RESUMO
The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.
Assuntos
Diferenciação Celular/fisiologia , Células Germinativas/citologia , Células Híbridas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Proliferação de Células , Células Cultivadas , Feminino , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/fisiologiaRESUMO
BACKGROUND: The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. METHODS: Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. RESULTS AND DISCUSSION: We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. CONCLUSION: Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.
Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Doenças do Cão/terapia , Distrofia Muscular Animal/terapia , Transplante de Células-Tronco , Dente Decíduo/citologia , Animais , Movimento Celular , Células Cultivadas , Criança , Pré-Escolar , Polpa Dentária/transplante , Doenças do Cão/sangue , Doenças do Cão/genética , Doenças do Cão/fisiopatologia , Cães , Distrofina/metabolismo , Imunofluorescência , Genótipo , Humanos , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/patologia , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologia , Dente Decíduo/transplanteRESUMO
Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 in after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Lisossomos/efeitos dos fármacos , Animais , Células CHO , Caspase 3 , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Cisteína Endopeptidases/metabolismo , Citosol , Lisossomos/metabolismo , Camundongos , Permeabilidade/efeitos dos fármacosRESUMO
Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.
Assuntos
Clonagem de Organismos/métodos , Células-Tronco Embrionárias/citologia , Oócitos/citologia , Espermatozoides/citologia , Animais , Diferenciação Celular , Técnicas Genéticas , Hormônios/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Oócitos/metabolismo , Tretinoína/metabolismoRESUMO
We report the isolation of a population of immature dental pulp stem cells (IDPSC), which express embryonic stem cell markers Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 as well as several other mesenchymal stem cell markers during at least 25 passages while maintaining the normal karyotype and the rate of expansion characteristic of stem cells. The expression of these markers was maintained in subclones obtained from these cells. Moreover, in vitrothese cells can be induced to undergo uniform differentiation into smooth and skeletal muscles, neurons, cartilage, and bone under chemically defined culture conditions. After in vivo transplantation of these cells into immunocompromised mice, they showed dense engraftment in various tissues. The relative ease of recovery and the expression profiles of various markers justify further exploration of IDPSC for clinical therapy.
Assuntos
Polpa Dentária/citologia , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Biomarcadores/análise , Técnicas de Cultura de Células , Diferenciação Celular , Divisão Celular , Criança , Pré-Escolar , Células Clonais , Meios de Cultura , Primers do DNA , Humanos , Imuno-Histoquímica , Cariotipagem , Fator 3 de Transcrição de Octâmero/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dente DecíduoRESUMO
Herein we report that crotamine, a small lysine- and cysteine-rich protein from the venom of the South American rattlesnake, can rapidly penetrate into different cell types and mouse blastocysts in vitro. In vivo crotamine strongly labels cells from mouse bone marrow and spleen and from peritoneal liquid, as shown by fluorescent confocal laser-scanning microscopy. Nuclear localization of crotamine was observed in both fixed and unfixed cells. In the cytoplasm, crotamine specifically associates with centrosomes and thus allows us to follow the process of centriole duplication and separation. In the nucleus, it binds to the chromosomes at S/G2 phase, when centrioles start dividing. Moreover, crotamine appears as a marker of actively proliferating cells, as shown by 5-BrdU cell-proliferation assay. Crotamine in the micromolar range proved nontoxic to any of the cell cultures tested and did not affect the pluripotency of ES cells or the development of mouse embryos.
