RESUMO
UNLABELLED: : Stem cell therapy has emerged as a new strategy for treatment of ischemic heart disease. Although umbilical cord-derived mesenchymal stromal cells (UC-MSCs) have been used preferentially in the acute ischemia model, data for the chronic ischemia model are lacking. In this study, we investigated the effect of UC-MSCs originated from Wharton's jelly in the treatment of chronic myocardial ischemia in a porcine model induced by ameroid constrictor. Four weeks after ameroid constrictor placement, the surviving animals were divided randomly into two groups to undergo saline injection (n = 6) or UC-MSC transplantation (n = 6) through the left main coronary artery. Two additional intravenous administrations of UC-MSCs were performed in the following 2 weeks to enhance therapeutic effect. Cardiac function and perfusion were examined just before and at 4 weeks after intracoronary transplantation. The results showed that pigs with UC-MSC transplantation exhibited significantly greater left ventricular ejection fraction compared with control animals (61.3% ± 1.3% vs. 50.3% ± 2.0%, p < .05). The systolic thickening fraction in the infarcted left ventricular wall was also improved (41.2% ± 3.3% vs. 46.2% ± 2.3%, p < .01). Additionally, the administration of UC-MSCs promoted collateral development and myocardial perfusion. The indices of fibrosis and apoptosis were also significantly reduced. Immunofluorescence staining showed clusters of CM-DiI-labeled cells in the border zone, some of which expressed von Willebrand factor. These results suggest that UC-MSC treatment improves left ventricular function, perfusion, and remodeling in a porcine model with chronic myocardial ischemia. SIGNIFICANCE: Ischemic heart disease is the leading cause of death worldwide. Many patients with chronic myocardial ischemia are not suitable for surgery and have no effective drug treatment; they are called "no-option" patients. This study finds that umbilical cord-derived mesenchymal stromal cells transplanted by intracoronary delivery combined with two intravenous administrations was safe and could significantly improve left ventricular function, perfusion, and remodeling in a large-animal model of chronic myocardial ischemia, which provides a new choice for the no-option patients. In addition, this study used clinical-grade mesenchymal stem cells with delivery and assessment methods commonly used clinically to facilitate further clinical transformation.
Assuntos
Circulação Coronária , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Infarto do Miocárdio/cirurgia , Cordão Umbilical/citologia , Função Ventricular Esquerda , Remodelação Ventricular , Geleia de Wharton/citologia , Proteínas Angiogênicas/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Circulação Colateral , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Células-Tronco Mesenquimais/metabolismo , Contração Miocárdica , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Neovascularização Fisiológica , Fenótipo , Recuperação de Função Fisiológica , Volume Sistólico , Suínos , Fatores de Tempo , Fator de von Willebrand/metabolismoRESUMO
Zero-dimensional fullerenes can modulate the biological behavior of a variety of cell lines. However, the effects and molecular mechanisms of proliferation and cardiomyogenic differentiation in brown adipose-derived stem cells (BADSCs) are still unclear. In this study, we report the initial biological effects of fullerene-C60 on BADSCs at different concentrations. Results suggest that fullerene-C60 has no cytotoxic effects on BADSCs even at a concentration of 100 µg/mL. Fullerene-C60 improves the MAPK expression level and stem cell survival, proliferation, and cardiomyogenesis. Further, we found that the fullerene-C60 modulates cardiomyogenic differentiation. Fullerene-C60 improves the expression of cardiomyocyte-specific proteins (cTnT and α-sarcomeric actinin). At elevated concentration, fullerene-C60 reduces the incidence of diminished spontaneous cardiac differentiation of BADSCs with time. At the genetic level, fullerene-C60 (5 µg/mL) also improves the expression of cTnT. In addition, fullerene-C60 promotes the formation of gap junction among cells. These findings have important implications for clinical application of fullerenes in the treatment of myocardial infarction.
Assuntos
Tecido Adiposo Marrom/citologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fulerenos/farmacologia , Coração/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco/citologia , Actinina/genética , Actinina/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Biomarcadores/análise , Western Blotting , Células Cultivadas , Fulerenos/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/fisiologia , Técnicas Imunoenzimáticas , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcômeros/genética , Sarcômeros/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Troponina T/genética , Troponina T/metabolismoRESUMO
Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the ß1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the ß1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.
