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Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.

Background In obstructive congenital hydrocephalus, cerebrospinal fluid accumulation is associated with high intracranial pressure and the presence of periventricular edema, ischemia/hypoxia, damage of the white matter, and glial reactions in the neocortex. The viability and short time effects of a therapy based on bone marrow-derived mesenchymal stem cells (BM-MSC) have been evaluated in such pathological conditions in the hyh mouse model. Methods BM-MSC obtained from mice expressing fluorescent mRFP1 protein were injected into the lateral ventricle of hydrocephalic hyh mice at the moment they present a very severe form of the disease. The effect of transplantation in the neocortex was compared with hydrocephalic hyh mice injected with the vehicle and non-hydrocephalic littermates. Neural cell populations and the possibility of transdifferentiation were analyzed. The possibility of a tissue recovering was investigated using 1H High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, thus allowing the detection of metabolites/osmolytes related with hydrocephalus severity and outcome in the neocortex. An in vitro assay to simulate the periventricular astrocyte reaction conditions was performed using BM-MSC under high TNFα level condition. The secretome in the culture medium was analyzed in this assay. Results Four days after transplantation, BM-MSC were found undifferentiated and scattered into the astrocyte reaction present in the damaged neocortex white matter. Tissue rejection to the integrated BM-MSC was not detected 4 days after transplantation. Hyh mice transplanted with BM-MSC showed a reduction in the apoptosis in the periventricular neocortex walls, suggesting a neuroprotector effect of the BM-MSC in these conditions. A decrease in the levels of metabolites/osmolytes in the neocortex, such as taurine and neuroexcytotoxic glutamate, also indicated a tissue recovering. Under high TNFα level condition in vitro, BM-MSC showed an upregulation of cytokine and protein secretion that may explain homing, immunomodulation, and vascular permeability, and therefore the tissue recovering. Conclusions BM-MSC treatment in severe congenital hydrocephalus is viable and leads to the recovery of the severe neurodegenerative conditions in the neocortex. NMR spectroscopy allows to follow-up the effects of stem cell therapy in hydrocephalus.

Mesenchymal stem cells (MSCs) are promising candidates for a range of tissue regeneration applications. Adequate scaffolds are necessary for their application in vivo, where interactions between cells and the surface material are critical. Arginine-glycine-aspartic acid tripeptides (RGD) were conjugated to polyamidoamine (PAMAM) dendrimers and used to pre-treat test surfaces. We demonstrate that pre-treatment with dendrimer-presented tripeptides efficiently increases MSC adhesion to a polystyrene test surface, and that treatment effectiveness is related to how tripeptides are presented by the dendrimer to the cell. We tested both R-G-D–dendrimer and dendrimer–R-G-D arrangements and found the former to be optimal in terms of surface adhesion.

Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is an immune-mediated multifactorial disease, with high level of prevalence in humans and dogs. Our aim in this study was to investigate the therapeutic effects of allogeneic adipose-derived mesenchymal stromal cells (Ad-MSCs) implanted around the lacrimal glands in 12 dogs (24 eyes) with KCS, which is refractory to current available treatments. Schirmer tear test (STT) and ocular surface integrity were assessed at 0 (before treatment), 3, 6, and 9 months after treatment. Average STT values and all clinical signs showed a statistically significant change(P<0.001)during the follow-up with reduction in all ocular parameters scored: ocular discharge, conjunctival hyperaemia, and corneal changes, and there were no signs of regression or worsening. Implanted cells were well tolerated and were effective reducing clinical signs of KCS with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that implantation of allogeneic Ad-MSCs around lacrimal glands has been found as an effective therapeutic alternative to treat dogs with KCS. These results could reinforce a good effective solution to be extrapolated to future studies in human.

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells. Open image in new window

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.

Background Feline eosinophilic keratitis (FEK) is a chronic keratopathy caused by a suspected immune mediated response to an unknown antigenic stimulus. The purpose of this study was to investigate the safety and therapeutic effects of allogeneic feline adipose-derived mesenchymal stromal cells (fAd-MSCs) implanted subconjunctival around the ocular surface lesion in five cats with FEK refractory to current available treatments. Results FEK was diagnosed by clinical appearance and evidence of eosinophil and/or mast cells in corneal cytology. Each animal was treated with two applications of 2 × 106 million of fAd-MSCs 2 months apart. Ocular surface integrity was assessed before treatment and at 1, 3, 6 and 11 months after treatment. Clinical signs showed a significant change during the follow-up with resolution of the corneal and conjunctiva lesions and there were no signs of regression or worsening. Conclusions Implanted cells were well-tolerated and effective reducing clinical signs of FEK with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that local implantation of allogeneic fAd-MSCs has been found as an effective therapeutic alternative to treat cats with FEK.

Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.

Today great hope is set on regenerative medicine in all medical fields. Leland Kaiser intro‐ duced the term “Regenerative Medicine” in 1992. He forecasted that a “new branch of medicine will develop that attempts to change the course of chronic diseases and in many instances will regenerate tired and failing organ systems” (Kaiser, 1992). Since then, scientists all over the world try to develop cell-based approaches to regenerate damaged tissues, or even substitute whole organs (Ehnert et al., 2009).