Assuntos
Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/toxicidade , Crotalus , Animais , Biomarcadores/análise , Carbocianinas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Centríolos/metabolismo , Centrossomo/metabolismo , Cromossomos de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Embrião não Mamífero , Humanos , Camundongos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
Acute renal failure (ARF) is a common renal disease that can lead to high mortality. Recovery from ARF occurs with the replacement of necrotic tubular cells by functional tubular epithelial cells and the normalization of microvascular endothelial cell function in the peritubular capillaries. Conventional therapeutic techniques are often ineffective against ARF. Hence, stem cell therapies, which act through multiple trophic and regenerative mechanisms, are encouraging. We investigated the homing of human immature dental pulp stem cells (IDPSCs) after endovenous (EV) or intraperitoneal (IP) injection, in immunocompetent Wistar rats with ARF induced by intramuscular injection of glycerol, without the use of immunosuppression. The cells, which had been cryopreserved for 6 years, were CD105(+), CD73(+), CD44(+), and partly, STRO-1(+) and CD146(+), and presented unaltered mesoderm differentiation potential. The presence of these cells in the tubular region of the kidney and in the peritubular capillaries was demonstrated. These cells accelerate tubular epithelial cell regeneration through significant increase of Ki-67-immunoreactive cells in damaged kidney. Flow cytometry analysis confirmed that IDPSCs home to the kidneys (EV 34.10% and IP 33.25%); a lower percentage of cells was found in the liver (EV 19.05% and IP 9.10%), in the muscles (EV 6.30% and IP 1.35%), and in the lungs (EV 2.0% and IP 1.85%). After infusion into rat, these cells express pericyte markers, such as CD146(+), STRO-1(+), and vascular endothelial growth factor (VEGF(+)). We found that IDPSCs demonstrate renotropic and pericyte-like properties and contributed to restore renal tubule structure in an experimental rat ARF model.
RESUMO
Mesenchymal stem cells (MSCs) due to their self-renewal potential and differentiation capacity are useful for tissue regeneration. Immunomodulatory and trophic properties of MSCs were demonstrated suggesting their use as medicinal signaling cells able to positively change local environment in injured tissue. Equine endometrosis is a progressive degenerative disease responsible for glandular alterations and endometrial fibrosis which causes infertility in mares. More precisely, this disease is characterized by phenotypic changes in the expression pattern of selected endometrial proteins. Currently, no effective treatment is available for endometrosis. Herein, we aimed at the evaluation of expression pattern of these proteins after allogeneic equine adipose tissue-derived multipotent mesenchymal stem cells (eAT-MSCs) infusion as well as at testing the capacity of these cells to promote endometrial tissue remodeling in mares with endometrosis. eAT-MSC (2 × 10(7)/animal) were transplanted into mares' uterus and control animals received only placebo. Uterine biopsies were collected before (day 0) and after (days 7, 21 and 60) cells transplantation. Conventional histopathology as well as expression analysis of such proteins as laminin, vimentin, Ki-67-antigen, α-smooth muscle actin (α-SMA) and cytokeratin 18 (CK18) have been performed before and after eAT-MSCs transplantation. We demonstrated that eAT-MSCs induced early (at day 7) remodeling of endometrial tissue microenvironment through changes observed in intra cellular and intra glandular localization of aforementioned proteins. We demonstrated that eAT-MSCs were able to positively modulate the expression pattern of studied secretory proteins as well as, to promote the induction of glandular epithelial cells proliferation suggesting local benefits to committed endometrial tissue environment after eAT-MSCs transplantation.
Assuntos
Endometriose/genética , Endometriose/patologia , Células-Tronco Mesenquimais/metabolismo , Transcriptoma , Animais , Biomarcadores , Biópsia , Endometriose/terapia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Perfilação da Expressão Gênica , Cavalos , Transplante de Células-Tronco Mesenquimais , Mucosa/metabolismo , Mucosa/patologiaRESUMO
Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources) has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP) with a more pronounced antifungal activity. In contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. In vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. The structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.
Assuntos
Peptídeos Penetradores de Células , Venenos de Crotalídeos , Sequência de Aminoácidos , Animais , Anti-Infecciosos , Antineoplásicos , Linhagem Celular , Crotalus , Humanos , Melanoma , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , América do SulRESUMO
Dental pulp (DP) can be extracted from child's primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2-5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3-4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2'-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their "stemness".
Assuntos
Separação Celular/métodos , Polpa Dentária/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Biomarcadores/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Criança , Condrogênese/efeitos dos fármacos , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Fatores de TempoRESUMO
OBJECTIVES: Selective anticancer cell activity for both cell-penetrating and cationic antimicrobial peptides has previously been reported. As crotamine possesses activities similar to both of these, this study investigates crotamine's anticancer toxicity in vitro and in vivo. RESEARCH DESIGN AND METHODS: In vitro cancer cell viability was evaluated after treatment with 1 and 5 µg/ml of crotamine. In vivo crotamine cytotoxic effects in C57Bl/6J mice bearing B16-F10 primary cutaneous melanoma were tested, with two groups each containing 35 mice. The crotamine-treated group received 1 µg/day of crotamine per animal, subcutaneously which was well tolerated; the untreated group received a placebo. RESULTS: Crotamine at 5 µg/ml was lethal to B16-F10, Mia PaCa-2 and SK-Mel-28 cells and inoffensive to normal cells. In vivo crotamine treatment over 21 days significantly delayed tumor implantation, inhibited tumor growth and prolonged the lifespan of the mice. Mice in the crotamine-treated group survived at significantly higher rates (n = 30/35) than those in the untreated group (n = 7/35) (significance calculated with the Kaplan-Meier estimator). The average tumor weight in the untreated group was 4.60 g but was only about 0.27 g in the crotamine-treated mice, if detectable. CONCLUSIONS: These data warrant further exploration of crotamine as a tumor inhibition compound.