Assuntos
Integrina beta1/metabolismo , Miócitos Cardíacos/citologia , Nanotubos de Carbono/química , Transdução de Sinais , Animais , Animais Recém-Nascidos , Cálcio/química , Adesão Celular , Sobrevivência Celular , Colágeno/química , Conexina 43/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Junções Comunicantes , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/efeitos dos fármacos , Nanomedicina/métodos , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Alicerces Teciduais/químicaRESUMO
Poly (N-isopropylacrylamide) (PNIPAAm) hydrogel was a widely used carrier in therapeutic agent delivery. However, its bioactivities for encapsulated cells were not satisfactory. In the study, we aimed to determine whether modification with single-wall carbon nanotubes (SWCNTs) could improve the bioactivitis, especially supportive adhesion of PNIPAAm to encapsulated cells and favor their efficacy in myocardial repair. A thermosensitive SWCNTs-modified PNIPAAm hydrogel (PNIPAAm/SWCNTs) were prepared by incorporating the SWCNTs into base PNIPAAm hydrogel. The bioactivities of the resulted hydrogel to brown adipose-derived stem cells (BASCs) were evaluated and compared with the base PNIPAAm hydrpgel in vitro. Then, the PNIPAAm-containing hydrogel was used as carrier for imtromyocardial delivery of BASCs in rats with myocardial infarction. The efficacy of PNIPAAm/SWCNTs hydrogel in stem cell-based myocardial repair was systematically evaluated. In vitro study showed that the PNIPAAm/SWCNTs hydrogel demonstrated significantly higher bioactivities to encapsulated BASCs compared with onefold PNIPAAm hydrogel, including promoting cell adhesion and proliferation. When used as carrier for intramyocardial delivery of BASCs after myocardial infarction, the PNIPAAm/SWCNTs hydrogel significantly enhanced the engraftment of seeding cells in infarct myocardium and augmented their therapeutic efficacies in myocardial infarction (MI). The data provided a supportive evidence for the myocardial application of the SWCNTs-modified hydrogel and offered a new perspective in development or improvement of cardiac tissue engineering scaffold.
Assuntos
Resinas Acrílicas/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Infarto do Miocárdio/terapia , Miocárdio/citologia , Nanotubos de Carbono/química , Transplante de Células-Tronco/métodos , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/química , Adesão Celular , Células Cultivadas , Masculino , Nanotubos de Carbono/ultraestrutura , Ratos , Ratos Sprague-Dawley , TemperaturaRESUMO
Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.
Assuntos
Colágeno/química , Laminina/química , Miócitos Cardíacos/citologia , Proteoglicanas/química , Engenharia Tecidual , Alicerces Teciduais , Junções Aderentes/metabolismo , Junções Aderentes/ultraestrutura , Animais , Biomarcadores/metabolismo , Caderinas/metabolismo , Microambiente Celular , Conexina 43/metabolismo , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Combinação de Medicamentos , Junções Comunicantes/metabolismo , Junções Comunicantes/ultraestrutura , Coração/anatomia & histologia , Coração/fisiologia , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/metabolismo , Placofilinas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To explore the inhibitory effects of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on the proliferation of peripheral blood mononuclear cells (PBMC) from spondyloarthritis (SpA) patients. METHODS: A total of 12 SpA patients at Chinese PLA General Hospital were recruited from May 2012 to October 2012. Information on demographic characteristics, disease and functional activity was collected. Isolated PBMC were stimulated by phytohemagglutinin (PHA, 1 µg/ml) in the presence or absence of hUCMSC.The proliferation of hUCMSC was suppressed by irradiation with Co60 (30 Gy) before co-culturing with PBMC. The proliferation of PBMC was determined by Cell Counting Kit-8 (CCK-8). Cell cycle profiles of PBMC were analyzed by flow cytometry. The association of inhibitory effect of hUCMSC with the disease and functional activity of SpA patients was examined. RESULTS: After coculturing with hUCMSC by cell-to-cell contact for 5 days, the proliferation of PBMC stimulated by PHA (1 µg/ml) was significantly inhibited by hUCMSC in a dose-dependent manner.The inhibition rate of the proliferation of PBMC cocultured with hUCMSC by cell-to-cell contact was higher than that by Transwell culture (57% ± 17% vs 32% ± 12%, P < 0.01). Compared to PBMC cultured alone, a larger number of PBMC cocultured with hUCMSC were in phase G1 (86% ± 3% vs 68% ± 5%, P < 0.01) while a lower number of cells in phases S and G2 (8% ± 3% vs 26% ± 5%, P < 0.01). No association was found between the inhibitory effect of hUCMSC and the disease and functional activity. CONCLUSION: The proliferation of PBMC from SpA patients may be inhibited by hUCMSC. And hUCMSC have therapeutic potentials for SpA patients.
