In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

In search of mechanisms associated with mesenchymal stem cell-based therapies for acute kidney injury

Acute kidney injury (AKI) is classically described as a rapid loss of kidney function. AKI affects more than 15% of all hospital admissions and is associated with elevated mortality rates. Although many advances have occurred, intermittent or continuous renal replacement therapies are still considered the best options for reversing mild and severe AKI syndrome. For this reason, it is essential that innovative and effective therapies, without side effects and complications, be developed to treat AKI and the end-stages of renal disease. Mesenchymal stem cell (MSC) based therapies have numerous advantages in helping to repair inflamed and damaged tissues and are being considered as a new alternative for treating kidney injuries. Numerous experimental models have shown that MSCs can act via differentiation-independent mechanisms to help renal recovery. Essentially, MSCs can secrete a pool of cytokines, growth factors and chemokines, express enzymes, interact via cell-to-cell contacts and release bioagents such as microvesicles to orchestrate renal protection. In this review, we propose seven distinct properties of MSCs which explain how renoprotection may be conferred: 1) anti-inflammatory; 2) pro-angiogenic; 3) stimulation of endogenous progenitor cells; 4) anti-apoptotic; 5) anti-fibrotic; 6) anti-oxidant; and 7) promotion of cellular reprogramming. In this context, these mechanisms, either individually or synergically, could induce renal protection and functional recovery. This review summarises the most important effects and benefits associated with MSC-based therapies in experimental renal disease models and attempts to clarify the mechanisms behind the MSC-related renoprotection. MSCs may prove to be an effective, innovative and affordable treatment for moderate and severe AKI. However, more studies need to be performed to provide a more comprehensive global understanding of MSC-related therapies and to ensure their safety for future clinical applications.

Experimental Basis and New Insights for Cell Therapy in Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease (COPD) can be briefly described as air flow limitation and chronic dyspnea associated to an inflammatory response of the respiratory tract to noxious particles and gases. Its main feature is the obstruction of airflow and consequent chronic dyspnea. Despite recent advances, and the development of new therapeutic, medical and clinical approaches, a curative therapy is yet to be achieved. Therapies involving the use of tissue-specific or donor derived cells present a promising alternative in the treatment of degenerative diseases and injuries. Recent studies demonstrate that mesenchymal stem cells have the capacity to modulate immune responses in acute lung injury and pulmonary fibrosis in animal models, as well as in human patients. Due to these aspects, different groups raised the possibility that the stem cells from different sources, such as those found in bone marrow or adipose tissue, could act preventing the emphysematous lesion progression. In this paper, it is proposed a review of the current state of the art and future perspectives on the use of cell therapy in obstructive lung diseases.

FGFR2 Mutation Confers a Less Drastic Gain of Function in Mesenchymal Stem Cells Than in Fibroblasts

Gain-of-function mutations in FGFR2 cause Apert syndrome (AS), a disease characterized by craniosynostosis and limb bone defects both due to abnormalities in bone differentiation and remodeling. Although the periosteum is an important cell source for bone remodeling, its role in craniosynostosis remains poorly characterized. We hypothesized that periosteal mesenchymal stem cells (MSCs) and fibroblasts from AS patients have abnormal cell phenotypes that contribute to the recurrent fusion of the coronal sutures. MSCs and fibroblasts were obtained from the periostea of 3 AS patients (S252W) and 3 control individuals (WT). We evaluated the proliferation, migration, and osteogenic differentiation of these cells. Interestingly, S252W mutation had opposite effects on different cell types: S252W MSCs proliferated less than WT MSCs, while S252W fibroblasts proliferated more than WT fibroblasts. Under restrictive media conditions, only S252W fibroblasts showed enhanced migration. The presence of S252W mutation increased in vitro and in vivo osteogenic differentiation in both studied cell types, though the difference compared to WT cells was more pronounced in S252W fibroblasts. This osteogenic differentiation was reversed through inhibition of JNK. We demonstrated that S252W fibroblasts can induce osteogenic differentiation in periosteal MSCs but not in MSCs from another tissue. MSCs and fibroblasts responded differently to the pathogenic effects of the FGFR2(S252W) mutation. We propose that cells from the periosteum have a more important role in the premature fusion of cranial sutures than previously thought and that molecules in JNK pathway are strong candidates for the treatment of AS patients.

Human Endometrial-Derived Mesenchymal Stem Cells Suppress Inflammation in the Central Nervous System of EAE Mice

FAPESP [2009/13109-5]

Exploring the Role of Soluble Factors Associated with Immune Regulatory Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are characterized as multipotent stromal cells with the capacity for both self-renewal and differentiation into mesodermal cell lineages. MSCs also have a fibroblast-like phenotype and can be isolated from several tissues. In recent years, researchers have found that MSCs secrete several soluble factors that exert immunosuppressive effects by modulating both innate (macrophages, dendritic and NK cells) and adaptive (B cells and CD4+ and CD8+ T cells) immune responses. This review summarizes the principal trophic factors that are related to immune regulation and secreted by MSCs under both autoimmune and inflammatory conditions. The understanding of mechanisms that regulate immunity in MSCs field is important for their future use as a novel cellular-based immunotherapy with clinical applications in several diseases.

