applications in various fields, such as biomedicine. Recent reports have revealed the severe toxic effects of these nanomaterials on cells and organs. In general, the chemical composition and surface chemistry of nanomaterials affect their biocompatibility. Therefore, the purpose of the present study was to evaluate the cytotoxicity and genotoxicity of graphene oxide (GO) synthesized by Hummer's method and functionalized by different amino acids such as lysine, methionine, aspartate, and tyrosine. The obtained nanosheets were identified by FT-IR, EDX, RAMAN, FE-SEM, and DLS techniques. In addition, trypan blue and Alamar blue methods were used to assess the cytotoxicity of mesenchymal stem cells extracted from human embryonic umbilical cord Wharton jelly (WJ-MSCs). The annexin V staining procedure was used to determine apoptotic and necrotic death. In addition, COMET and karyotyping techniques were used to assess the extent of DNA and chromosome damage. The results of the cytotoxicity assay showed that amino acid modifications significantly reduced the concentration-dependent cytotoxicity of GO to varying degrees. The GO modified with aspartic acid had the lowest cytotoxicity. There was no evidence of chromosomal damage in the karyotyping method, but in the comet assay, the samples modified with tyrosine and lysine showed the greatest DNA damage and rate of apoptosis. Overall, the aspartic acid-modified GO caused the least cellular and genetic damage to WJ-MSCs, implying its superior biomedical applications such as cell therapy and tissue engineering over GO.

Symphytum officinale augments osteogenesis in human bone marrow-derived mesenchymal stem cells in vitro as they differentiate into osteoblasts.

Mesenchymal stem cells (MSCs) are multipotent stem cells possessing regenerative potential. Symphytum officinale (SO) is a medicinal plant and in homoeopathic literature, believed to accelerate bone healing. This study aimed to determine if homoeopathic doses of SO could augment osteogenesis in MSCs as they differentiate into osteoblasts in vitro. Bone marrow samples were obtained from patients who underwent bone grafting procedures (n = 15). MSCs were isolated, expanded and characterized by flow cytometry (CD90, CD105). Cytotoxicity of SO was evaluated by MTT assay. Osteogenic differentiation was induced in MSCs with β-glycerophosphate, ascorbic acid and dexamethasone over 2 weeks. Different homoeopathic doses of SO (MT, 3C, 6C, 12C and 30C) were added to the basic differentiation medium (BDM) and efficiency of MSCs differentiating into osteoblasts were measured by evaluating expression of Osteocalcin using flow cytometry, and alkaline phosphatase activity using ELISA. Gene expression analyses for osteoblast markers (Runx-2, Osteopontin and Osteocalcin) were evaluated in differentiated osteoblasts using qPCR. Flow cytometry (CD90, CD105) detected MSCs isolated from bone marrow (93-98%). MTT assay showed that the selected doses of SO did not induce any cytotoxicity in MSCs (24 hours). The efficiency of osteogenic differentiation (2 weeks) for different doses of Symphytum officinale was determined by flow cytometry (n = 10) for osteoblast marker, Osteocalcin, and most doses of Symphytum officinale enhanced osteogenesis. Interestingly, gene expression analysis for Runx-2 (n = 10), Osteopontin (n = 10), Osteocalcin (n = 10) and alkaline phosphatase activity (n = 8) also showed increased osteogenesis with the addition of Symphytum officinale to BDM, specially mother tincture. Our findings suggest that homoeopathic dose (specially mother tincture) of Symphytum officinale has the potential to enhance osteogenesis.

Effect of metformin and celecoxib on cytotoxicity and release of GDF-15 from human mesenchymal stem cells in high glucose condition.

Mesenchymal stem cells (MSCs) have therapeutic potential for treatment of diabetes. However, in vitro behavior of MSCs in high glucose condition as well as presence of glucose lowering agents is not fully understood. Because MSCs have an important role in tissue repair, we examined the effects of metformin and celecoxib on viability of MSCs in different glucose conditions. MSCs, from umbilical cord blood, were cultured in normoglycemic (glucose 5.5mM), midglycemic (glucose 10mM), and hyperglycemic (glucose 25mM) conditions, and the cell viability was evaluated by MTT assay. The cytotoxicity and secretion of GDF-15 were further tested in MSCs treated with metformin and celecoxib in various glucose concentrations. Our results showed that high glucose condition lowered viability of MSCs. Metformin treatment also inhibited proliferation of MSCs, but its toxicity was not changed in high glucose condition. Celecoxib induced cytotoxicity in MSCs, and the toxicity was increased in high glucose condition. Metformin and celecoxib induced release from MSCs; however, high glucose inhibited the metformin-induced GDF-15 release. These findings suggested that metformin did not increase the cytotoxicity of high glucose condition in MSCs. Moreover, celecoxib treatment in diabetic condition can reduce the viability of MSCs to proliferate and regenerate perhaps via change in release of GDF-15.

