RESUMO
Periodontitis gradually damages the hard and soft tissues surrounding the tooth, leading to tooth loss. In recent years, the use of biomaterials in periodontitis treatment has expanded, including gels, nanoparticles, microparticles, fibers, and membranes. Among these, membranes have more clinical applications. Due to the ability of the piezoelectric material to regenerate damaged tissues, the aim of this study was to create piezoelectric composite membranes. To achieve this, Barium titanate powder (BaTiO3 powder)-a piezoelectric substance-was synthesized using the hydrothermal method and analyzed with X-ray diffraction (XRD) and Field emission scanning electron microscopy (FESEM). Four types of membranes were fabricated using solvent casting method: three composite membranes with chitosan matrix and BaTiO3 fillers (at 3%, 6%, and 9% weight), and one chitosan membrane without BaTiO3. The microstructure of the membrane surfaces, agglomeration of BaTiO3 in membranes, and hydrophilicity, antibacterial, and electrical properties of the membrane were also investigated. The results indicated that membranes containing 3 and 6% BaTiO3 had suitable surface structure for the periodontitis treatment. Agglomeration of BaTiO3 particles was higher in the membrane containing 9% BaTiO3. The large amount of BaTiO3 improved the antibacterial properties of the membranes. Additionally, the membranes containing BaTiO3 had high electrical properties, especially those with 3% and 6% BaTiO3. Therefore, composite membranes containing BaTiO3, especially membranes containing 6% BaTiO3, are more favorable options than those without BaTiO3 for periodontitis treatment.
Assuntos
Quitosana , Periodontite , Humanos , Pós , Periodontite/terapia , Materiais Biocompatíveis , Antibacterianos/uso terapêuticoRESUMO
Multiple Sclerosis (MS) is the most common demyelinating disease with inflammatory demyelination in the central nerve system. Besides the defect in the myelin repair process, the balance change in inflammatory and anti- inflammatory cytokines is one of the most significant factors in MS pathogenesis. This study aimed at evaluating the effects of co-overexpressing beta interferon (IFN-ß) and Leukemia inhibitory factor (LIF) in human adipose-derived stem cells (IFN-ß/LIF-hADSCs) on the experimental autoimmune encephalomyelitis (EAE). 12 days after the induction of EAE on female mice C57Bl/6 with MOG35-55 and the emergence of primary clinical signs, the IFN-ß/LIF-hADSCs were injected into the mice tail vein of the EAE mice. The mice were sacrificed after 32 days and the spinal cords of the experimental groups were dissected out for the histopathologic and real-time RT-PCR studies. Here, we showed that the clinical scores and infiltration of mononuclear cells of treated mice with IFN-ß/LIF-hADSCs were decreased significantly. Demyelination and the number of Olig2+ and MBP+ cells were significantly increased in the test (IFN-ß/LIF-hADSCs) group. The findings revealed that the pattern of inflammatory and anti- inflammatory cytokines gene expression in the IFN-ß/LIF-hADSCs group was reversed compared to the control group. Overexpression of LIF as a neurotrophic and IFN-ß as an anti-inflammatory cytokine in hADSCs increases the immunomodulatory effect of hADSCs reduces the extent of demyelination, improves the number of Olig2+ cells, and also increases the amount of MBP protein which can increase the production of myelin in EAE model. This, besides hADSCs capacity for proliferation and differentiation, might enhance the treatment efficacy and provide a promising candidate for stem cell-based gene therapy of MS therapy in the future.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Humanos , Animais , Camundongos , Feminino , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/terapia , Fator Inibidor de Leucemia/metabolismo , Interferon beta/metabolismo , Medula Espinal/metabolismo , Células-Tronco/metabolismo , Citocinas/metabolismo , Camundongos Endogâmicos C57BLRESUMO
The common retinal diseases are age-related macular degeneration (AMD) and retinitis pigmentosa (RP). They are usually associated with the dysfunction of retinal pigment epithelial (RPE) cells and degeneration of underlying Bruch's membrane. The RPE cell transplantation is the most promising therapeutic option to restore lost vision. This study aimed to construct an ultrathin porous fibrous film with properties similar to that of native Bruch's membrane as carriers for the RPE cells. Human amniotic membrane powder (HAMP)/Polycaprolactone (PCL) scaffolds containing different concentrations of HAMP were fabricated by electrospinning technique. The results showed that with increasing the concentration of HAMP, the diameter of fibers increased. Moreover, hydrophilicity and degradation rate were improved from 119° to 92° and 14 to 56% after 28 days immersion in phosphate-buffered saline (PBS) solution, respectively. All scaffolds had a porosity above 85%. Proper cell adhesion was obtained one day after culture and no toxicity was observed. However, after seven days, the rate of growth and proliferation of ARPE-19 cells, a culture model of RPE, on the PCL-30HAMP scaffold (HAMP concentration in PCL 7.2% by weight) was higher compared to other scaffolds. These results indicated that PCL-30HAMP fibrous scaffold has a great potential to be used in retinal tissue engineering applications.