Assuntos
Antineoplásicos/uso terapêutico , Venenos de Crotalídeos/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transplante de NeoplasiasRESUMO
IMPORTANCE OF THE FIELD: Molecules isolated from animals, insects, plants or microorganisms can provide prototypes for design of biopharmaceutical products. Some venom toxins and their derivatives are used in medicine, while others provide templates for development of new drugs. AREAS COVERED IN THIS REVIEW: The mild toxin, crotamine, a small basic low-molecular-weight polypeptide purified from the venom of a South American rattlesnake, Crotalus durissus terrificus. Crotamine was discovered more than 50 years ago and only in the past six years has its exceptional biological versatility been demonstrated. Particularly, its cell-penetrating ability, which allows crotamine to cross cell membranes and to accumulate in the nucleus; its use for intracellular vesicle tracking and as a cell cycle marker and its capability for delivering DNA into replicating mammalian cells. Both antimicrobial action and potential selective antitumor activity of crotamine have also been found. WHAT THE READER WILL GAIN: Multidisciplinary approaches and pathways of discovery placed crotamine in a rare category of versatile biomolecules, in which concentration, molecular target preference, structural ancestry and specificity toward biological membranes play an integral role. TAKE HOME MESSAGE: Crotamine is a druggable peptide with high potential for use as an imaging agent for detecting dividing cells, for intracellular delivery of hydrophilic biomolecules, and as an alternative chemotherapeutic compound against aggressive types of cancer.
Assuntos
Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Biomarcadores/química , Biomarcadores/metabolismo , Ciclo Celular/fisiologia , Membrana Celular/metabolismo , Crotalus , Humanos , América do SulRESUMO
Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.
Assuntos
Células-Tronco Embrionárias/citologia , Neurônios/citologia , Esferoides Celulares/citologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Corpos Embrioides/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Neurônios/fisiologia , Esferoides Celulares/metabolismo , Esferoides Celulares/fisiologia , Fatores de TempoRESUMO
Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19-24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.
Assuntos
Modelos Animais de Doenças , Expressão Gênica , Síndrome de Marfan/genética , Proteínas dos Microfilamentos/genética , Animais , Sequência de Bases , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Genótipo , Humanos , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.
Assuntos
Polpa Dentária/transplante , Necrose da Cabeça do Fêmur/cirurgia , Transplante de Células-Tronco Mesenquimais , Animais , Materiais Biocompatíveis/efeitos adversos , Modelos Animais de Doenças , Humanos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Distribuição Aleatória , Ovinos , Transplante HeterólogoRESUMO
PURPOSE: To determine the outcome of the use of a tissue-engineered cell sheet composed of human undifferentiated immature dental pulp stem cells (hIDPSC) for ocular surface reconstruction in an animal model of total limbal stem cell deficiency (LSCD). METHODS: LSCD was induced by the application of 0.5 M NaOH to the right eye of rabbits for 25 seconds (mild chemical burn [MCB]) and for 45 seconds (severe chemical burn [SCB]). After 1 month, a superficial keratectomy was performed to remove the fibrovascular pannus that covered the animals' burned corneas. A tissue-engineered hIDPSC sheet was transplanted onto the corneal bed and then covered with deepithelialized human amniotic membrane (AM). In the respective control groups, the denuded cornea was covered with AM only. After 3 months, a detailed analysis of the rabbit eyes was performed with regard to clinical aspect, histology, electron microscopy, and immunohistochemistry. RESULTS: Corneal transparency of the rabbit eyes that underwent hIDPSC transplantation was improved throughout the follow-up, while the control corneas developed total conjunctivalization and opacification. Rabbits from the MCB group showed clearer corneas with less neovascularization. The clinical data were confirmed by histologic analysis that showed healthy uniform corneal epithelium, especially in the MCB group. The presence of hIDPSC was detected using an anti-hIDPSC antibody. The corneal tissue also showed positive immunostaining with anti-human antibodies. In the control corneas, none of these antigens were detected. CONCLUSIONS: Overall, these data showed that transplantation of a tissue-engineered hIDPSC sheet was successful for the reconstruction of corneal epithelium in an animal model of LSCD.