Assuntos
Proliferação de Células , Leucócitos Mononucleares/citologia , Células-Tronco Mesenquimais/citologia , Espondilartrite/patologia , Adulto , Ciclo Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Masculino , Cordão Umbilical/citologiaRESUMO
Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC-derivates), iPSC-derivated cardiomyocyte (iPSC-CMs) were subcutaneously injected into the back of nude mice. Non-invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC-derivates and iPSC-CMs survived in receipts. Both iPSCs and iPSC-derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC-CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC-derivates transplanted mice, but not in iPSC-CM transplanted ones. In vitro study showed the long-term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC-like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage-specific differentiation is necessary prior to therapeutic use of iPSCs.
Assuntos
Transformação Celular Neoplásica/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Teratoma/patologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Transformação Celular Neoplásica/metabolismo , Genes Reporter , Sobrevivência de Enxerto , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Injeções Subcutâneas , Luciferases de Vaga-Lume , Camundongos , Camundongos Nus , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Teratoma/metabolismoRESUMO
In this study, the inhibitory effect of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on interleukin-17 (IL-17) production in peripheral blood T cells from patients with spondyloarthritis (SpA) were investigated, in order to explore the therapeutic potential of hUCMSC in the SpA. Peripheral blood mononuclear cells (PBMNC) were isolated from patients with SpA (n = 12) and healthy subjects (n = 6). PBMNC were cultured in vitro with hUCMSC or alone. The expression of IL-17 in CD4(+) T cells or γ/δ T cells were determined in each subject group by flow cytometry. IL-17 concentrations in PBMNC culture supernatants were measured by ELISA. The results indicated that the proportion of IL-17-producing CD4(+) T cells and IL-17-producing γ/δ T cells of SpA patients were 4.5 folds and 5 folds of healthy controls [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (0.75 ± 0.25)%, P < 0.01; CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.06 ± 0.02)%, P < 0.01]. After co-culture of PBMNC in patients with hUCMSC, the increased proportions of CD3(+)CD4(+)IL-17(+) cells and CD3(+)γδTCR(+)IL-17(+) cells in SpA patients were inhibited significantly by hUCMSC [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (1.81 ± 0.59)% (P < 0.01); CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.16 ± 0.06)% (P < 0.01]. In response to phytohemagglutinin (PHA, 1 µg/ml), PBMNC from SpA patients secreted more IL-17 than that from healthy control [(573.95 ± 171.68) pg/ml vs (115.53 ± 40.41) pg/ml (P < 0.01)]. In the presence of hUCMSC, PBMNC of SpA patients produced less amount of IL-17 [(573.95 ± 171.68) pg/ml vs (443.20 ± 147.94) pg/ml, (P < 0.01)]. It is concluded that the IL-17 production in peripheral blood T cells from SpA patients can be inhibited by hUCMSC, which have therapeutic potential for SpA.
