Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells

Colorectal cancer (CRC) is a disease whose genesis may include metabolic dysregulation. Cancer stem cells are attractive targets for therapeutic interventions since their aberrant expansion may underlie tumor initiation, progression, and recurrence. To investigate the actions of metabolic regulators on cancer stem cell-like cells (CSC) in CRC, we determined the effects of soybean-derived bioactive molecules and the anti-diabetes drug metformin (MET), alone and together, on the growth, survival, and frequency of CSC in human HCT116 cells. Effects of MET (60 μM) and soybean components genistein (Gen, 2 μM), lunasin (Lun, 2 μM), β-conglycinin (β-con, 3 μM), and glycinin (Gly, 3 μM) on HCT116 cell proliferation, apoptosis, and mRNA/protein expression and on the frequency of the CSC CD133(+)CD44(+) subpopulation by colonosphere assay and fluorescence-activated cell sorting/flow cytometry were evaluated. MET, Gen, and Lun, individually and together, inhibited HCT116 viability and colonosphere formation and, conversely, enhanced HCT116 apoptosis. Reductions in frequency of the CSC CD133(+)CD44(+) subpopulation with MET, Gen, and Lun were found to be associated with increased PTEN and reduced FASN expression. In cells under a hyperinsulinemic state mimicking metabolic dysregulation and without and with added PTEN-specific inhibitor SF1670, colonosphere formation and frequency of the CD133(+)CD44(+) subpopulation were decreased by MET, Lun and Gen, alone and when combined. Moreover, MET + Lun + Gen co-treatment increased the pro-apoptotic and CD133(+)CD44(+)-inhibitory efficacy of 5-fluorouracil under hyperinsulinemic conditions. Results identify molecular networks shared by MET and bioavailable soy food components, which potentially may be harnessed to increase drug efficacy in diabetic and non-diabetic patients with CRC.

Mesenchymal stem cell enhances chondral defects healing in horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of power doppler for the assessment of vascularization in equine tendinitis treated with mesenchymal stem cells

Tendinous lesions are very common in athlete horses. The process of tendon healing is slow and the quality of the new tissue is often inferior to the original, leading in many cases to recurrence of the lesion. One of the main reasons for the limited healing capacity of tendons is its poor vascularization. At present, cell therapy is used in equine practice for the treatment of several disorders including tendinitis, desmitis and joint disease. However, there is little information regarding the mechanisms of action of these cells during tissue repair. It is known that Mesenchymal Stem Cells (MSCs) release several growth factors at the site of implantation, some of which promote angiogenesis. Comparison of blood flow using power Doppler ultrasonography was performed after the induction superficial digital flexor tendon tendinitis and implantation of adipose tissue-derived MSCs in order to analyze the effect of cell therapy on tendon neovascularization. For quantification of blood vessel histopathological examinations were conducted. Increased blood flow and number of vessels was observed in treated tendons up to 30 days after cell implantation, suggesting promotion of angiogenesis by the cell therapy.

One-hundred days of monitoring of patients undergoing hematopoietic stem cell transplantation: events of metabolic syndrome

Objective This study assessed early manifestations of metabolic syndrome determinants in patients submitted to hematopoietic stem cell transplantation. Methods Twenty-seven individuals participated in the study (20 with autologous and 7 with allogeneic hematopoietic stem cell transplantation). Anthropometric variables and biochemical indicators of lipid and glucose metabolism were determined before and 100 days after hematopoietic stem cell transplantation.Results The mean total cholesterol (p=0.086), very low density lipoprotein-cholesterol (p=0.069) and triglycerides (p=0.086) of all patients did not change significantly between the two study periods, but when the patients were separated by type of hematopoietic stem cell transplantation, triglycerides and very low density lipoprotein-cholesterol were close to the critical level of significance for individuals with allogeneic hematopoietic stem cell transplantation (p=0.060) and total cholesterol was significant in individuals with autologous hematopoietic stem cell transplantation (p=0.027). Anthropometric variables did not change significantly between before and 100 days after hematopoietic stem cell transplantation. Conclusion Metabolic syndrome risk factors may be associated with lipid metabolism in the early phase of allogeneic and autologous hematopoietic stem cell transplantation.

Physical and mechanical characteristic of particleboards produced with residues of sugarcane and stem leaves of bamboo bonded with castor oil adhesive

In this research the aim was produce a particleboard with alternative materials and evaluated its physical and mechanical characteristics. The raw materials used are residues from sucarcane bagasse (SC) (Saccharum officinarum) and stem leaves of bamboo (B) (Dendrocalamus giganteus), bonded with a bi component adhesive based on castor oil. It was produced particleboards with five different traces: 100% SC, 75% SC+25% B, 50% SC+50% B, 25% SC +75%B and 100 % B. Their physical and mechanical characteristics were evaluated accordingly to Brazilian standard NBR 14810-3. Regarding the results obtained, it can be detached that for physical and mechanical evaluation it is evident a negative relation among the amount the sugarcane bagasse and their physical and mechanical characteristics, that is particleboards with low concentrations of sugarcane bagasse had better results. However all particleboards could be recommended for use as sealing particleboards in the segment of civil construction.

