Common chromosomal imbalances and stemness-related protein expression markers in endometriotic lesions from different anatomical sites: the potential role of stem cells

Endometriosis is a multifactorial gynecological disease characterized by the presence of functional endometrium-like tissue in ectopic sites. Several studies have focused on elucidating the immunological, endocrine, environmental and genetic factors involved in endometriosis. However, its pathogenesis is still unclear. High-resolution comparative genomic hybridization was applied to screen for genomic imbalances in laser microdissected stromal and epithelial cells from 20 endometriotic lesions and three samples of eutopic endometrium derived from eight patients. The expression of seven stemness-related markers (CD9, CD13, CD24, CD34, CD133, CD117/c-Kit and Oct-4) in endometrial tissue samples was evaluated by immunohistochemistry. Samples of eutopic endometrium showed normal genomic profiles. In ectopic tissues, an average of 68 genomic imbalances was detected per sample. DNA losses were more frequently detected and involved mainly 3p, 5q, 7p, 9p, 11q, 16q, 18q and 19q. Many of the genomic imbalances detected were common to endometriotic stroma and epithelia and also among different endometriotic sites from the same patient. These findings suggested a clonal origin of the endometriotic cells and the putative involvement of stem cells. Positive immunostaining for CD9, CD34, c-Kit and Oct-4 markers was detected in isolated epithelial and/or stromal cells in eutopic and ectopic endometrium in the majority of cases. The presence of shared genomic alterations in stromal and epithelial cells from different anatomical sites of the same patient and the expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells.

Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation

Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios.

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.

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.

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.

Topoisomerase expression in oral squamous cell carcinoma: relationship with cancer stem cells profiles and lymph node metastasis

BACKGROUND: The relationship between predictive proteins and tumors presenting cancer stem cells (CSCs) profiles in oral tumors is still poorly understood. This study aims to identify the relationship between topoisomerases I, II alpha, and III alpha and putative CSCs immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. METHODS: The following data were retrieved from 127 patients: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, and survival. An immunohistochemical study for topoisomerases I, II alpha, and III alpha was performed in a tissue microarray containing 127 paraffin blocks of OSCCs. RESULTS: In univariate analysis, topoisomerases expression showed significant differences according to CSCs profiles and p53 immunoexpression, but not with survival. Topoisomerases II alpha and III alpha also showed significant relationship with lymph node metastasis. The multivariate test confirmed these associations. CONCLUSIONS: The results that all topoisomerases correlates with OSCC CSCs may indicate a role for topoisomerases in head and neck carcinogenesis. Notwithstanding, it is plausible that other members of topoisomerases family could represent novel therapeutical targets in oral squamous cell carcinoma. J Oral Pathol Med (2012) 41: 762-768

In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

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

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Derivation and characterization of progenitor stem cells from canine allantois and amniotic fluids at the third trimester of gestation

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells. (C) 2012 Elsevier Ltd. All rights reserved.

Protective Effects of Human Amniotic Fluid Stem Cells in a Model of Aorta Allograft Vasculopathy in Rats

Background. Chronic allograft vasculopathy (CAV) is an important cause of graft loss. Considering the immune inflammatory events involved in the development of CAV, therapeutic approaches to target this process are of relevance. Human amniotic fluid derived stem cells (hAFSCs), a class of fetal, pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, display immunomodulatory properties. hAFSCs express mesenchymal and embryonic markers, show high proliferation rates; however, they do not induce tumor formation, and their use does not raise ethical issues. Thus, we sought to investigate the effect of hAFSC on CAV in a model of aorta transplantation. Methods. Orthotopic aorta transplantation was performed using Fisher (F344) rats as donors and Lewis rats as recipients. Rats were divided into three groups: syngeneic (SYNG), untreated F344 receiving aorta from F344 (n = 8); allogeneic (ALLO), Lewis rats receiving allogeneic aorta from F344 (n = 8); and ALLO + hAFSC, ALLO rats treated with hAFSC (10(6) cells; n = 8). Histological analysis and immunohistochemistry were performed 30 days posttransplantation. Results. The ALLO group developed a robust aortic neointimal formation (208.7 +/- 25.4 gm) accompanied by a significant high number of ED1(+) (4845 +/- 841 cells/mm(2)) and CD43(+) cells (4064 +/- 563 cells/mm(2)), and enhanced expression of a-smooth muscle actin in the neointima (25 +/- 6%). Treatment with hAFSC diminished neointimal thickness (180.7 +/- 23.7 mu m) and induced a significant decrease of ED1(+) (1100 +/- 276 cells/mm(2)), CD43(+) cells (1080 +/- 309 cells/mu m(2)), and alpha-smooth muscle actin expression 8 +/- 3% in the neointima. Conclusions. These preliminary results showed that hAFSC suppressed inflammation and myofibroblast migration to the intima, which may contribute to ameliorate vascular changes in CAV.

Altered Oxygen Metabolism Associated to Neurogenesis of Induced Pluripotent Stem Cells Derived From a Schizophrenic Patient

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions. and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.