Assuntos
Interleucina-17/metabolismo , Células-Tronco Mesenquimais , Espondilartrite/sangue , Linfócitos T/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Contagem de Linfócitos , Espondilartrite/metabolismo , Espondilartrite/terapia , Cordão Umbilical/citologiaRESUMO
Telocyte (TC) as a special stromal cell exists in mammary gland and might play an important role in the balance of epithelium-stroma of mammary gland. Considering that different types of breast interstitial cells influence the development and progression of breast cancer, TCs may have its distinct role in this process. We here studied the roles of TCs in the self-assembly of reconstituted breast cancer tissue. We co-cultured primary isolated TCs and other breast stromal cells with breast cancer EMT-6 cells in collagen/Matrigel scaffolds to reconstitute breast cancer tissue in vitro. Using histology methods, we investigated the immunohistochemical characteristics and potential functions of TCs in reconstituted breast cancer tissue. TCs in primary mammary gland stromal cells with long and thin overlapping cytoplasmic processes, expressed c-kit/CD117, CD34 and vimentin in reconstitute breast cancer tissue. The transmission electron microscopy showed that the telocyte-like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells through membrane-to-membrane contact. Compared with cancer tissue sheets of EMT-6 alone, PCNA proliferation index analysis and TUNEL assay showed that TCs and other breast stromal cells facilitated the formation of typical nest structure, promoted the proliferation of breast cancer cells, and inhibited their apoptosis. In conclusion, we successfully reconstituted breast cancer tissue in vitro, and it seems to be attractive that TCs had potential functions in self-assembly of EMT-6/stromal cells reconstituted breast cancer tissue.
Assuntos
Comunicação Celular , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Colágeno , Combinação de Medicamentos , Feminino , Expressão Gênica , Laminina , Glândulas Mamárias Animais/fisiologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Proteoglicanas , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Células Estromais/patologia , Células Estromais/fisiologia , Técnicas de Cultura de Tecidos , Vimentina/genética , Vimentina/metabolismoRESUMO
This study was purposed to optimize the culture conditions of the human amniotic epithelium cells (hAEC) in vitro, and detect the expression of hAEC pluripotent markers. Amnion tissues were separated from the underlying chorion through the spongy layer immediately after elective cesarean section of healthy pregnancy women at term. After the subsequent exposure to trypsin digestion, hAEC were cultured in DMEM with different supplements. The growth and proliferation potential of hAEC was evaluated, and the expression of cultured hAEC pluripotent markers was detected by using flow cytometry and immunohistochemistry methods. The results indicated that when being cultured in the mediums similar to that of embryonic stem cell culture supplemented with 10 ng/ml EGF, the hAEC grew better and the time for passage was shortened. In addition, compared to other culture conditions, under this condition, the cells could be passaged up to 5 times as much without obvious morphological changes, and the pluripotent marker SSEA-4 was detected in the cultured cells by flow cytometry. Meanwhile, the detection of immunofluorescence showed the expression of vimentin in cultured hAEC was strengthened as compared with primary cells. It is concluded that the culture condition similar to that for embryonic stem cells supplemented with EGF facilitates the proliferation and passage of hAEC in vitro.
Assuntos
Âmnio/citologia , Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Células-Tronco/metabolismo , Âmnio/metabolismo , Diferenciação Celular , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Humanos , Gravidez , Células-Tronco/citologiaRESUMO
The concept of regenerating diseased myocardium by implanting engineered heart tissue (EHT) is intriguing. Yet it was limited by immune rejection and difficulties to be generated at a size with contractile properties. Somatic cell nuclear transfer is proposed as a practical strategy for generating autologous histocompatible stem (nuclear transferred embryonic stem [NT-ES]) cells to treat diseases. Nevertheless, it is controversial as NT-ES cells may pose risks in their therapeutic application. EHT from NT-ES cell-derived cardiomyocytes was generated through a series of improved techniques in a self-made mould to keep the EHTs from contraction and provide static stretch simultaneously. After 7 days of static and mechanical stretching, respectively, the EHTs were implanted to the infarcted rat heart. Four weeks after transplantation, the suitability of EHT in heart muscle repair after myocardial infarction was evaluated by histological examination, echocardiography and multielectrode array measurement. The results showed that large (thickness/diameter, 2-4 mm/10 mm) spontaneously contracting EHTs was generated successfully. The EHTs, which were derived from NT-ES cells, inte grated and electrically coupled to host myocardium and exerted beneficial effects on the left ventricular function of infarcted rat heart. No teratoma formation was observed in the rat heart implanted with EHTs for 4 weeks. NT-ES cells can be used as a source of seeding cells for cardiac tissue engineering. Large contractile EHT grafts can be constructed in vitro with the ability to survive after implantation and improve myocardial performance of infarcted rat hearts.