JOURNAL ARTICLE; Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells which are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and non-adherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4 and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent, hematopoietic, mesenchymal and endothelial specific markers in vitro and can undergo osteogenic differentiation in vivo. Yes

1.1 The definition of MSCs Bone marrow (BM) was the first tissue described as a source of plastic-adherent, fibroblastlike cells that develops colony-forming unit fibroblastic (CFU-F) when seeded in tissue culture plates (Friedenstein et al., 1982; 1987). These cells, originally designated stromal cells, elicited much attention, and the main goal of thousands of studies conducted using these cells was to find an ultimate pure cell population that could be further utilized for regenerative purposes. In these studies, cells were isolated using several methods and were given names such as mesenchymal stem cells (MSCs), mesenchymal progenitors, stromal stem cells, among others. Lately, a committee of the International Society for Cytotherapy suggested the name “multipotent mesenchymal stromal cells” (Dominici et al., 2006). However, most scientists have been referring to them simply as “MSCs”. The precise definition of these cells remains a matter of debate. Nevertheless, to date MSCs are widely defined as a plastic-adherent cell population that, under closely controlled conditions, can be directed to differentiate in vitro into cells of osteogenic, chondrogenic, adipogenic, myogenic, tenogenic, or hematopoietic-supportive stromal lineages (Pittenger et al., 1999; Javazon et al., 2004; Alonso et al., 2008; Prockop, 2009) (Fig. 1). As part of their stem cell nature, MSCs proliferate and give rise to daughter cells that have the same pattern of gene expression and phenotype and, therefore, maintain the “stemness”

Recent studies have demonstrated that adipose-derived adult stromal cells (ADASCs) offer great promise for cell-based therapies due to their ability to differentiate towards bone, cartilage and fat [corrected] The objective of this study was to investigate whether type I collagen would elicit in vivo bone formation of passaged rat adipose-derived adult stromal cells (ADASC) placed extraskeletally.After expansion for 1-4 passages (P), cells were incubated in osteogenic medium containing dexamethasone, ascorbic acid and beta-glycerol phosphate for 2-4 weeks. Undifferentiated cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and von Kossa staining as well as by gene expression of ALP, osteopontin (OP), osteonectin (ON), osteocalcin (OC), collagen I (colI), collagen II (colII), bone sialoprotein (BSP), periostin (Postn), runx2, osterix (Osx), sox9, msx1 and msx2. Diffusion chambers were filled with 1x10(6) cells mixed with or without type I collagen gel and implanted subcutaneously into rats. Controls included chambers exposed to (1) undifferentiated cells (with or without collagen, (2) collagen without cells and (3) empty chambers (n=5 per group).Four weeks after implantation, in vivo bone and cartilage formation was demonstrated in implants containing 4-week osteo-induced P1 and P4 cells wrapped in the collagen gel, as confirmed by Goldner's trichrome and Alcian blue staining, respectively. Newly formed bone stained positive for type I collagen. Control implants had no bone or cartilage and were primarily filled with fibrous tissue at that time interval.Recent studies have demonstrated that ADASC offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage and fat. However, the influence of different matrices on the in vivo osteogenic capability of ADASC is not fully understood. These findings suggest that type I collagen may support the survival and expression of osteogenic and chondrogenic phenotypes in passaged rat ADASC in vivo.

We report the first clinical case of transplantation of autologous bone marrow-derived cells in vitro exposed to a novel recombinant human transforming growth factor (rhTGF)-β1 fusion protein bearing a collagen-binding domain (rhTGF-β1-F2), dexamethasone (DEX) and β-glycerophosphate (β-GP). When such culture-expanded cells were loaded into porous ceramic scaffolds and transplanted into the bone defect of a 69-year-old man, they differentiated into bone tissue. Marrow cells were obtained from the iliac crest and cultured in collagen gels impregnated with rhTGF-β1-F2. Cells were selected under serum-restricted conditions in rhTGF-β1-F2-containing medium for 10 days, expanded in 20% serum for 22 days and osteoinduced for 3 additional days in DEX/β-GP-supplemented medium. We found that the cell number harvested from rhTGF-β1-F2-treated cultures was significantly higher (2.3- to 3-fold) than that from untreated cultures. rhTGF-β1-F2 treatment also significantly increased alkaline phosphatase activity (2.2- to 5-fold) and osteocalcin synthesis, while calcium was only detected in rhTGF-β1-F2-treated cells. Eight weeks after transplantation, most of the scaffold pores were filled with bone and marrow tissue. When we tested the same human cells treated in vitro in a rat model using diffusion chambers, there was subsequent development of cartilage and bone following the subcutaneous transplantation of rhTGF-β1-F2-treated cells. This supports the suggestion that such cells were marrow-derived cells, with chondrogenic and osteogenic potential, whereas the untreated cells were not under the same conditions. The ability for differentiation into cartilage and bone tissues, combined with an extensive proliferation capacity, makes such a marrow-derived stem cell population valuable to induce bone regeneration at skeletal defect sites.

Introduction Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). Methods MDMSCs were cultured as a monolayer at a density of 1 × 105 cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression. Results The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1. Conclusions The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage.