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Helena Debiazi Zomer,1,2 Kelly CS Roballo,2 Thais Borges Lessa,2 Fabiana Fernandes Bressan,2 Natália Nardeli Gonçalves,2 Flávio Vieira Meirelles,2,3 Andrea Gonçalves Trentin,1 Carlos Eduardo Ambrósio2,3 1Department of Cell Biology, Embryology and Genetic, Faculty of Biological Sciences, Santa Catarina Federal University (UFSC), Florianópolis, Brazil; 2Department of Surgery, Sector Anatomy, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil; 3Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil Introduction: Owing to their similarity with humans, rabbits are useful for multiple applications in biotechnology and translational research from basic to preclinical studies. In this sense, mesenchymal stem cells (MSCs) are known for their therapeutic potential and promising future in regenerative medicine. As many studies have been using rabbit adipose-derived MSCs (ASCs) as a model of human ASCs (hASCs), it is fundamental to compare their characteristics and understand how distinct features could affect the translation to human medicine.Objective: The aim of this study was to comparatively characterize rabbit ASCs (rASCs) and hASCs to further uses in biotechnology and translational studies.Materials and methods: rASCs and hASCs were isolated and characterized by their immunophenotype, differentiation potential, proliferative profile, and nuclear stability in vitro.Results and discussion: Both ASCs presented differentiation potential to osteocytes, chondrocytes, and adipocytes and shared similar immunophenotype expression to CD105+, CD34–, and CD45–, but rabbit cells expressed significantly lower CD73 and CD90 than human cells. In addition, rASCs presented greater clonogenic potential and proliferation rate than hASCs but no difference in nuclear alterations.Conclusion: The distinct features of rASCs and hASCs can positively or negatively affect their use for different applications in biotechnology (such as cell reprogramming) and translational studies (such as cell transplantation, tissue engineering, and pharmacokinetics). Nevertheless, the particularities between rabbit and human MSCs should not prevent rabbit use in preclinical models, but care should be taken to interpret results and properly translate animal findings to medicine.Keywords: characterization, comparison, MSC, iPS, immunophenotype, proliferation

Background: Although the paracrine effects of mesenchymal stem/stromal cells (MSCs) have been recognized as crucial mediators of their regenerative effects on tissue repair, the potential of MSC secretomes as effective substitutes for cellular therapies remains underexplored., Methods: In this study, we compared MSCs from the human dermis (DSCs) and adipose tissue (ASCs) with their secretomes regarding their efficacy for skin wound healing using a translationally relevant murine model., Results: Proteomic analysis revealed that while there was a substantial overlap in protein composition between DSC and ASC secretomes, specific proteins associated with wound healing and angiogenesis were differentially expressed. Despite a similar angiogenic potential in vivo, DSC and ASC secretomes were found to be less effective than cells in accelerating wound closure and promoting tissue remodeling., Conclusions: Overall, secretome-treated groups showed intermediary results between cells- and control-treated (empty scaffold) groups. These findings highlight that although secretomes possess therapeutic potential, their efficacy might be limited compared to cellular therapies. This study contributes to the growing understanding of MSC secretomes, emphasizes the need for further protocol optimization, and offers insights into their potential applications in regenerative medicine., (© 2024. The Author(s).)

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies., (© 2022. The Author(s).)

Body fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies. In vitro amplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, and these had higher proliferative potential, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-β-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and γ-H2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This systematic review aimed to determine the effects of intra-articular administration of mesenchymal stem cells from adipose tissue in dogs with hip joint osteoarthritis (OA). Clinical trials were systematically reviewed, using PubMed, EMBASE, Cochrane Library, LILACS, Web of Science, Scopus, Open Grey, Google Scholar, and ProQuest Dissertation and Thesis without publication year restrictions. References were screened and selected based on predefined eligibility criteria by two independent reviewers, according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Clinical outcomes were assessed quantitatively using clinical pain scores, physical examination, imaging examination, questionnaire responses, pain in manipulation, gait analysis, range of joint motion, and adverse effects. The risk of bias was assessed using the Joanna Briggs Institute Critical Appraisal Checklist. Out of 1483 articles, six met the inclusion criteria for qualitative analysis, with two randomized controlled trials and four before-and-after studies. All studies reported significantly better clinical outcomes in the adipose tissue stem cells (ADSC) group with improvements in pain and function and decreased evidence of hip OA. The risk of bias was categorized as high in the before-and-after studies and moderate to high in the randomized studies. The studies were considered heterogeneous owing to clinical results and methodology. Because of this heterogeneity, it was not possible to perform meta-analysis. Assessments of ADSC reports yielded positive clinical effects that showed improvements in pain and function and decreased evidence of hip osteoarthritis. More high-level, larger-cohort dog studies that utilize standardized protocols are needed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Neural crest cells possess characteristics of stem cells including plasticity and ability to differentiate into various cell types. HNK1 and Sox10 are markers of neural crest cell progenitors that have been demonstrated in osteoblasts during osteogenesis of the maxilla and mandible. We investigated the presence of Sox10 and HNK1 during regeneration of mandibular bone defects. Defects were created in mandibles of rats. Samples of these defects were collected at 7, 14 and 28 days post-surgery; bone regeneration was observed during this period. Immunohistochemical analysis revealed expression of HNK1 and Sox10 in osteoblasts, osteocytes and osteogenic cells, whereas osteoclasts were unstained. HNK1 expression was increased in osteoblasts and osteocytes over time and SOX10 expression was found in osteoblasts and osteogenic cells at 7, 14 and 28 days post-surgery. HNK1 and Sox10 are present in osteoblasts, osteocytes and osteogenic cells during mandible bone regeneration.