Assuntos
Âmnio , Epitélio Pigmentado da Retina , Lâmina Basilar da Corioide , Colágeno/metabolismo , Células Epiteliais , Humanos , Poliésteres/metabolismo , Pós , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
AIMS: The degeneration of retinal neurons which occurs in many neurodegenerative diseases of retina such as retinitis pigmentosa and aged-related macular degeneration, is a progressive phenomenon and leads to permanent visual disability. Aside from their economic and social impact, those who suffer from these diseases have a poor quality of life due to the lack of cures. Researchers have turned to stem cell therapies as a potential solution to this global health crisis. Mesenchymal stem cells (MSCs) and their paracrine agents such as conditioned medium (CM) and exosomes (Exo) have been applied to treat different retinal disorders. This study compared the therapeutic effects of human adipose mesenchymal stem cells (hADSCs) and their secretome on an in vivo model of sodium iodate retinal neurodegeneration. MAIN METHODS: We analyzed the expression of retinal cells' specific mRNAs by RT-PCR and proteins by immunostaining as well as performing visual cliff avoidance test as a functional evaluation technique. There were four therapeutic groups in this study: hADSC, hADSC-CM, hADSC-Exo and hADSC-Exo + CM. KEY FINDINGS: Although all groups showed different therapeutic effects on various retinal cells, the results of hADSC-CM were most striking, especially in terms of photoreceptor regeneration and retinal function. SIGNIFICANCE: The findings of present study demonstrated the different effects of MSC-based therapies on various retinal cells which could be helpful in designing more precise treatments that suit to each neurodegenerative disease mechanism and the cells involved. It also suggests that CM might be a better choice due to its multifactorial characteristic.
Assuntos
Células-Tronco Mesenquimais , Doenças Neurodegenerativas , Degeneração Retiniana , Idoso , Animais , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Humanos , Iodatos , Células-Tronco Mesenquimais/metabolismo , Doenças Neurodegenerativas/metabolismo , Qualidade de Vida , Ratos , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Degeneração Retiniana/terapiaRESUMO
AIMS: Among all the treatments for Multiple Sclerosis, stem cell transplantation, such as ADSCs, has attracted a great deal of scientific attention. On the other hand, Edaravone, as an antioxidant component, in combination with stem cells, could increase the survival and differentiation potential of stem cells. MAIN METHODS: 42 rats were divided into: Control, Cuprizone (CPZ), Sham, Edaravone (Ed), hADSCs, and Ed/hADSCs groups. Following induction of cuprizone, induced MS model, behavioral tests were designed to evaluate motor function during. Luxal fast blue staining was done to measure the level of demyelination and remyelination. Immunofluorescent staining was used to evaluate the amount of MBP, OLIG2, and MOG proteins. The mRNA levels of human MBP, MOG, and OLIG2 and rat Mbp, Mog, and Olig2 were determined via RT-PCR. KEY FINDINGS: Flow cytometry analysis exhibited that the extracted cells were positive for CD73 (93.8 ± 3%) and CD105 (91.6 ± 3%), yet negative for CD45 (2.06 ± 0.5%). Behavioral tests, unveiled a significant improvement in the Ed (P < 0.001), hADSCs (P < 0.001), and Ed/hADSCs (P < 0.001) groups compared to the others. In the Ed/hADSCs group, the myelin density was significantly higher than that in the Ed treated and hADSCs treated groups (P < 0.01). Edaravone and hADSCs increased the expression of Mbp, Mog, and Olig2 genes in the cuprizone rat models. Moreover, significant differences were seen between the Ed treated and hADSCs treated groups and the Ed/hADSCs group (P < 0.05 for Mbp and Olig2 and P < 0.01 for Mog). SIGNIFICANCE: Edaravone in combination with hADSCs reduced demyelination and increased oligodendrogenesis in the cuprizone rat models.