Vitamin D: Correlation with biochemical and body composition changes in a southern Brazilian population and induction of cytotoxicity in mesenchymal stem cells derived from human adipose tissue

Studies have shown that metabolic disorders, serum inflammatory markers and weight gain (obesity) are correlated with vitamin D deficiency. Therefore, the present study correlated the serum calcidiol (s25(OH)D3) levels in a sample of individuals from southern Brazil with variables related to metabolic disorders, obesity and lifestyle habits and assessed the cytotoxic effect of calcitriol on adipose tissue-derived mesenchymal stem cells (ADSCs). The results showed a 79.23% prevalence of hypovitaminosis D in the study population and a correlation (p<0.05) between a low serum vitamin D concentration and an elevated low-density lipoprotein cholesterol (LDL-c) level. Univariate linear regression analysis using 25(OH)D3 as a regressor showed a negative association (p<0.05) with an indoor work environment (β=−2.305), increased body fat (β=−0.095), age (β=−0.065) and high-density lipoprotein cholesterol (HDL-c; β=−0.109). An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed with ADSCs using five calcitriol concentrations (15.625, 31.25, 62.5, 125 and 250nM) indicated cytotoxic potential (p<0.05) at the 62.5nM concentration at 48 and 72h and at the 125 and 250nM concentrations at 24, 48 and 72h. The results reported herein corroborate one another and suggest a key association between vitamin D deficiency and the development of obesity because ADSCs are involved in adipose tissue hyperplasia and differentiate into adipocytes that can sequester the bioavailable vitamin D necessary for homeostasis.

Local anesthetic cytotoxicity on human mesenchymal stem cells during chondrogenic differentiation.

This study was to investigate the cytotoxic potency of local anesthetics on human mesenchymal stem cells during chondrogenesis. Aggregates were created from density-gradient centrifugation-separated bone marrow-derived mesenchymal stem cells. After 7, 14, and 21days, aggregates were analyzed histologically and immunohistochemically and exposed to equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1h. Cell viability, apoptosis, and necrosis were determined using live-dead and caspase staining. Additionally, following a 1-h exposure on day 7, aggregates were cultured under chondrogenic conditions until day 21 to assess the effects of local anesthetics on differentiation potency of mesenchymal stem cells. In the course of chondrogenesis, mesenchymal stem cells were embedded in varying amount and structure of cartilage-specific extracellular matrix. Contents of sulfated glycosaminoglycan, type I and II collagen increased from day 7 to day 21. Compared to control, death rates of mesenchymal stem cells were significantly elevated 1day after treatment at 7 and 14days. Four days after exposure, death rates were 13-15% at 7 and 11-17% at 14days. Mesenchymal stem cell viability in aggregates at 21days was unchanged to controls. The width of the superficial aggregate zone containing stem cell necrosis decreased with elongated differentiation time. Apoptosis rates were elevated in the edge regions of aggregates, reaching maximum values 4days after treatment. Local anesthetic exposure on day 7 reduced Collagen II, but not DNA contents in aggregates at 21days. Bupivacaine, ropivacaine, and mepivacaine did not differ in mesenchymal stem cell cytotoxicity in aggregates. Local anesthetic exposure results in cytotoxicity of mesenchymal stem cells undergoing chondrogenesis, especially in superficial layers. Therefore, induced cell damage should be avoided during chondrogenesis of mesenchymal stem cells, particularly early after cartilage repair.

Inhibitory effect of mesenchymal stem cells on lymphocyte proliferation

We investigated the mechanism underlying the inhibitory effect of rat mesenchymal stem cells (MSCs) on non-specific mitogen-stimulated lymphocytes (LCs) and lymphoblasts (LBs). We used MSCs of passages 2-8 prepared from Sprague-Dawley (SD) rats. LCs were isolated from the spleens of SD rats. Mixed LCs reactions of mitomycin C-treated MSCs with concanavalin A (ConA)-stimulated LCs or LBs were performed, and the proliferation inhibition effect was tested by MTS assay. The cytotoxicity of MSCs against naïve and ConA-stimulated LBs was detected, after co-culturing for 24 h, by lactate dehydrogenase release assay. The rate of apoptosis of ConA-stimulated LBs was measured by flow cytometry after incubation with MSCs for 9 h in the ratio 10:1. The MSCs were treated with Fas ligand (FasL), transforming growth factor (TGF)-beta, and interleukin (IL)-10 blocking antibodies and co-cultured with ConA-stimulated LBs to observe the apoptosis and growth inhibitory effect. The main outcomes were bone marrow-derived adherent CD29+, CD44+, CD45-, CD54+, CD95+, and SH-2+ MSCs. FasL, TGF-beta, and IL-10 production by MSCs were visualized by immunocytochemical analysis. MSCs exhibited a dose-dependent growth inhibitory effect on ConA-stimulated LCs and LBs. When treated with anti-FasL and anti-IL-10 blocking antibodies, the inhibitory effect of MSCs on LBs proliferation, and the effect of apoptosis induction on LBs decreased. Anti-TGF-beta blocking antibody treatment did not significantly influence MSCs. Therefore, the inhibitory effects of MSCs against activated LBs were significantly stronger than that against naïve LCs. FasL and IL-10, rather than TGF-beta, play important roles in the immunosuppressive effects of MSCs.

Mesenchymal stem cellular adhesion and cytotoxicity study of random biopolyester scaffolds for tissue engineering

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rabbits and inoculated respectively on 3D scaffolds of poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) (PHBV), poly(butylenes succinate) (PBS) and different blends (100/0, 80/20, 50/50, 20/80, 0/100) (Wt%) in vitro. It was found that the (50/50) blends possessed the best performance on adhesion and cytotoxicity of MSCs. The scanning electronic microscopy (SEM) results showed that the (50/50) blends had the appropriate roughness for MSCs to attach and grow, which may be used as a suitable biomaterial to create small caliber vascular grafts.