The antioxidant trolox inhibits aging and enhances prostaglandin E-2 secretion in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been widely used in regenerative medicine and clinical therapy due to their capabilities of proliferation, differentiation, and immune regulation. However, during in vitro expansion, MSCs are prone to aging, which largely limits their application. Prostaglandin E-2 (PGE-2) is a key effector secreted by MSCs to exert immunomodulatory effects. By screening the compound library for PGE-2 secretion, the antioxidant trolox was verified as a stimulator of MSCs to secrete PGE-2. The effect of antioxidant trolox on biological characteristics of MSCS, including aging, proliferation, and gene expression, was examined. The results demonstrated that trolox can resist aging, promote proliferation, and enhance PGE-2 secretion of MSCs without affecting their surface marker expression. Furthermore, trolox treatment up-regulates miR-17-92 clusters in MSCs and may contribute to its anti-aging effects. Thus, trolox addition might be beneficial for MSCs expansion and their application.     

The RhoA nuclear localization changes in replicative senescence: New evidence from <i>in vitro</i> human mesenchymal stem cells studies

All non-immortalized mesenchymal stem cells have a limited proliferative potential, that is, replicative senescence (RS) is an integral characteristic of the life of all mesenchymal stem cells (MSCs). It is known that one of the important signs of RS is a decrease of cell motility, and that violations of migration processes contribute to the deterioration of tissue regeneration. Therefore, the characterization of the properties of the cell line associated with RS is a prerequisite for the effective use of MSCs in restorative medicine. One of the key proteins regulating cell motility is the small GTPase RhoA. The main purpose of this work was to study the nuclear-cytoplasmic redistribution of the RhoA protein during RS in MSC lines recently obtained and characterized in our laboratory. The study found that a comparative analysis of the intracellular localization of RhoA in three cell lines (MSCWJ-1, FetMSC, DF2) showed a decrease in the nuclear localization of RhoA during RS.     

Applications of scaffolds: Tools for enhancing the immunomodulation of mesenchymal stromal cells

Exogenously delivered mesenchymal stromal cells (MSCs) are therapeutically beneficial owing to their paracrine effect; they secrete various cytokines, nucleic acids, and proteins. Multiple bioengineering techniques can help MSC cultures to release secretomes by providing stem cell niche-like conditions (both structurally and functionally). Various scaffolds mimic the natural extracellular matrix (ECM) using both natural and synthetic polymers, providing favorable environments for MSC proliferation and differentiation. Depending on material properties, either topographically or elastically structured scaffolds can be fabricated. Three-dimensional scaffolds have tunable substrate rigidities and structures, aiding MSC cultivation. Decellularized ECM-derived hydrogels are similar to the natural ECM, thus improving the paracrine effects of MSCs. Here, we discuss recent research on the application of scaffolds to maximize the immunomodulatory function of MSCs.     

Phenotypic,morphological, and metabolic characterization of vascular-spheres from human vascular mesenchymal stem cells

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography–mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44⁺, CD105^low, CD90^low, exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.     

Immunophenotypic,immunocytochemistry, ultrastructural,and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium‐rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech. 76:618–624, 2013. © 2013 Wiley Periodicals, Inc.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Mesenchymal stem cells,secretome and biomaterials in <i>in-vivo</i> animal models: Regenerative medicine application in cutaneous wound healing

The treatment of nonhealing and chronic cutaneous wounds still needs a clinical advancement to be effective.Both mesenchymal stem cells (MSCs), obtained from different sources, and their secretome derived thereof (especiallyexosomes) can activate signaling pathways related to promotion of cell migration, vascularization, collagen deposition,and inflammatory response demonstrating prohealing, angiogenetic and anti-scarring capacities. On the other hand,biodegradable biomimetic scaffolds can facilitate endogenous cell attachment and proliferation as well as extracellularmatrix production. In this Review, we revise the complex composites made by biomimetic scaffolds, mainly hydrogels,and MSC-derived exosomes constructed for cutaneous wound healing. Studies demonstrate that there exists asynergistic action of scaffolds with encapsulated exosomes, displaying a sustained release profiles to facilitate longlasting healing effects. It can be envisioned that dressings made by biomimetic hydrogels and MSC-derived exosomeswill be clinically applied in the near future for the effective treatment of nonhealing and chronic wounds.     

Immunophenotypic evaluation,and physiological and laboratory correlations of hematopoietic stem cells from umbilical cord blood

The use of umbilical cord blood stem cells is an efficient alternative for the transplantation of hematopoietic progenitor cells. A number of factors can influence the volume and amount of CD34⁺ cells, which are considered as immature and capable of proliferation. Quantification of CD34⁺ cells, evaluation of CD38 and c-kit molecules on these cells, as well as correlations of such factors as maternal age, gestational age, newborn sex and weight, umbilical cord length, placental weight with increased volume and concentration of immature cells, among others, were performed in 70 blood samples from term newborns. The mean volume of umbilical cord blood collected was 53.8±33.6 mL, where 30.96±18.9 CD34⁺/µL UCB cells were found, of which 16.66±8.32% were CD34⁺ CD38- cells, and 47.23±24.0% were CD34⁺ CD117^- cells. Newborn weight and placental weight were positively correlated with increased volume of collected UCB. The volume of collected blood was found to affect the absolute count of CD34⁺ cells and the relative value of these among total nucleated cells, as well as the percentage of CD34⁺CD117⁺ and CD34⁺CD117^- cells. CD34⁺ cells were positively correlated with leukocytes, and gestational age was negatively correlated with the number of CD34⁺ cells. Our results confirm the importance of the accurate quantification of CD34⁺ cells and their subsets, and that many factors may be related to the higher number of hematopoietic stem cells, which are crucial for successful transplantation.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

<i>miR-103-3p</i> regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs(miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic andosteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified.Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold inMDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenicdifferentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the oppositeresults in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantlyimpacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation whileweakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.