Assuntos
Células-Tronco Embrionárias/citologia , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/fisiologia , Regeneração , Engenharia Tecidual/métodos , Transplante de Tecidos , Animais , Coração/fisiologia , Camundongos , Contração Miocárdica , Técnicas de Transferência Nuclear , Ratos , Transplante AutólogoRESUMO
The transplantation of embryonic stem cells could improve cardiac function but was limited by immune rejection as well as low cell retention and survival within the ischemic tissues. The somatic cell nuclear transfer (SCNT) is practical to generate autologous histocompatible stem (nuclear-transferred embryonic stem [NTES]) cells for diseases, but NTES may be arguably unsafe for therapeutic application. The temperature-responsive chitosan hydrogel is a suitable matrix in cell transplantation. As the scaffold, chitosan hydrogel was coinjected with NTES cells into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were performed to determine the structure and functional consequences of transplantation. The myocardial performance in SCNT- and fertilization-derived mouse ES cell transplantation with chitosan hydrogel was also compared. The results showed that both the 24-h cell retention and 4-week graft size were significantly greater in the NTES + chitosan group than that of NTES + phosphate-buffered saline (PBS) group (p < 0.01). The NTES cells might differentiate into cardiomyocytes in vivo. The heart function improved significantly in the chitosan + NTES group (fractional shortening: 28.7% +/- 2.8%) compared with that of PBS + NTES group (fractional shortening: 25.2% +/- 2.9%) at 4 weeks after transplantation (p < 0.01). In addition, the arteriole/venule densities within the infarcted area improved significantly in the chitosan + NTES group (280 +/- 17/mm(2)) compared with that of PBS + NTES group (234 +/- 16/mm(2)) at 4 weeks after transplantation (p < 0.01). There was no difference in the myocardial performance in SCNT- and fertilization-derived mouse ES cell transplantation with chitosan hydrogel. The NTES cells with chitosan hydrogel have been proved to possess therapeutic potential to improve the function of infarcted heart. Thus the method of in situ injectable tissue engineering is promising clinically.
Assuntos
Células-Tronco Embrionárias/transplante , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Engenharia Tecidual/métodos , Animais , Sequência de Bases , Técnicas de Cultura de Células , Diferenciação Celular , Quitosana , Primers do DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Fertilização , Hidrogéis , Camundongos , Contração Miocárdica , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Transferência Nuclear , Ratos , Ratos Sprague-Dawley , Temperatura , Alicerces Teciduais , UltrassonografiaRESUMO
OBJECTIVE: To culture rabbit endometrial cells by using sex steroids to provide adequate seeding cells for endometrium reconstruction and uterine tissue engineering. DESIGN: Prospective experimental study. SETTING: Beijing Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences. ANIMAL(S): New Zealand rabbit and Kunming white strain mice. INTERVENTION(S): Rabbits were primed with pregnant mare serum gonadotropin and hCG. Endometrial cells were cultured with E(2) and P(4) of different concentrations. The endometrium was reconstructed by using endometrial cells as seeding cells and collagen-basement membrane matrix as scaffolds. MAIN OUTCOME MEASURE(S): Assay with 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, immunofluorescence staining, flow cytometric analysis, hematoxylin and eosin and immunohistochemical staining, and developmental rate of embryos. RESULT(S): The expression patterns of estrogen receptor and P receptor of rabbit endometrium were different before and after treatment with pregnant mare serum gonadotropin-hCG. One hundred nanomolar E(2) with 10 nmol/L P(4) facilitated the proliferation of epithelial cells whereas 100 nmol/L P(4) facilitated that of stromal cells. The epithelial cells could be stable if cultured for seven or eight passages. Cells in the epithelial layer of the reconstructed endometrium were cytokeratin positive. Some showed columnar morphology akin to the luminal epithelium in vivo. Reconstructed endometrium could improve the developmental rate and quality of one-cell mice embryos. CONCLUSION(S): Rabbit endometrial cells could be cultured with a long-standing proliferation capability by sex steroids and applied in uterine tissue engineering. Reconstructed endometrium with proliferated endometrial cells was akin to native endometrium in structure and function.