Assuntos
Tecido Adiposo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Edaravone/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Esclerose Múltipla , Oligodendroglia/metabolismo , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Xenoenxertos , Humanos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/terapia , RatosRESUMO
Human adipose stem cells (hADSCs) are proper cell sources for tissue regeneration. They mainly mediate their therapeutic effects through paracrine factors as exosomes. The exosomes contents are protein, lipid and RNA. Exosomes are effective in restoring the function of neurons and astrocytes in neurodegenerative diseases, and improve the therapeutic outcomes. We investigated the effect of hADSCs derived exosomes on survival and neural differentiation of PC12 cells in vitro. The isolated hADSCs, were characterized by flow cytometry. Exosomes were separated from hADSC-condition medium using Exo-spinTM kit and characterized by DLS and TEM. Then acridine orange staining was performed to confirm entrance of exosomes into PC12 cells. PC12 cells were treated with culture medium containing NGF and exosome. Cell viability was assessed by MTT assay, and neural differentiation by ICC technique and qRT-PCR. TEM and DLS data confirmed the isolation of exosomes according to their size (30-100 nm) and acridine orange staining indicated entrance of exosomes to target cells. MTT assay showed that cell viability was significantly increased in exosome treated group. ICC technique revealed that the expression of Map2 was superior in the exosome treated group. Based on qRT-PCR data, Map2 and ß-tub III gene expression was increased in the exosome treated group. Significant expression of Gfap was seen in the NGF and NGF/EXO treated groups. Present study indicated that hADSCs derived exosomes might enhance cell viability and promote neuronal differentiation and expression of mature neural marker in PC12 cells.
Assuntos
Diferenciação Celular/fisiologia , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adipócitos , Tecido Adiposo/citologia , Animais , Astrócitos , Proliferação de Células , Sobrevivência Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Exossomos/fisiologia , Humanos , Neurônios/metabolismo , Neurônios/fisiologia , Células PC12 , Ratos , Células-Tronco/metabolismo , CicatrizaçãoRESUMO
Multiple sclerosis (MS) is an inflammatory demyelination disease in the central nervous system (CNS) characterized by incomplete endogenous remyelination in the chronic phase. A shift of the balance between pro and anti-inflammatory cytokines is one of the important markers in the pathogenesis of MS. This study aimed to evaluate the effects of human adipose derived stem cells (hADSCs) overexpressing interleukin 11 and interleukin 13 (IL-11, 13-hADSCs) on the experimental autoimmune encephalomyelitis (EAE), an animal model of MS.12 days after immunization of C57Bl/6 female mice with MOG35-55 and initial clinical symptoms appearance, the IL-11, 13-hADSCs were injected via the tail vein into the EAE mice. Then, the mice were sacrificed at 30 days post-immunization (DPI) and the spinal cords of experimental groups were extracted for histopathological and real-time RT-PCR studies.The results indicated that the clinical scores and mononuclear cells infiltration into the spinal cords of EAE mice were significantly reduced in mice treated with IL-11, 13-hADSCs. Likewise, the remyelination and oligodendrogenesis were significantly enhanced in the mentioned treatment group. Real-time results demonstrated that pro/anti-inflammatory cytokine genes expression was reversed in IL-11, 13-hADSCs treatment group in comparison to the untreated EAE group.Expression of IL-11 as a neurotrophic cytokine and IL-13 as an anti-inflammatory cytokine by hADSCs could increase the immunomodulatory and neuroprotective effects of hADSCs and be a powerful candidate in stem cell therapy for future treatment of MS.