The process of wound healing restores skin homeostasis but not full functionality; thus, novel therapeutic strategies are needed to accelerate wound closure and improve the quality of healing. In this context, tissue engineering and cellular therapies are promising approaches. Although sharing essential characteristics, mesenchymal stromal cells (MSCs) isolated from different tissues might have distinct properties. Therefore, the aim of this study was to comparatively investigate, by a mouse model in vivo assay, the potential use of dermal-derived MSCs (DSCs) and adipose tissue-derived MSCs (ASCs) in improving skin wound healing. Human DSCs and ASCs were delivered to full-thickness mouse wounds by a collagen-based scaffold (Integra Matrix). We found that the association of both DSCs and ASCs with the Integra accelerated wound closure in mice compared with the biomaterial only (control). Both types of MSCs stimulated angiogenesis and extracellular matrix remodeling, leading to better quality scars. However, the DSCs showed smaller scar size,superior extracellular matrix deposition, and greater number of cutaneous appendages. Besides, DSCs and ASCs reduced inflammation by induction of macrophage polarization from a pro-inflammatory (M1) to a pro-repair (M2) phenotype. In conclusion, both DSCs and ASCs were able to accelerate the healing of mice skin wounds and promote repair with scars of better quality and more similar to healthy skin than the empty scaffold. DSCs associated with Integra induced superior overall results than the Integra alone, whereas scaffolds with ASCs showed an intermediate effect, often not significantly better than the empty biomaterial., (Copyright © 2020 International Society for Cell and Gene Therapy. All rights reserved.)

Neural crest cells (NCCs) comprise a population of multipotent progenitors and stem cells at the origin of the peripheral nervous system (PNS) and melanocytes of skin, which are profoundly influenced by microenvironmental factors, among which is basic fibroblast growth factor 2 (FGF2). In this work, we further investigated the role of this growth factor in quail trunk NC morphogenesis and demonstrated its huge effect in NCC growth mainly by stimulating cell proliferation but also reducing cell death, despite that NCC migration from the neural tube explant was not affected. Moreover, following FGF2 treatment, reduced expression of the early NC markers Sox10 and FoxD3 and improved proliferation of HNK1-positive NCC were observed. Since these markers are involved in the regulation of glial and melanocytic fate of NC, the effect of FGF2 on NCC differentiation was investigated. Therefore, in the presence of FGF2, increased proportions of NCCs positives to the melanoblast marker Mitf as well as NCCs double stained to Mitf and BrdU were recorded. In addition, treatment with FGF2, followed by differentiation medium, resulted in increased expression of melanin and improved proportion of melanin-pigmented melanocytes without alteration in the glial marker Schwann myelin protein (SMP). Taken together, these data further reveal the important role of FGF2 in NCC proliferation, survival, and differentiation, particularly in melanocyte development. This is the first demonstration of FGF2 effects in melanocyte commitment of NC and in the proliferation of Mitf-positive melanoblasts. Elucidating the differentiation process of embryonic NCCs brings us a step closer to understanding the development of the PNS and then undertaking the search for advanced technologies to prevent, or treat, injuries caused by NC-related disorders, also known as neurocristopathies.

Novel strategies combining cell therapy, tissue engineering, and regenerative medicine have been developed to treat major skin wounds. Although mesenchymal stromal cells (MSCs) from different tissues have similar stem cell features, such as self-renewing mesodermal differentiation potential and expression of immunophenotypic markers, they also have distinct characteristics. Therefore, we aimed to characterize the application of MSCs derived from the dermis and adipose tissue (DSCs and ASCs, respectively) in cutaneous wound healing by in vitro approaches. Human DSC and ASC were obtained and evaluated for their isolation efficiency, stemness, proliferative profile, and genetic stability over time in culture. The ability of wound closure was first assessed by direct cell scratch assay. The paracrine effects of DSC- and ASC-conditioned medium in dermal fibroblasts and keratinocytes and in the induction of tubule formation were also investigated. Although the ASC isolation procedures resulted in 100 times more cells than DSC, the latter had a higher proliferation rate in culture. Both presented low frequency of nuclear alterations over time in culture and showed similar characteristics of stem cells, such as expression of immunophenotypic markers and differentiation potential. DSCs showed increased healing capacity, and their conditioned media had greater paracrine effect in closing the wound of dermal fibroblasts and keratinocytes and in inducing angiogenesis. In conclusion, the therapeutic potential of MSCs is influenced by the obtainment source. Both ASCs and DSCs are applicable for skin wound healing; however, DSCs have an improved potential and should be considered for future applications in cell therapy., (© 2019 John Wiley & Sons, Ltd.)

This study aimed to evaluate the use of human dental pulp stem cells (hDPSCs) in non-critical-sized mandibular bone defects in rats. hDPSCs from permanent teeth were isolated and engrafted in mandibular bone defects in rats for 7, 14, and 28 days; bone defects without cells formed the control group. Samples were evaluated by scanning electron microscopy (SEM), light microscopy (hematoxylin and eosin staining), and the regeneration area was measured by the Image J program. Before surgery procedures, the human dental pulp cells were characterized as dental pulp stem cells: fusiform morphology, plastic-adherent; expression of CD105, CD73, and CD90; lack of expression of CD45 and CD34, and differentiated into osteoblasts, adipocytes, and chondroblasts. The results indicated that within 7 days the control group presented a pronounced bone formation when compared with the treated group (p < 0.05). After 14 days, the treated group showed an increase in bone formation, but with no statistical difference among the groups (p > 0.05). In the final evaluated period there was no difference between the control group and the treated group (p > 0.05). There was a significant difference between 7 and 14 days (p < 0.05) and between 7 and 28 days (p < 0.05) in the treated group. In conclusion, there is no evidence that the use of hDPSCs in the conditions of this study could improve bone formation in non-critical-sized mandibular bone defects., (© 2019 S. Karger AG, Basel.)