Assuntos
Doenças da Córnea/cirurgia , Polpa Dentária/citologia , Limbo da Córnea/cirurgia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Procedimentos Cirúrgicos Oftalmológicos , Engenharia Tecidual , Animais , Queimaduras Químicas/patologia , Queimaduras Químicas/cirurgia , Técnicas de Cultura de Células , Modelos Animais de Doenças , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/patologia , Queimaduras Oculares/cirurgia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Limbo da Córnea/ultraestrutura , Microscopia Eletrônica de Transmissão , Coelhos , Procedimentos de Cirurgia Plástica , Transplante HeterólogoRESUMO
In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.
Assuntos
Tecido Adiposo/citologia , Criopreservação , Células-Tronco/citologia , Adipogenia , Animais , Proliferação de Células , Separação Celular , Forma Celular , Condrogênese , Cavalos , OsteogêneseRESUMO
The main aim of this study is to evaluate the capacity of human dental pulp stem cells (hDPSC), isolated from deciduous teeth, to reconstruct large-sized cranial bone defects in nonimmunosuppressed (NIS) rats. To our knowledge, these cells were not used before in similar experiments. We performed two symmetric full-thickness cranial defects (5 x 8 mm) on each parietal region of eight NIS rats. In six of them, the left side was supplied with collagen membrane only and the right side (RS) with collagen membrane and hDPSC. In two rats, the RS had collagen membrane only and nothing was added at the left side (controls). Cells were used after in vitro characterization as mesenchymal cells. Animals were euthanized at 7, 20, 30, 60, and 120 days postoperatively and cranial tissue samples were taken from the defects for histologic analysis. Analysis of the presence of human cells in the new bone was confirmed by molecular analysis. The hDPSC lineage was positive for the four mesenchymal cell markers tested and showed osteogenic, adipogenic, and myogenic in vitro differentiation. We observed bone formation 1 month after surgery in both sides, but a more mature bone was present in the RS. Human DNA was polymerase chain reaction-amplified only at the RS, indicating that this new bone had human cells. The use of hDPSC in NIS rats did not cause any graft rejection. Our findings suggest that hDPSC is an additional cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.
Assuntos
Doenças Ósseas/cirurgia , Polpa Dentária/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Osso Parietal/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Adipogenia/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Células Cultivadas , Criança , Colágeno , Craniotomia , Modelos Animais de Doenças , Fibroblastos/citologia , Humanos , Masculino , Membranas Artificiais , Células-Tronco Mesenquimais/citologia , Desenvolvimento Muscular/fisiologia , Osteogênese/fisiologia , Ratos , Ratos Wistar , Dente Decíduo/citologiaRESUMO
In order to increase the amount of available bone where dental implants must be placed, the present study has associated platelet-rich plasma (PRP) and mononuclear cells (MNCs) from bone marrow aspirate and bone scaffold (BS) in 32 patients aged between 45 and 75 years old. The MNC attainment and the adherence to the BS were confirmed through histology, cell culture, and scanning electron microscopy. The clinical results, analyzed by computed tomography, have showed that the scaffolds were well integrated and adapted to the cortical bone. We can conclude that the process of healing observed in the patients was due to the presence of mesenchymal stem cell in MNC fraction in the bone grafts.
Assuntos
Regeneração Óssea , Transplante Ósseo/métodos , Implantes Dentários , Transplante de Células-Tronco Hematopoéticas , Plasma Rico em Plaquetas/fisiologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucócitos Mononucleares/transplante , Leucócitos Mononucleares/ultraestrutura , Masculino , Maxila/diagnóstico por imagem , Maxila/cirurgia , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Osseointegração , Engenharia Tecidual , Tomografia Computadorizada por Raios X , Transplante HomólogoRESUMO
Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.