Assuntos
Tecido Adiposo/patologia , Células-Tronco Adultas/fisiologia , Encefalomielite Autoimune Experimental/terapia , Interleucina-11/metabolismo , Interleucina-13/metabolismo , Esclerose Múltipla/terapia , Transplante de Células-Tronco , Adulto , Células-Tronco Adultas/transplante , Animais , Modelos Animais de Doenças , Feminino , Humanos , Imunomodulação , Interleucina-11/genética , Interleucina-13/genética , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Fármacos Neuroprotetores , Fragmentos de Peptídeos/imunologia , Adulto JovemRESUMO
Gold nanoparticles (AuNPs) have been proposed as useful medical carriers in the field of regenerative medicine. This study aimed to assess the direct conjugation ability of retinoic acid (RA) with AuNPs and to develop a strategy to differentiate the human adipose-derived stromal/stem cells (hADSCs) into neurons using AuNPs-RA. The physical properties of this nanocarrier were characterized using FT-IR, TEM, and FE-SEM. Moreover, the efficiency of RA conjugation on AuNPs was determined at 99% using UV-Vis spectroscopy. According to the MTT assay, an RA concentration of 66 µM caused a 50% inhibition of cell viability and AuNPs were not cytotoxic in concentrations below 5 µg/ml. Real-time PCR and immunocytochemistry proved that AuNPs-RA is able to increase the expression of neuronal marker genes and the number of neuronal protein (GFAP and MAP2)-positive cells, 14 days post-induction of hADSCs. Taken together, these results confirmed that the AuNPs-RA promote the neuronal differentiation of hADSCs.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Nanopartículas Metálicas/química , Células-Tronco Neurais/citologia , Tretinoína/farmacologia , Sobrevivência Celular , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Ouro/química , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Nanopartículas Metálicas/efeitos adversos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Nanoconjugados/efeitos adversos , Nanoconjugados/química , Células-Tronco Neurais/metabolismo , Neurogênese , Tretinoína/efeitos adversosRESUMO
Lignin displays attractive properties in peripheral nerve applications. Here, aligned polycaprolactone (PCL) fibers with various percentages of lignin nanoparticles were fabricated using the electrospinning method. The morphologies, contact angles, mechanical properties, in vitro degradation, and water uptake of the PCL/lignin fibers were characterized. Cell viability and adhesion of PC12 and human adipose-derived stem cells (hADSCs) were studied employing MTT assay and SEM, respectively. SEM, immunocytochemistry, and Real-Time PCR were utilized to characterize neural differentiation and neurite length of PC12 and hADSCs. To further study on lignin effect on nerve regeneration, in vivo studies were performed. The results indicated that all nanocomposite fibers were smooth and bead-free. With increasing the lignin content, the water contact angle decreased while in vitro degradation, water uptake, and Young's modulus increased compared to the PCL fibers. Cell viability, and differentiation along with neurite length extension were promoted by increasing lignin content. The neural markers expression for differentiated cells were upregulated by the increase of lignin percent. In vivo investigation also demonstrates that sample groups incorporating 15% lignin nanoparticles showed better regeneration among others. Therefore, PCL with 15% of lignin nanoparticles shows great potential to be applied for nerve regeneration.
Assuntos
Lignina/química , Nanopartículas/química , Regeneração Nervosa , Poliésteres/química , Adulto , Animais , Células Cultivadas , Módulo de Elasticidade , Humanos , Masculino , Nanopartículas/uso terapêutico , Crescimento Neuronal , Células PC12 , Traumatismos dos Nervos Periféricos/terapia , Ratos , Ratos WistarRESUMO
Stem cells have been long looked at as possible therapeutic vehicles in regenerative medicine largely due to their multi-lineage differentiation potential and paracrine actions. Therefore, development of new procedures for the differentiation of stem cells into different cell types holds great potential for opening new opportunities in regenerative medicine. In addition to various methods for inducing stem cell differentiation, the utilization of nanomaterials for differentiation of stem cells has recently received considerable attention and has become a potential tool for such purpose. Multiple lines of evidence revealed that nanomaterial-based scaffolds, inorganic nanoparticles (NPs), and biodegradable polymers have led to significant progress in regulation of stem cell differentiation. Several studies indicated that different NPs including selenium, gold, graphene quantum dots (QDs) and silica could be employed for the regulation of differentiation of stem cells such as human mesenchymal stem cells (hMSCs). In addition, magnetic core-shell NPs could be applied for the regulation of neural stem cell (NSC) differentiation. Taken together, these findings suggested that NPs are potential candidates which could be utilized for the differentiation of stem cells into various cell types such as neural cells. Herein, we summarized the application of NPs for differentiation of stem cells into various cells in particular neural cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Nanopartículas Metálicas/química , Células-Tronco Neurais/metabolismo , Pontos Quânticos/química , HumanosRESUMO