The developmental mechanisms that control the building of the complex head of vertebrates and particularly, facial skeletogenesis, remain poorly known. Progenitor cells derived from the embryonic neural crest (NC) are the major constituents and players of facial tissue development. Deciphering the cellular and molecular machinery that controls NC cell (NCC) differentiation into bone, cartilage, fat and other mesenchymal tissues, is thus a main issue for understanding vertebrate facial variations. In this work, we investigated the effects of fibroblast growth factor 8 (FGF8) and Sonic Hedgehog (Shh), two signaling molecules essential for craniofacial development, on the in vitro differentiation and multipotentiality of mesencephalic NCCs (MNCCs) isolated from the quail embryo. Comparison of distinct temporal treatments with FGF8 and/or Shh showed that both promoted chondrogenesis of MNCCs by increasing the amount and size of cartilage nodules. Higher rates of chondrogenesis were observed when MNCCs were treated with FGF8 during the migration phase, thus mimicking the in vivo exposure of migrating NCCs to FGF8 secreted by the isthmic brain signaling center. An in vitro cell cloning assay revealed that, after concomitant treatment with FGF8 and Shh, about 80% of NC progenitors displayed chondrogenic potential, while in untreated cultures, only 18% exhibited this potential. In addition, colony analysis showed for the first time the existence of a highly multipotent progenitor able to clonally give rise to adipocytes in addition to other cephalic NC phenotypes (i.e. glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes) (GNMFCA progenitor). This progenitor was observed only when clonal cultures were treated with both FGF8 and Shh. Several other types of multipotent cells, which generated four, five or six distinct phenotypes, accounted for 55% of the progenitors in FGF8 and Shh treated cultures, versus 13,5% in the untreated ones. Together, these data reveal an essential role for both FGF8 and Shh together in maintenance of MNCC multipotentiality by favoring the development of NC progenitors endowed with a broad array of mesectodermal potentials., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The neural crest (NC) is one of the most fascinating structures during embryonic development. Unique to vertebrate embryos, these cells give rise to important components of the craniofacial skeleton, such as the jaws and skull, as well as melanocytes and ganglia of the peripheral nervous system. Worldwide, several groups have been studying NC development and specifically in the Latin America (LA) they have been growing in numbers since the 1990s. It is important for the world to recognize the contributions of LA researchers on the knowledge of NC development, as it can stimulate networking and improvement in the field. We developed a database of LA publications on NC development using ORCID and PUBMED as search engines. We thoroughly describe all of the contributions from LA, collected in five major topics on NC development mechanisms: i) induction and specification; ii) migration; iii) differentiation; iv) adult NC; and, v) neurocristopathies. Further analysis was done to correlate each LA country with topics and animal models, and to access collaboration between LA countries. We observed that some LA countries have made important contributions to the comprehension of NC development. Interestingly, some LA countries have a topic and an animal model as their strength; in addition, collaboration between LA countries is almost inexistent. This review will help LA NC research to be acknowledged, and to facilitate networking between students and researchers worldwide., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Carrageenan is a thermoreversible polymer of natural origin widely used in food and pharmaceutical industry that presents a glycosaminoglycan-like structure. Herein, we show that kappa-type carrageenan extracted by a semi-refined process from the red seaweed Kappaphycus alvarezii displayed both chemical and structural properties similar to a commercial carrageenan. Moreover, both extracted carrageenan hydrogel and commercial carrageenan hydrogel can serve as a scaffold for in vitro culture of human skin-derived multipotent stromal cells, demonstrating considerable potential as cell-carrier materials for cell delivery in tissue engineering. Skin-derived multipotent stromal cells cultured inside the carrageenan hydrogels showed a round shape morphology and maintained their growth and viability for at least one week in culture. Next, the effect of the extracted carrageenan hydrogel loaded with human skin-derived multipotent stromal cells was evaluated in a mouse model of full-thickness skin wound. Macroscopic and histological analyses revealed some pointed ameliorated features, such as reduced inflammatory process, faster initial recovery of wounded area, and improved extracellular matrix deposition. These results indicate that extracted carrageenan hydrogel can serve as a scaffold for in vitro growth and maintenance of human SD-MSCs, being also able to act as a delivery system of cells to wounded skin. Thus, evaluation of the properties discussed in this study contribute to a further understanding and specificities of the potential use of carrageenan hydrogel as a delivery system for several applications, further to skin wound healing.

Despite the great progress in translational research concerning skin wound healing in the last few decades, no animal model fully predicts all clinical outcomes. The mouse is the most commonly used model, as it is easy to maintain and standardize, and is economically accessible. However, differences between murine and human skin repair, such as the contraction promoted by panniculus carnosus and the role of specific niches of skin stem cells, make it difficult to bridge the gap between preclinical and clinical studies. Therefore, this review highlights the particularities of each species concerning skin morphophysiology, immunology, and genetics, which is essential to properly interpret findings and translate them to medicine., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Bentonites are commonly used as feed additives to reduce the bioavailability and thus the toxicity of aflatoxins by adsorbing the toxins in the gastrointestinal tract. Aflatoxins are particular harmful mycotoxins mainly found in areas with hot and humid climates. They occur in food and feedstuff as a result of fungal contamination before and after harvest. The aim of this study was to modify Brazilian bentonite clay by incorporation of zinc (Zn) ions in order to increase the adsorption capacity and consequently reduce the toxicity of aflatoxins. The significance of Zn intercalating conditions such as concentration, temperature and reaction time were investigated. Our results showed that the Zn treatment of the bentonite increased the aflatoxin B 1 (AFB 1 ) adsorption and that Zn concentration had a negative effect. Indeed, temperature and time had no significant effect in the binding capacity. The modified bentonite (Zn-Bent1) was not cytotoxic to either fibroblasts (3T3) nor epithelial colorectal adenocarcinoma cells (Caco-2) cell lines. Interestingly, Zn-Bent1 has higher protective effect against AFB 1 induced cytotoxicity than the unmodified bentonite. In conclusion, the Zn modified bentonite, Zn-Bent1, represent an improved tool to prevent aflatoxicosis in animals fed on AFB 1 contaminated feed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Spinal cord injury (SCI) is a devastating neurologic disorder with significant impacts on quality of life, life expectancy, and economic burden. Although there are no fully restorative treatments yet available, several animal and small-scale clinical studies have highlighted the therapeutic potential of cellular interventions for SCI. Mesenchymal stem cells (MSCs)-which are conventionally isolated from the bone marrow-recently emerged as promising candidates for treating SCI and have been shown to provide trophic support, ameliorate inflammatory responses, and reduce cell death following the mechanical trauma. Here we evaluated the human skin as an alternative source of adult MSCs suitable for autologous cell transplantation strategies for SCI. We showed that human skin-derived MSCs (hSD-MSCs) express a range of neural markers under standard culture conditions and are able to survive and respond to neurogenic stimulation in vitro. In addition, using histological analysis and behavioral assessment, we demonstrated as a proof-of-principle that hSD-MSC transplantation reduces the severity of tissue loss and facilitates locomotor recovery in a rat model of SCI. Altogether, the study provides further characterization of skin-derived MSC cultures and indicates that the human skin may represent an attractive source for cell-based therapies for SCI and other neurological disorders. Further investigation is needed to elucidate the mechanisms by which hSD-MSCs elicit tissue repair and/or locomotor recovery.