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non-osteogenic medium with or without purmorphamine (2 mu M) for periods of up to 14 days. Purmorphamine up-regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204208, 2012. (C) 2011 Wiley Periodicals, Inc.

Human Fallopian Tube Mesenchymal Stromal Cells Enhance Bone Regeneration in a Xenotransplanted Model

We have recently reported that human fallopian tubes, which are discarded during surgical procedures of women submitted to sterilization or hysterectomies, are a rich source of human fallopian tube mesenchymal stromal cells (htMSCs). It has been previously shown that human mesenchymal stromal cells may be useful in enhancing the speed of bone regeneration. This prompted us to investigate whether htMSCs might be useful for the treatment of osteoporosis or other bone diseases, since they present a pronounced capacity for osteogenic differentiation in vitro. Based on this prior knowledge, our aim was to evaluate, in vivo, the osteogenic capacity of htMSCs to regenerate bone through an already described xenotransplantation model: nonimmunosuppressed (NIS) rats with cranial defects. htMSCs were obtained from five 30-50 years old healthy women and characterized by flow cytometry and for their multipotenciality in vitro capacity (osteogenic, chondrogenic and adipogenic differentiations). Two symmetric full-thickness cranial defects on each parietal region of seven NIS rats were performed. The left side (LS) of six animals was covered with CellCeram (Scaffdex)-a bioabsorbable ceramic composite scaffold that contains 60% hydroxyapatite and 40% beta-tricalciumphosphate-only, and the right side (RS) with the CellCeram and htMSCs (10(6) cells/scaffold). The animals were euthanized at 30, 60 and 90 days postoperatively and cranial tissue samples were taken for histological analysis. After 90 days we observed neobone formation in both sides. However, in animals euthanized 30 and 60 days after the procedure, a mature bone was observed only on the side with htMSCs. PCR and immunofluorescence analysis confirmed the presence of human DNA and thus that human cells were not rejected, which further supports the imunomodulatory property of htMSCs. In conclusion, htMSCs can be used successfully to enhance bone regeneration in vivo, opening a new field for future treatments of osteoporosis and bone reconstruction.

Local Injections of Adipose-Derived Mesenchymal Stem Cells Modulate Inflammation and Increase Angiogenesis Ameliorating the Dystrophic Phenotype in Dystrophin-Deficient Skeletal Muscle

The effects of adipose-derived mesenchymal stem cells (ADMSC) transplantation on degeneration, regeneration and skeletal muscle function were investigated in dystrophin-deficient mice (24-week-old). ADMSC transplantation improved muscle strength and, resistance to fatigue. An increase in fiber cross-sectional area and in the number of fibers with centralized nuclei and augment of myogenin content were observed. In ADMSC-treated muscles a decrease in muscle content of TNF-alpha, IL-6 and oxidative stress measured by Amplex(A (R)) reagent were observed. The level of TGF-beta 1 was lowered whereas that of VEGF, IL-10 and IL-4 were increased by ADMSC treatment. An increase in markers of macrophage M1 (CD11 and F4-80) and a decrease in T lymphocyte marker (CD3) and arginase-1 were also observed in ADMSCs-treated dystrophic muscle. No change was observed in iNOS expression. Increased phosphorylation of Akt, p70S6k and 4E-BP1 was found in dystrophic muscles treated with ADMSC. These results suggest that ADMSC transplantation modulates inflammation and improves muscle tissue regeneration, ameliorating the dystrophic phenotype in dystrophin-deficient mice.

TWIST and p-Akt immunoexpression in normal oral epithelium, oral dysplasia and in oral squamous cell carcinoma

Objectives: The aim of this study was to evaluate the immunoexpression of TWIST and p-Akt proteins in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), correlating their expressions with the histological features of the lesions. Study design: Immunohistochemical studies were carried out on 10 normal oral epithelium, 30 OL and 20 OSCC formalin-fixed, paraffin-embedded tissue samples. Immunoperoxidase reactions for TWIST and p-Akt proteins were applied on the specimens and the positivity of the reactions was calculated for 1000 epithelial cells. Results: Kruskal-Wallis and Dunn's post tests revealed a significant difference in TWIST and p-Akt immunoexpression among normal oral mucosa, OL and OSCC. In addition, a significant positive correlation was found between TWIST and p-Akt expressions according to the Pearson's correlation test. Conclusions: The results obtained in the current study suggest that TWIST and p-Akt may participate of the multi-step process of oral carcinogenesis since its early stages.

Molecular Analysis of the Differentiation Potential of Murine Mesenchymal Stem Cells from Tissues of Endodermal or Mesodermal Origin

Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription-polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow-derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue-derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.

Human Adipose-Derived Mesenchymal Stromal Cells Injected Systemically Into GRMD Dogs Without Immunosuppression Are Able to Reach the Host Muscle and Express Human Dystrophin

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting I in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2 x 10(6), CELL) intravenously 3 h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-beta, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema. (c) 2012 Elsevier B.V. All rights reserved.

Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (beta-TCP) (60: 40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100 degrees C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/beta-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study