Electrospinning of natural and synthetic polymers open a new practical approach to tissue engineering by producing fibers. In this study, aligned electrospun poly(vinyl alcohol) (PVA)-poly(glycerol sebacate) (PGS) fibers with various percentages of lignin (0, 1, 3, and 5%wt) fabricated for nerve tissue engineering. The effect of the different amount of lignin on the morphology and diameter of the fibers was investigated by scanning electron microscopy (SEM). The physicochemical properties of fibers were studied using FTIR, tensile strain, contact angle, water uptake, and degradation test. MTT assay and SEM were employed to evaluate PC12 cell proliferation and adhesion, respectively. Immunocytochemistry and gene expression were utilized to study how the lignin affected on cell differentiation. The results revealed the smooth with a uniform diameter of the fabricated fibers, and the increased amount of lignin reduced the fiber diameter from 530 to 370â¯nm. The modulus of elasticity increased from 0.1 to 0.4â¯MPa by increasing the lignin percentage. The PC12 cell culture indicated that the lignin enhanced cell proliferation. The mRNA expression level for Gfap, ß-Tub III, and Map2 and immunocytochemistry (Map2) revealed the positive effect of lignin on neural cell differentiation. Finally, the results suggest PVA-PGS/5% lignin as a promising material for nerve tissue engineering.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Decanoatos/química , Glicerol/análogos & derivados , Lignina/química , Nanofibras/administração & dosagem , Nanofibras/química , Tecido Nervoso/efeitos dos fármacos , Polímeros/química , Álcool de Polivinil/química , Animais , Linhagem Celular Tumoral , Elasticidade/efeitos dos fármacos , Glicerol/química , Células PC12 , Ratos , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Retinal degenerative diseases lead to blindness due to poorly regenerative potential of the retina. Recently, cell therapy is more considered for degenerative diseases. Autologous mesenchymal stem cells derived from adipose tissue are a suitable source for this purpose. Therefore, we conducted a stepwise efficient method to differentiate human adipose-derived stem cells (hADSCs) into retinal precursor-like cells in vitro. We compared two differentiation protocols, monolayer and hanging drop cultures. Through the defined medium and 3D hanging drop culture method, we could achieve up to 75% retinal precursor gene expression profile (PAX6, RAX, CHX10, and CRX) from hADSCs. By imitation of in vivo development, for direct conversion of stem cells into retinal cells, the suppression of the BMP, Nodal, and Wnt signaling pathways was carried out by using three small molecules. The hADSCs were primarily differentiated into anterior neuroectodermal cells by expression of OTX2, SIX3, and Β-TUB III and then the differentiated cells were propelled into the retinal cells. According to our data from real-time PCR, RT-PCR, immunocytochemistry, and functional assay, it seems that the hanging drop method improved retinal precursor differentiation yield which these precursor-like cells respond to glutamate neurotransmitter. Regarding the easy accessibility and immunosuppressive properties of hADSCs and more efficient hanging drop method, this study may be useful for future autologous cell therapy of retinal degenerative disorders.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Neurais/citologia , Neurônios Retinianos/citologia , Adulto , Benzamidas/farmacologia , Células Cultivadas , Dioxóis/farmacologia , Humanos , Isoquinolinas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neurais/metabolismo , Cultura Primária de Células/métodos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Neurônios Retinianos/metabolismo , TranscriptomaRESUMO
Recently, stem-cell therapy as a promising therapeutic alternative is considered to treat retinal degenerative diseases. Here, we used small molecules and concentrated conditioned medium selectively enriched with Amicon filter units from human adipose-derived stem cells (hADSC-CM) containing various neurotrophic factors to induce hADSCs toward eye field neuroectoderm (EFN). For induction of stem cells, hADSC-CM and small molecules CKI-7, SB431542 and LDN193189 as inhibitors of Wnt, Nodal and BMP4 signaling pathways were used, respectively. We found the highest expression of ß-TUB III as a neural marker in the group in which small molecules and conditioned medium were applied simultaneously. Moreover, EFN markers SIX3, PAX6 and RAX had higher expression in the presence of a conditioned medium. However, the superior expression of ENF marker OTX2 was seen in the small molecules group. Our results indicated that neurotrophic factors present in hADSCs-CM could induce hADSCs into EFN cells. Therefore, a more thorough study of these factors and their effects in hADSC-CM might pave the way for cellular and non-cellular therapy in retinal degenerative diseases.