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies-a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.

Bentonite clays exhibit high adsorptive capacity for contaminants, including aflatoxin B1 (AFB1), a mycotoxin responsible for causing severe toxicity in several species including pigs, poultry and man. Organophilic treatments is known to increase the adsorption capacity of bentonites, and the primary aim of this study was to evaluate the ability of Brazilian bentonite and two organic salts - benzalkonium chloride (BAC) and cetyltrimethylammonium bromide (CTAB) to adsorb AFB1. For this end, 2(2) factorial designs were used in order to analyze if BAC or CTAB was able to increase AFB1 adsorption when submitted in different temperature and concentration. Both BAC and CTAB treatment (at 30°C and 2% of salt concentration) were found to increase the adsorption of AFB1 significantly compared with untreated bentonite. After organophilic bentonite treatments with BAC or CTAB, a vibration of CH stretch (2850 and 2920cm(-1)) were detected. A frequency of the SiO stretch (1020 and 1090cm(-1)) was changed by intercalation of organic cation. Furthermore, the interlayer spacing of bentonite increases to 1.23nm (d001 reflection at 2θ=7.16) and 1.22 (d001 reflection at 2θ=7.22) after the addition of BAC and CTAB, respectively. Another aim of the study was to observe the effects of these two bentonite salts in neural crest stem cell cultures. The two materials that were created by organophilic treatments were not found to be toxic to stem cells. Furthermore the results indicate that the two materials tested may protect the neural crest stem cells against damage caused by AFB1., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Bentonites are clays that highly adsorb aflatoxin B1 (AFB1) and, therefore, protect human and animal cells from damage. We have recently demonstrated that bentonite protects the neural crest (NC) stem cells from the toxicity of AFB1. Its protective effects are due to the physico-chemical properties and chemical composition altered by heat treatment. The aim of this study is to prepare and characterize the natural and thermal treatments (125 to 1000 °C) of bentonite from Criciúma, Santa Catarina, Brazil and to investigate their effects in the AFB1 adsorption and in NC cell viability after challenging with AFB1. The displacement of water and mineralogical phases transformations were observed after the thermal treatments. Kaolinite disappeared at 500 °C and muscovite and montmorillonite at 1000 °C. Slight changes in morphology, chemical composition, and density of bentonite were observed. The adsorptive capacity of the bentonite particles progressively reduced with the increase in temperature. The observed alterations in the structure of bentonite suggest that the heat treatments influence its interlayer distance and also its adsorptive capacity. Therefore, bentonite, even after the thermal treatment (125 to 1000 °C), is able to increase the viability of NC stem cells previously treated with AFB1. Our results demonstrate the effectiveness of bentonite in preventing the toxic effects of AFB1., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cell differentiation is essential for maxillaries and tooth development. Facial mesenchymal tissue is formed by neural crest cells (NC). These cells are highly migratory, giving rise to various cell types, considered with a high level of plasticity, indicating that they contain progenitor cells with a great power of differentiation. In this study, it was analyzed the presence of NC cell progenitors and mesenchymal stem cells (MSC) during maxillaries osteogenesis and odontogenesis in rats. Histological slides were collected in two phases: embryonic age of 15 and 17 days; 2, 4 and 7 days after birth. Immunohistochemistry for MSC markers (Osterix) and NC cells (Sox10, HNK1) was performed. The results showed positive expression for Osterix and HNK1 in undifferentiated ectomesenchymal cells in early and late stages; Sox10 was present only in early stages in undifferentiated cells. All markers were present in differentiated cells. Although the experiments performed do not allow us to explain a possible role for Osx, HNK1 and Sox10 in both differentiated and undifferentiated cells during osteogenesis and odontogenesis, it had shown important results not yet described: the presence of HNK1 and Sox10 in osteoblasts and odontoblasts in late development stages and in the tooth germ epithelial cells and ameloblasts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Thyroid hormones (THs) play key roles in brain development and function. The lack of THs during childhood is associated with the impairment of several neuronal connections, cognitive deficits and mental disorders. Several lines of evidence point to astrocytes as TH targets and as mediators of TH action in the central nervous system; however, the mechanisms underlying these events are still not completely known. In this review, we focus on advances in our understanding of the effects of THs on astroglial cells and the impact of these effects on neurone-astrocyte interactions. First, we discuss the signalling pathways involved in TH metabolism and the molecular mechanisms underlying TH receptor function. Then, we discuss data related to the effects of THs on astroglial cells, as well as studies regarding the generation of mutant TH receptor transgenic mice that have contributed to our understanding of TH function in brain development. We argue that astrocytes are key mediators of hormone actions on development of the cerebral cortex and cerebellum and that the identification of the molecules and pathways involved in these events might be important for determining the molecular-level basis of the neural deficits associated with endocrine diseases., (© 2015 British Society for Neuroendocrinology.)

Strategies for skin regeneration have been developed to provide effective treatment for cutaneous wounds and disease. Dermal substitutes have been used to cover the lesion to facilitate cell colonization, thereby promoting dermal regeneration. However, very little is known about Pelnac matrix especially at histological level. Therefore, the present work carried out an experimental in vivo comparative analysis between Pelnac and Integra, the most used dermal templates, in a mouse model of full-thickness skin wounds. Histological sections performed at the 3rd, 6th and 9th days after surgery were analyzed with regard to inflammatory response and vascularization. Both templates were completely incorporated in all animals at the end of the analyzed period. Pelnac-treated animals displayed reduced granulation tissue during the first 6 days of treatment compared to the animals treated with Integra at the same time period. The number of inflammatory cells (neutrophils) was similar in both groups during the period, significantly reducing at the end of inflammatory phase (9th day of treatment) consistent with the progression of healing process. In addition, the density of blood vessels was also statistically similar in both matrices. Therefore, the two dermal templates displayed comparable biological behavior in tissue repair. It is noteworthy that this is the first experimental study comparing Pelnac and Integra dermal templates with focus on full-thickness skin wounds.

Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF2) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF2, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins βIII-tubulin and nestin, as well as upregulation of the pan neuronal marker βIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF-FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF-FGF2 in neuronal differentiation protocols., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Experimental ischemia results in cortical brain lesion followed by ischemic stroke. In this study, focal cerebral ischemia was induced in mice by occlusion of the middle cerebral artery. We studied cortical layers I, II/III, V and VI in the caudal forelimb area (CFA) and medial agranular cortex (AGm) from control and C57BL/6 mice induced with ischemic stroke. Based on our analysis of CFA and AGm motor cortex, significant differences were observed in the numbers of neurons, astrocytes and microglia in the superficial II/III and deep V cortical layers. Cellular changes were more prominent in layer V of the CFA with nuclear pyknosis, chromatin fragmentation, necrosis and degeneration, as well as, morphological evidence of apoptosis, mainly in neurons. As result, the CFA was more severely impaired than the AGm in this focal cerebral ischemic model, as evidenced by the proliferation of astrocytes, potentially resulting in neuroinflammation by microglia-like cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and disease. Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their prolonged self-renewal capacity, multipotentiality, and ability to release active molecules important for tissue repair. In this paper, we show that human skin-derived mesenchymal stromal cells (SD-MSCs) display similar characteristics to the multipotent MSCs. We also evaluate their growth in a three-dimensional (3D) culture system with dermal substitutes (Integra and Pelnac). When cultured in monolayers, SD-MSCs expressed mesenchymal markers, such as CD105, Fibronectin, and α-SMA; and neural markers, such as Nestin and βIII-Tubulin; at transcriptional and/or protein level. Integra and Pelnac equally supported the adhesion, spread and growth of human SD-MSCs in 3D culture, maintaining the MSC characteristics and the expression of multilineage markers. Therefore, dermal substitutes support the growth of mesenchymal stromal cells from human skin, promising an effective tool for tissue engineering and regenerative technology.

The neural crest (NC) is an attractive system for investigating the mechanisms underlying cell lineage diversification in higher vertebrates. The NC contains a mixed population of already defined precursors and multipotent cells that can give rise to a great variety of cell types, including glial cells and neurons of the peripheral nervous system, melanocytes, and smooth muscle cells (SMCs). Microenvironmental factors, such as the fibroblast growth factor 2 (FGF2), found along migratory paths and in target tissues, strongly influence the fate of multipotent NC precursors. We have previously demonstrated that the FGF2 promotes the differentiation of NC cells to glial phenotypes, while the epidermal growth factor induces NC differentiation to neurons and melanocytes. In the present study, we used mass cultures and single-cell culture assays to demonstrate that FGF2 influences NC cell differentiation and increases the proportion of multipotent progenitors. Furthermore, we demonstrate for the first time that avian tripotent glial, melanocyte and smooth muscle NC progenitors, as well as bipotent melanocyte and smooth muscle NC progenitors, are capable of self-renewal. FGF2 significantly stimulated the self-renewal of bipotent progenitor cells with glial cells and SMC potentials. These cells propagated for many generations and behaved as stem cells. These results suggest an important role of FGF2 in maintaining the stemness of avian NC cells.

Mesenchymal stem cells (MSCs) are multipotent stem cells with clinical interest. It has been reported that MSCs can be isolated from the human term placenta. We investigated the ability of human placenta-derived MSCs to differentiate into a neural phenotype in coculture assays with astrocytes obtained from neonatal rats. Placenta-derived MSCs were cocultured on a confluent monolayer of astrocytes obtained from the rat cerebellum to evaluate the differences in morphology. The extracellular matrix (ECM) produced by astrocytes as well as the growth factors produced by the astrocyte-conditioned medium were evaluated. The expression of the neural markers glial fibrillate acid protein (GFAP) and Nestin was studied in MSCs by immunocytochemistry. MSCs were able to respond to the astrocyte niche in coculture assays. They expressed the neural markers GFAP, Nestin, or β-Tubulin III, followed by an outgrowth of cell processes. The ECM from astrocytes was not effective in inducing the neural phenotype in MSCs, although the expression of β-Tubulin III was observed. When MSCs were cocultured with cerebellar astrocytes from newborn rats, a neural phenotype was achieved. This was determined by immunocytochemistry to GFAP, Nestin, or β-Tubulin III and by morphological changes. It was achieved without the addition of exogenous differentiation factors. This demonstrates that placenta-derived MSCs may be able to differentiate into neural cell types when in direct contact with a neural environment.

The neural crest (NC) corresponds to a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potentials. The derivatives of the NC at trunk level include neurons and glial cells of the peripheral nervous system. Despite the well-known influence of aflatoxins on the development of cancer, the issue of whether they also influence NC cells has not been yet addressed. In the present work, we have investigated the effects of aflatoxin B(1) on quail NC cells and the concomitant effects of the flavonoid hesperidin associated with this mycotoxin. We show for the first time that aflatoxin B(1) decreases the viability and the total number of glial and neuronal cells/field, although their proportions in relation to the total number of cells were not altered. Therefore, aflatoxin has no effect on NC differentiation. However, this compound was able to reduce NC proliferation and NC survival. Furthermore, the co-administration of hesperidin, a well-known polyphenolic protector of cell death, partially prevented the effect of aflatoxin B(1) . Taken together, our results demonstrate that aflatoxin B(1) is toxic to NC cells, an effect partially prevented by the flavonoid hesperidin. This study may contribute to the understanding of the effects of these compounds during early embryonic development and offer potentially more assertive diets and treatments for pregnant animals., (© 2013 International Federation for Cell Biology.)