Development of a cryopreservation protocol for equine mesenchymal stem cells obtained from umbilical cord

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Is Competition Making a Comeback? Discovering Methods to Keep Female Adolescents Engaged in STEM: A Phenomenological Approach

The decreasing number of women who are graduating in the Science, Technology, Engineering and Mathematics (STEM) fields continues to be a major concern. Despite national support in the form of grants provided by National Science Foundation, National Center for Information and Technology and legislation passed such as the Deficit Reduction Act of 2005 that encourages women to enter the STEM fields, the number of women actually graduating in these fields is surprisingly low. This research study focuses on a robotics competition and its ability to engage female adolescents in STEM curricula. Data have been collected to help explain why young women are reticent to take technology or engineering type courses in high school and college. Factors that have been described include attitudes, parental support, social aspects, peer pressure, and lack of role models. Often these courses were thought to have masculine and “nerdy” overtones. The courses were usually majority male enrollments and appeared to be very competitive. With more female adolescents engaging in this type of competitive atmosphere, this study gathered information to discover what about the competition appealed to these young women. Focus groups were used to gather information from adolescent females who were participating in the First Lego League (FLL) and CEENBoT competitions. What enticed them to participate in a curriculum that data demonstrated many of their peers avoided? FLL and CEENBoT are robotics programs based on curricula that are taught in afterschool programs in non-formal environments. These programs culminate in a very large robotics competition. My research questions included: What are the factors that encouraged participants to participate in the robotics competition? What was the original enticement to the FLL and CEENBoT programs? What will make participants want to come back and what are the participants’ plans for the future? My research mirrored data of previous findings such as lack of role models, the need for parental support, social stigmatisms and peer pressure are still major factors that determine whether adolescent females seek out STEM activities. An interesting finding, which was an exception to previous findings, was these female adolescents enjoyed the challenge of the competition. The informal learning environments encouraged an atmosphere of social engagement and cooperative learning. Many volunteers that led the afterschool programs were women (role models) and a majority of parents showed support by accommodating an afterschool situation. The young women that were engaged in the competition noted it was a friendly competition, but they were all there to win. All who participated in the competition had a similar learning environment: competitive but cooperative. Further research is needed to determine if it is the learning environment that lures adolescent females to the program and entices them to continue in the STEM fields or if it is the competitive aspect of the culminating activity. Advisors: James King and Allen Steckelberg

Human immature dental pulp stem cells share key characteristic features with limbal stem cells

Objectives: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. Materials: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. Results and conclusions: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta 1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.

Thalidomide plus dexamethasone as a maintenance therapy after autologous hematopoietic stem cell transplantation improves progression-free survival in multiple myeloma

Despite the good response of stem cell transplant (SCT) in the treatment of multiple myeloma (MM), most patients relapse or do not achieve complete remission, suggesting that additional treatment is needed. We assessed the impact of thalidomide in maintenance after SCT in untreated patients with MM. A hundred and eight patients (<70 years old) were randomized to receive maintenance with dexamethasone (arm A; n = 52) or dexamethasone with thalidomide (arm B; n = 56; 200 mg daily) for 12 months or until disease progression. After a median follow-up of 27 months, an intention to treat analysis showed a 2-year progression-free survival (PFS) of 30% in arm A (95% CI 2238) and 64% in arm B (95% CI 5771; P = 0.002), with median PFS of 19 months and 36 months, respectively. In patients who did not achieve at least a very good partial response, the PFS at 2 years was significantly higher when in use of thalidomide (19 vs. 59%; P = 0.002). Overall survival at 2 years was not significantly improved (70 vs. 85% in arm A and arm B, respectively; P = 0.27). The addition of thalidomide to dexamethasone as maintenance improved the PFS mainly in patients who did not respond to treatment after SCT. Am. J. Hematol. (c) 2012 Wiley Periodicals, Inc.

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic beta cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-xL and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-xL genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-xL and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background: The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods: A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results: Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion: We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.

Plasticity of stomatal distribution pattern and stem tracheid dimensions in Podocarpus lambertii: an ecological study

Leaf and wood plasticity are key elements in the survival of widely distributed plant species. Little is known, however, about variation in stomatal distribution in the leaf epidermis and its correlation with the dimensions of conducting cells in wood. This study aimed at testing the hypothesis that Podocarpus lambertii, a conifer tree, possesses a well-defined pattern of stomatal distribution, and that this pattern can vary together with the dimensions of stem tracheids as a possible strategy to survive in climatically different sites. Leaves and wood were sampled from trees growing in a cold, wet site in south-eastern Brazil and in a warm, dry site in north-eastern Brazil. Stomata were thoroughly mapped in leaves from each study site to determine a spatial sampling strategy. Stomatal density, stomatal index and guard cell length were then sampled in three regions of the leaf: near the midrib, near the leaf margin and in between the two. This sampling strategy was used to test for a pattern and its possible variation between study sites. Wood and stomata data were analysed together via principal component analysis. The following distribution pattern was found in the south-eastern leaves: the stomatal index was up to 25 higher in the central leaf region, between the midrib and the leaf margin, than in the adjacent regions. The inverse pattern was found in the north-eastern leaves, in which the stomatal index was 10 higher near the midrib and the leaf margin. This change in pattern was accompanied by smaller tracheid lumen diameter and length. Podocarpus lambertii individuals in sites with higher temperature and lower water availability jointly regulate stomatal distribution in leaves and tracheid dimensions in wood. The observed stomatal distribution pattern and variation appear to be closely related to the placement of conducting tissue in the mesophyll.

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.