Assuntos
Tecido Adiposo/citologia , Proteínas do Olho/metabolismo , Células-Tronco Mesenquimais/fisiologia , Doenças Retinianas/terapia , Adulto , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Meios de Cultivo Condicionados , Feminino , Humanos , Distribuição Aleatória , Transplante de Células-TroncoRESUMO
BACKGROUND: Nowadays, cartilage tissue engineering is the best candidate for regeneration of cartilage defects. This study evaluates the function of herbal extracts icariin (ICA), the major pharmacological constituent of herba Epimedium, compared with transforming growth factor ß3 (TGFß3) to prove its potential effect for cartilage tissue engineering. MATERIALS AND METHODS: ICA, TGFß3, and TGFß3 + ICA were added fibrin-cell constructions derived from adipose tissue stem cells. After 14 days, cell viability analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide assay and the expression of cartilage genes was evaluated with real-time polymerase chain reaction (RT-PCR). RESULTS: The results showed ICA, TGFß3, and TGFß3 + ICA increased the rate of proliferation and viability of cells; but there were no significant differences between them (P > 0.05). Furthermore, quantitative RT-PCR analysis demonstrated that cooperation of ICA with TGFß3 showed a better effect in expression of cartilaginous specific genes and increased Sox9, type II collagen, and aggrecan expression significantly. Furthermore, the results of the expression of type I and X collagens revealed that TGFß3 increased the expression of them (P < 0.01); However, treatment with ICA + TGFß3 down regulated the expression of these genes significantly. CONCLUSION: The results indicated ICA could be a potential factor for chondrogenesis and in cooperation with TGFß3 could reduce its hypertrophic effects and it is a promising factor for cartilage tissue engineering.
RESUMO
Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, ß-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders.
Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Placa Neural/fisiologia , Adulto , Antígenos CD/metabolismo , Células Cultivadas , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteína Homeobox SIX3RESUMO
Retinal disease caused by retinal cell apoptosis leads to irreversible vision loss. Stem cell investigation efforts have been made to solve and cure retinal disorders. There are several sources of stem cells which have been used in these experiments. Numerous studies demonstrated that transplanted stem cells can migrate into and integrate in different layers of retina. Among these, mesenchymal stem cells (MSCs) were considered a promising source for cell therapy. Here, we review the literature assessing the potential of MSCs to differentiate into retinal cells in vivo and in vitro as well as their clinical application. However, more investigation is required to define the protocols that optimize stem cell differentiation and their functional integration in the retina.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Retina/citologia , Animais , Diferenciação Celular , Humanos , Retina/crescimento & desenvolvimento , Doenças Retinianas/terapiaRESUMO
There is wide interest in application of adult stem cells due to easy to obtain with a minimal patient discomfort, capable of producing cell numbers in large quantities and their immunocompatible properties without restriction by ethical concerns. Among these stem cells, multipotent mesenchymal stem cells (MSCs) from human adipose tissue are considered as an ideal source for various regenerative medicine. In spite of mesodermal origin of human adipose-derived stem cells (hADSCs), these cells have differentiation potential toward mesodermal and non-mesodermal lineages. Up to now, several studies have shown that hADSCs can undergo transdifferentiation and produce cells outside of their lineage, especially into neural cells when they are transferred to a specific cell environment. The purpose of this literature review is to provide an overview of the existing state of knowledge of the differentiation potential of hADSCs, specifically their ability to give rise to neuronal cells. The following review discusses different protocols considered for differentiation of hADSCs to neural cells, the neural markers that are used in each procedure and possible mechanisms that are involved in this differentiation.