The neural crest (NC) is composed of highly multipotent precursor cells able to differentiate into both neural and mesenchymal phenotypes. Until now, most studies focusing on NC cell differentiation have been performed with traditional two-dimensional (2D) cell culture systems. However, such culture systems do not reflect the complex three-dimensional (3D) microenvironments of in vivo NC cells. To address this limitation, we have developed a method of Matrigel™ coating to create 2D and 3D microenvironments in the same culture well. When we performed cultures of trunk neural crest cells (TNCCs) on three different lots of basement membrane matrix (Matrigel™), we observed that all analyzed Matrigel™ lots were equally efficient in allowing the appearance of glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes. We further observed that chondrocytes were found predominantly in the 3D microenvironment, whereas smooth muscle cells were almost exclusively located in the 2D microenvironment. Glial cells were present in both environments, but with broader quantities on the 2D surface. Melanocytes and neurons were equally distributed in both 2D and 3D microenvironments, but with distinct morphologies. It is worth noting the higher frequency of chondrocytes detected in this study using the 3D Matrigel™ microenvironment compared to previous reports of chondrogenesis obtained from TNCCs on traditional 2D cultures. In conclusion, Matrigel™ represents an attractive scaffold to study NC multipotentiality and differentiation, since it permits the appearance of the major NC phenotypes.

The neural crest (NC) corresponds to a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potentials. The derivatives of the NC at trunk level include neurons and glial cells of the peripheral nervous system in addition to melanocytes, smooth muscle cells and some endocrine cells. Environmental factors control the fate decisions of NC cells. Despite the well-known influence of flavonoids on the central nervous system, the issue of whether they also influence NC cells has not been yet addressed. Flavonoids are polyphenolic compounds that are integral components of the human diet. The biological activities of these compounds cover a very broad spectrum, from anticancer and antibacterial activities to inhibition of bone reabsorption and modulation of inflammatory response. In the present work, we have investigated the actions of the flavonoids hesperidin, rutin and quercetin on NC cells of quail, in vitro. We show for the first time, that hesperidin and rutin increase the viability of trunk NC cells in culture, without affecting cell differentiation and proliferation. The molecular mechanism of this action is dependent on ERK2 and PI3K pathways. Quercetin had no effect on NC progenitors. Taken together, these results suggest that flavonoids hesperidin and rutin increase NC cell survival, which may be useful against the toxicity of some chemicals during embryonic development.

Astrocytes clearly play a role in neuronal development. An indirect mechanism of thyroid hormone (T3) in the regulation of neuronal development mediated by astrocytes has been proposed. T3 alters the production and organization of the extracellular matrix (ECM) proteins and proteoglycans, producing a high-quality substrate for neuronal differentiation. The present study investigated the effect of hypothyroidism on the astrocyte production of fibronectin (FN) and laminin (LN) as well as their involvement in neuronal growth and neuritogenesis. Our results demonstrated that the amount of both FN and LN were significantly reduced in cultures of hypothyroid astrocytes from rat cerebellum compared with normal cells. This effect was accompanied by reduced numbers of neurons and neuritogenesis. Similarly, the proportions of neurons and neurons with neurites were reduced in cultures on ECM prepared from hypothyroid astrocytes in comparison with normal cells. The proportion of both normal and hypothyroid neurons is strongly reduced in astrocyte ECM compared with cocultures on astrocyte monolayers, suggesting that extracellular factors other than ECM proteins are involved in this process. Moreover, treatment of hypothyroid astrocytic cultures with T3 restored the area of both FN and LN immunostaining to normal levels and partially reestablished neuronal survival and neuritogenesis. Taken together, our results demonstrated that hypothyroidism involves impairment of the astrocytic microenvironment and affects the production of ECM proteins. Thus, hypothyroidism is implicated in impaired neuronal development.

Proliferation of neural crest (NC) stem cells and their subsequent differentiation into different neural cell types are key early events in the development of the peripheral nervous system. Soluble growth factors present at the sites where NC cells migrate are critical to the development of NC derivatives in each part of the body. In the present study, we further investigate the effect of microenvironmental factors on quail trunk NC development. We show for the first time that EGF induces differentiation of NC to the neuronal and melanocytic phenotypes, while fibroblast growth factor 2 (FGF2) promotes NC differentiation to Schwann cells. In the presence of both EGF and FGF2, the neuronal differentiation predominates. Our results suggest that FGF2 stimulates gliogenesis, while EGF promotes melanogenesis and neurogenesis. The combination of both growth factors stimulates neurogenesis. These findings suggest that these two growth factors may play an important role in the fate decision of NC progenitors and in the development of the peripheral nervous system.

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.

Thyroid hormone (T(3)) regulates the growth and differentiation of rat cerebellar astrocytes. Previously, we have demonstrated that these effects are due, at least in part, to the increased expression of extracellular matrix molecules and growth factors, such as fibroblast growth factor-2. T(3) also modulates neuronal development in an astrocyte-mediated manner. In the mammalian central nervous system, excitatory neurotransmission is mediated mainly by glutamate. However, excessive stimulation of glutamate receptors can lead to excitotoxicity and cell death. Astrocytic glutamate transporters, GLT-1 and GLAST, play an essential role in the clearance of the neuronal-released glutamate from the extracellular space and are essential for maintaining physiological extracellular glutamate levels in the brain. In the present study, we showed that T(3) significantly increased glutamate uptake by cerebellar astrocytes compared with control cultures. Inhibitors of glutamate uptake, such as L-PDC and DL-TBOA, abolished glutamate uptake on control or T(3)-treated astrocytes. T(3) treatment of astrocytes increased both mRNA levels and protein expression of GLAST and GLT-1, although no significant changes on the distribution of these transporters were observed. The gliotoxic effect of glutamate on cultured cerebellar astrocytes was abolished by T(3) treatment of astrocytes. In addition, the neuronal viability against glutamate challenge was enhanced on T(3)-treated astrocytes, showing a putative neuroprotective effect of T(3). In conclusion, our results showed that T(3) regulates extracellular glutamate levels by modulating the astrocytic glutamate transporters. This represents an important mechanism mediated by T(3) on the improvement of astrocytic microenvironment in order to promote neuronal development and neuroprotection., ((c) 2008 Wiley-Liss, Inc.)