Assuntos
Proliferação de Células/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Endométrio/fisiologia , Hormônios Esteroides Gonadais/farmacologia , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Endométrio/citologia , Endométrio/metabolismo , Feminino , Camundongos , Técnicas de Cultura de Órgãos/métodos , Gravidez , Coelhos , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Alicerces Teciduais , Útero/citologia , Útero/fisiologiaRESUMO
OBJECTIVE: This study attempted to reconstruct engineered uterine tissues (EUTs) containing smooth muscle layer, akin to the normal uterine wall. METHODS: EUTs were reconstructed by seeding epithelial cells on top of the constructed stromal layer over smooth muscle layer. A self-made mold was used to keep the EUTs from contraction. At the same time, it provided static stretch to the EUTs. After 14 days of culture, the structure of the EUTs was analyzed histologically and immunohistochemically, or by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of integrin beta3 subunit, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and HOXA-10 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ability of the EUTs supporting the development of embryos was estimated by coculturing embryos on the EUTs. We also tried a new method to reconstruct EUTs by mixing epithelial cell and stromal cells (1:2) in collagen/Matrigel to form an endometrial layer and putting it on top of the smooth muscle layer. The self-assembling ability of the endometrial epithelial cells and stromal cells in the reconstructed EUTs was analyzed histologically and immunohistochemically. RESULTS: The results found that the constructed EUTs with the first and the second method showed three-layered structures. The epithelial layer, stromal layer, and smooth muscle layer were stained by cytokeratin 18, vimentin, and alpha-actin, respectively. TEM showed that the cells in the EUTs reconstructed by the first method were attached to each other by apical tight junctions and rivet-like desmosomes. SEM showed protruded pinopodes, microvilli, and cilium of epithelial cells. The RT-PCR analysis showed that integrin beta3 subunit, HB-EGF, and HOXA-10 were expressed in EUTs. The coculture system of EUTs improved the development rate and quality of murine embryo significantly in comparison with those of control Chatot Ziomek Bavister culture. In the EUTs reconstructed by the second method, the epithelial cells demonstrated self-assembling ability and formed epithelial cell layer on top of the stromal layer and glandular tube-like structures in the stromal layer. Columnar epithelial cells existed in some parts of the epithelial layer. CONCLUSION: We engineered EUTs containing smooth muscle layer by two methods. The reconstructed EUTs could support the development of embryos. The epithelial cells showed self-assembling ability in the EUTs.
Assuntos
Colágeno/metabolismo , Laminina/metabolismo , Músculo Liso/fisiologia , Proteoglicanas/metabolismo , Engenharia Tecidual , Alicerces Teciduais , Útero/fisiologia , Animais , Combinação de Medicamentos , Embrião de Mamíferos/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Camundongos , Músculo Liso/ultraestrutura , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Útero/citologia , Útero/ultraestruturaRESUMO
Transplantation of embryonic stem cells (ESCs) can improve cardiac function in treatment of myocardial infarction. The low rate of cell retention and survival within the ischemic tissues makes the application of cell transplantation techniques difficult. In this study, we used a temperature-responsive chitosan hydrogel (as scaffold) combined with ESCs to maintain viable cells in the infarcted tissue. Temperature-responsive chitosan hydrogel was prepared and injected into the infarcted heart wall of rat infarction models alone or together with mouse ESCs. The result showed that the 24-h cell retention and 4 week graft size of both groups was significantly greater than with a phosphate buffered saline control. After 4 weeks of implantation, heart function, wall thickness, and microvessel densities within the infarct area improved in the chitosan + ESC, chitosan, and ESC group more than the PBS control. Of the three groups, the chitosan + ESC performed best. Results of this study indicate that temperature-responsive chitosan hydrogel is an injectable scaffold that can be used to deliver stem cells to infarcted myocardium. It can also increase cell retention and graft size. Cardiac function is well preserved, too.