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Adultas/citologia , Neurônios/citologia , Medicina Regenerativa/métodos , Tecido Adiposo/metabolismo , Adulto , Células-Tronco Adultas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Transdiferenciação Celular , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Retinal pigment epithelium (RPE) is a hexagonal monolayer of pigmented cells located between the neural retina and the choroid with an essential role for visual function. So, isolation, propagation and maintenance of their functional integrity of RPE are crucial for research in vitro which next used for cell transplantation. The evaluation of features of RPE cells as a sheet after 14 days has not been reported yet. This study aimed to examine and compare three protocols for RPE isolation from rabbit eyes and obtain a proper protocol, which illustrated isolated RPE cells as a sheet cause to preserve their characterize even after 2 weeks. MATERIALS AND METHODS: RPE cells were prepared from eyes of 24 rabbit eyes. After enucleating of eyes, anterior segment discarded and posterior segment cut to small pieces. Two of these procedures are based on the enzymatic digestion, but third protocol based on mechanical dissection. The culture cells harvested and morphological feature of cells assessed by phase-contrast microscope and then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. RESULTS: Evaluation of morphological feature showed that isolation of RPE cells as a sheet lead to preserve their hexagonal morphology. Immunocytochemistry and RT-PCR assessment demonstrated RPE cell cultured in sheet maintained their phenotypic feature, tight junction and the distribution of actin and cytokeratin filament. Comparison of three protocols showed that dissociation of RPE cells as a sheet was superior in the preserve of RPE characteristic. CONCLUSIONS: Isolation of RPE cells as a sheet maintains the integrity of these cells, this procedure promising a therapeutic approach, which is important for some retinal diseases.
RESUMO
BACKGROUND: Close interaction between retinal pigment epithelium (RPE) and photoreceptors plays an essential role in visual function. The objective of this study is to determine the effects of RPE cells in the differentiation of progenitor derived human embryonic stem cells (hESC) into retinal cells; we developed in vitro co-culture models and compare these models to investigate in which model the expression of photoreceptor markers is superior. It seems the effects of RPE cells on differentiation of retinal progenitor cells (RPCs) through the cell-to-cell contact or with the use of insert and compare of these methods has not been reported yet. METHODS: Initially, retinal progenitors (RPs) were differentiated from hESC. After isolation of RPE sheet from rabbit eyes, demonstrated these cells maintains the integrity and feature after 2 weeks. Next, we examined the induction of photoreceptors by the co-culture of RPE through insert in 1 week and 2 weeks (indirect) or without insert by the cell-to-cell contact (direct). The differentiation of retinal cells was verified by protein and gene expression in these three methods. The adherent cells were morphologically examined using phase contrast microscopy and characterized by immunofluorescent staining and reverse transcription.polymerase chain reaction (RT-PCR). RESULTS: Evaluation of immunostaining showed that hESC, highly (>80%) can be directed to the RPs fate. Upon co-culture of RPCs with RPE sheet using insert for 2 weeks or by the cell-to-cell contact, these cells differentiated to neural retina and expressed photoreceptor-specific markers. However, in direct co-culture, some mature photoreceptor markers like arrestin expressed in compare with indirect co-culture. CONCLUSIONS: The expression of late photoreceptor marker could be improved when RPE cells seeded on RPCs in compare with the use of insert.
RESUMO
Transplantation of retinal cells has recently provided a promising therapeutic approach for retinal degeneration. Here, we differentiated initially retinal progenitors (RPs) from adherent feeder-free human embryonic stem cells (hESCs) with the use of defined media supplemented with a specific combination of growth factors. The differentiated RPs highly (>80%) expressed related molecular features that included Six3 at an early stage in addition to Crx, Rx, Pax6, Otx2, and Chx10 at later stage. Next, we examined the induction of photoreceptors by Shh and/or the coculture of rabbit retinal pigmented epithelium with hESCs-derived RPs. The differentiation of retinal cells was demonstrated by protein and gene expression in all groups. However, S-Opsin, a cone photoreceptor marker, had higher expression in the presence of Shh, whereas expressions of Gli and Hes1 decreased in the same group. Finally, hESC-derived RPs were treated with Shh transplanted into the subretinal space of sodium iodate-injected albino-type adult rabbits and analyzed 4 weeks later. Transplanted retinal cells survived, migrated into retinal layers, and restored a small but significant B-wave. The grafted cells expressed photoreceptor markers, S-Opsin and Rhodopsin. Our results indicate that putative hESC-derived retinal cells express related genes and proteins. Further, our results show that retinal-like cells can be useful replacements for photoreceptors in retinal diseases.