Background and Objective: The periodontal ligament is a specialized connective tissue, derived from dental follicle and originated from neural crest cells. Recently it has been suggested, based on animal models, that periodontal ligament could be a niche for neural crest stem cells. However, there is still little knowledge on this subject. The identification of neural crest adult stem cells has received much attention based on its potential in tissue regeneration. The objective of the present work was to verify the human periodontal ligament as a niche for neural crest stem cells., Material and Methods: Cells from human periodontal ligament were isolated from 10 teeth of seven individuals (periodontal ligament pool group) and also from four teeth of one individual (periodontal ligament single group), after enzymatic digestion. The cells were cultured in specific inductive medium. Analyses of protein and gene expression were performed through immunocytochemistry and reverse transcription-polymerase chain reaction techniques, respectively., Results: Mesodermal phenotypes (adipogeneic, osteogenic and myofibroblastic) were identified after culture in inductive medium. Immunocytochemistry analyses showed the presence of the nestin marker of neural stem cells and also markers of undifferentiated neural crest cells (HNK1, p75). When cultured in inductive medium that allowed neural differentiation, the cells showed markers for beta-tubulin III, neurofilament M, peripherin, microtubule-associated protein 2 and protein zero. The results were similar between the two study groups (the periodontal ligament pool group and the periodontal ligament single group)., Conclusion: This research provides evidence that human periodontal ligament, in addition to its mesodermal derivatives, produces neural crest-like cells. Such features suggest a recapitulation of their embryonic state. The human periodontal ligament revealed itself as a viable alternative source for possible primitive precursors to be used in stem-cell therapies.

Cell interaction with extracellular matrix is a crucial event for various biological processes, including tumor progression. Although not exclusively, these interactions are frequently mediated by bidirectional signaling receptors known as integrins. Using a human histiocytic lymphoma-derived cell line (U-937), we evaluated the effects of ECM proteins and their integrin-type receptors in the regulation of cell attachment, proliferation, migration and survival. Fibronectin induces higher cell attachment in vitro when compared to laminin. Fibronectin also promotes a decrease in cell migration but do not modulate cell proliferation and death. Pre-incubation of U-937 cells with VLA-5 antagonistic peptides inhibited attachment of the cells to fibronectin-coated substrates. In a second vein, we observed that lymph node specimens obtained from diagnosed patient for true histiocytic lymphoma had greater deposition of fibronectin (but not laminin) around malignant clones. These results suggest that fibronectins play a relevant role in the establishment and progression of true histiocytic lymphoma cells.

Thyroid hormone (T(3)) plays an essential role in the central nervous system development. Astrocytes mediate many of the T(3) effects in the growth and differentiation of cerebellum. In culture, T(3) induces cerebellar astrocytes to secrete growth factors, mainly FGF(2), and alters the expression and organization of the extracellular matrix (ECM) proteins, laminin, and fibronectin. In addition, T(3)-treated astrocytes promote neuronal differentiation. In this study, we have investigated whether other ECM molecules, such as syndecans, are involved in T(3) action. Thus, we analyzed the expression of syndecans (1-4) by RT-PCR in astrocyte cultures from cerebellum, cortex, and hippocampus of newborn rats. Our results showed that syndecans (1-4) are expressed in astrocytes of cerebellum and cortex, whereas in hippocampus only syndecans 2 and 4 were detected. Semi-quantitative RT-PCR analysis revealed the reduced expression of syndecans 1, 2, and 4, and increased expression of syndecan 3 in hypothyroid cerebellum, when compared to the euthyroid tissue. Furthermore, we observed a reduced expression of syndecans 2 and 3 in T(3)-treated cerebellar astrocytes, when compared to control cultures. This balance of proteoglycans may be involved in T(3) action mediated by FGF(2) signaling, possibly affecting the formation of the trimeric signaling receptor complex composed by syndecan/FGF/FGF-receptor (FGFR), which is essential for FGFR dimerization, activation, and subsequent cell signaling.

Guanine derivatives (GD) have been shown to exert relevant extracellular effects as intercellular messengers, neuromodulators in the central nervous system, and trophic effects on astrocytes and neurons. Astrocytes have been pointed out as the major source of trophic factors in the nervous system, however, several trophic effects of astrocytic-released soluble factors are mediated through modulation of extracellular matrix (ECM) proteins. In this study, we investigated the effects of guanosine-5'-monophosphate (GMP) and guanosine (GUO) on the expression and organization of ECM proteins in cerebellar astrocytes. Moreover, to evaluate the effects of astrocytes pre-treated with GMP or GUO on cerebellar neurons we used a neuron-astrocyte coculture model. GMP or GUO alters laminin and fibronectin organization from a punctate to a fibrillar pattern, however, the expression levels of the ECM proteins were not altered. Guanine derivatives-induced alteration of ECM proteins organization is mediated by activation of mitogen activated protein kinases (MAPK), CA(2+)-calmodulin-dependent protein kinase II (CaMK-II), protein kinase C (PKC), and protein kinase A (PKA) pathways. Furthermore, astrocytes treated with GMP or GUO promoted an increased number of cerebellar neurons in coculture, without altering the neuritogenesis pattern. No proliferation of neurons or astrocytes was observed due to GMP or GUO treatment. Our results show that guanine derivatives promote a reorganization of the ECM proteins produced by astrocytes, which might be responsible for a better interaction with neurons in cocultures.

Pb(II) is a neurotoxic pollutant that produces permanent cognitive deficits in children. Pb(II) can modulate cell signaling pathways and cell viability in a variety of cell types. However, these actions are not well demonstrated on glial cells, which represent an important target for metals into the central nervous system. The present work was undertaken to determine the ability of Pb(II) in modulating the activity of mitogen activated protein kinases (MAPKs) in cultures of C6 rat glioma cells, a useful functional model for the study of astrocytes. Additionally, cell viability was analyzed by measurement of MTT reduction. Cells were exposed to lead acetate 0.1, 1, 10 microM for 24 and 48 h. MAPKs activation - in particular ERK1/2, p38(MAPK) and JNK1/2 - were analyzed by western blotting. Results showed that 10 microM Pb(II) treatment for 24 h caused a discrete stimulation of p38(MAPK) phosphorylation. However, 1 and 10 microM Pb(II) treatment for 48 h provoked a significant stimulation in the phosphorylation state of p38(MAPK) and JNK1/2. The phosphorylation state of ERK1/2 was not modified by any Pb(II) treatment. Moreover, data indicate that at 48 h treatment even 1 microM Pb(II) can be cytotoxic, causing impairment on cell viability. Therefore, depending on a long incubation period, a significant concomitant activation of p38(MAPK) and JNK1/2 by Pb(II) took place in parallel with the impairment of C6 glioma cells viability.