Assuntos
Quitosana/farmacologia , Células-Tronco Embrionárias/transplante , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Recuperação de Função Fisiológica/efeitos dos fármacos , Temperatura , Laranja de Acridina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Indóis/metabolismo , Injeções , Camundongos , Microvasos/citologia , Microvasos/efeitos dos fármacos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Compostos Orgânicos/metabolismo , Propídio/metabolismo , Ratos , Ratos Sprague-Dawley , UltrassonografiaRESUMO
Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.
Assuntos
Reatores Biológicos , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Técnicas de Transferência Nuclear , Animais , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Células-Tronco Embrionárias/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologiaRESUMO
Neuronal PC12 cells induced by nerve growth factor (NGF) have been considered to be postmitotic and lack the ability to divide. However, in this study, we not only detected DNA synthesis but also observed cell division in some morphologically differentiated neuronal PC12 cells bearing long neurites. More interestingly, in addition to the division of perikaryon, the neurites located on the division site of the cell membrane also divided into two parts and were allocated to the two daughter cells. These results demonstrate that the morphologically differentiated neuronal PC12 cells still retain the ability to divide. This is the first report that neuronal PC12 cells as well as their neurites can divide.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Replicação do DNA/fisiologia , Fator de Crescimento Neural/farmacologia , Neurônios/citologia , Animais , Replicação do DNA/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Células PC12 , RatosRESUMO
The purpose of this study has been to investigate the possible effects of the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs). Autologous bone marrow was aspirated from the iliac crest of male sheep. MSCs were purified, expanded, and labeled with the fluorescent dye PKH26. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The cells labeled with red fluorescent PKH26 in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Animais , Células da Medula Óssea/fisiologia , Técnicas de Cocultura , Colágeno Tipo II/metabolismo , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Varredura , Ovinos , Líquido Sinovial/fisiologia , Membrana Sinovial/fisiologia , Engenharia TecidualRESUMO
In order to explore if mature neurons derived from neural stem cells have the potentiality to divide, we utilized the chemical digestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured for about eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizing the method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At the same time, PCNA+NF-160 (or Chat, GABA, GAD) double label were used to investigate if the dividing-neurons were mature ones. After the neural stem cells were induced to differentiate in vitro for eight days, they possessed the shape and character of mature neurons. The differentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon,there were a large amount of dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF were added into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, the number of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive. Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. After PCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned results indicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions. Mature neurons still have the potential to divide, proliferate and self-renew.
Assuntos
Diferenciação Celular , Neurônios/citologia , Células-Tronco/citologia , Animais , Encéfalo/citologia , Divisão Celular , Separação Celular , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fotografação/métodos , Antígeno Nuclear de Célula em Proliferação/farmacologia , Ratos , Ratos WistarRESUMO
At present, the most popular biomaterials used in cartilage tissue engineering are synthetic polymers. However, problems-such as acidic by-product accumulation and side effects in local or systemic inflammatory reactions during in vivo degradation-are drawing much attention. The polymers are also highly hydrophobic and degrade within 4 weeks, allowing insufficient time to support neocartilage formation. All these have made polymers less promising in clinical application. In this study, we tested a new bioceramic scaffold made of artificial synthesized powder of beta-tricalcium phosphate (beta-TCP) in a sheep model. Osteochondral defects were filled with a bioceramic-chondrocyte construct and neocartilage tissue completely resurfaced the cartilage defects after 24 weeks. Typical hyaline cartilage structure was generated in the engineered cartilage. Biodegradation of bioceramic was notable, leading to bioceramic fragmentation and particle formation. Numerous ceramic particles (size, 0.5-1.9 microm) and numerous macrophages were observed at the ceramic-tissue interface as well as in the marrow tissue. No macrophages were visible in the neocartilage tissue. Although long-term in vivo study is needed to further determine the pathological sequences of the beta-TCP-based cartilage construct, this study suggests that this bioceramic might be used to repair chondral or osteochondral defects and could be used as a scaffold for cartilage tissue engineering.