The stimulatory input of extracellular matrix (ECM) components has been implicated in the invasive properties of glioma cells. It has been demonstrated that undersulfation of glycosaminoglycans (GAGs) promoted by sodium chlorate (SC) treatment reduces C6 glioma cell proliferation and adhesion to ECM molecules, in-vitro. In the present study, the authors investigated the involvement of GAG undersulfation in glioma cell growth in the brain parenchyma. The in-vitro treatment of C6 cells with SC and subsequent intracerebral inoculation in vehicle containing SC resulted in a reduced proportion of animals bearing glioma and a reduced tumor mass diameter. It also promoted longer animal survival. Intracerebral inoculation of SC-treated C6 cells in vehicle without SC or the SC treatment after intracerebral implantation of untreated C6 cells did not result in any reduction of tumor growth. Alterations in clinical, hematological and behavioral parameters in the open field were observed near the point of death when tumors presented a greater size. The results suggest an important role of GAGs in glioma growth which possibly affects cell proliferation and/or interactions with the normal tissue environment.

Astrocyte cells clearly play a role in neural development, but nowadays their total action is seen as a far wider one. Recent findings consider them as stem cells, involved in the control of most facets of functional neural networks. Astrocytes play a central role in thyroid hormone metabolism in the brain, being the principal transporters of thyroxine from the blood, responsible for its conversion to 3,5,3'-triiodothyronine and hence supplying the neural tissues with the biologically active form of the hormone. Specific thyroid hormone transporters play an essential role in this regulatory system. The presence of thyroid hormone receptors has been demonstrated in cultured astrocytes. Furthermore, thyroid hormone regulates several aspects of astrocyte differentiation and maturation, including the production of extracellular matrix proteins and growth factors, and thus controls neuronal growth and neuritogenesis. Therefore, astrocytes are currently suggested as important mediators of thyroid hormone in neuronal development.

Purpose: The aim of this work was to evaluate the pulmonary antimetastatic activity and the systemic toxicity of camptothecin-loaded microspheres., Methods: PCL microspheres containing camptothecin (CPT) were prepared by the emulsion solvent/evaporation method and characterized according to their encapsulation efficiency, particle size, morphology, and drug release. The ability of CPT to inhibit the lung metastasis was verified using an experimental mouse model intravenously injected with metastatic B16- F10 melanoma cells. The microspheres and the free drug were given intraperitoneally at a dose of 7 mg/kg at intervals of three or five days for 24 days. The systemic toxicity of CPT was evaluated by weight measurements, survival and hemograms of the animals., Results: The encapsulation efficiency was nearly 80%. The drug release was complete after 72 hours, but the burst effect increased from 7% to 35% with the increase in CPT content in the particles. It was observed during the in vivo essays that all groups treated with CPT had a decrease of nearly 70% in the number of lung metastases. However, systemic toxicity was verified in animals that received the free drug., Conclusion: Camptothecin-loaded microspheres demonstrated similar therapeutic efficacy when compared to those of the free drug, but the toxicity was significantly reduced.

Background: Adhesion to extracellular matrix (ECM) components has been implicated in the proliferative and invasive properties of tumor cells. We investigated the ability of C6 glioma cells to attach to ECM components in vitro and described the regulatory role of glycosaminoglycans (GAGs) on their adhesion to the substrate, proliferation and migration., Results: ECM proteins (type IV collagen, laminin and fibronectin) stimulate rat C6 glioma cell line adhesion in vitro, in a dose-dependent manner. The higher adhesion values were achieved with type IV collagen. Exogenous heparin or chondroitin sulfate impaired, in a dose-dependent manner the attachment of C6 glioma cell line to laminin and fibronectin, but not to type IV collagen. Dextran sulfate did not affect C6 adhesion to any ECM protein analyzed, indicating a specific role of GAGs in mediating glioma adhesion to laminin and fibronectin. GAGs and dextran sulfate did not induce C6 glioma detachment from any tested substrate suggesting specific effect in the initial step of cell adhesion. Furthermore, heparin and chondroitin sulfate impaired C6 cells proliferation on fibronectin, but not on type IV collagen or laminin. In contrast, both GAGs stimulate the glioma migration on laminin without effect on type IV collagen or fibronectin., Conclusion: The results suggest that GAGs and proteoglycans regulate glioma cell adhesion to ECM proteins in specific manner leading to cell proliferation or cell migration, according to the ECM composition, thus modulating tumor cell properties.

Thyroid hormone deficiency during the critical period of neural differentiation produces permanent and severe alterations in the morphology and function of the nervous system leading to cretinism. Perinatal hypothyroidism results in permanent alterations of hippocampal synaptic functions in adult rats consequently causing learning and memory impairment. Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that regulate essential cellular activities ranging from gene expression, mitosis, programmed cell death to plasticity and memory formation, but their involvement in perinatal hypothyroidism is not determined. The present work was designed to investigate MAPKs phosphorylation in hippocampus of congenital neonatal hypothyroid rats. Congenital hypothyroidism promotes an increase in extracellular signal-regulated kinases 1/2 (ERK 1/2) phosphorylation (+50%) and a decrease in p38(MAPK) phosphorylation (-50%) without changing in Jun N-terminal kinases (JNK) phosphorylation. Therefore, the congenital hypothyroidism model disturbs ERK 1/2 and p38(MAPK) phosphorylation pathways causing an important molecular alteration in the hippocampus. This event might be related, at least partially, to the deficits in hippocampal development and cognitive functions due neonatal congenital hypothyroidism.