RESUMO
Permanent degeneration and loss of dopaminergic (DA) neurons in substantia nigra is the main cause of Parkinson's disease. Considering the therapeutic application of stem cells in neurodegeneration, we sought to examine the neurogenic differentiation potential of the newly introduced neural crest originated mesenchymal stem cells (MSCs), namely, trabecular meshwork-derived mesenchymal stem cells (TM-MSCs) compared to two other sources of MSCs, adipose tissue-derived stem cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs). The three types of cells were therefore cultured in the presence and absence of a neural induction medium followed by the analysis of their differentiation potentials. Our results showed that TM-MSCs exhibited enhanced neural morphologies as well as higher expressions of MAP2 as the general neuron marker and Nurr-1 as an early DA marker compared to the adipose tissue-derived mesenchymal stem cells (AD-MSCs) and bone marrow-derived stem cells (BMSCs). Also, analysis of Nurr-1 immunostaining showed more intense Nurr-1 stained nuclei in the neurally induced TM-MSCs compared to those in the AD-MSCs, BMSCs, and noninduced control TM-MSCs. To examine if Wnt/beta-catenin pathway drives TM-MSCs towards a DA fate, we treated them with the Wnt agonist (CHIR, 3 µM) and the Wnt antagonist (IWP-2, 3 µM). Our results showed that the expressions of Nurr-1 and MAP2, as well as the Wnt/beta-catenin target genes, c-Myc and Cyclin D1, were significantly increased in the CHIR-treated TM-MSCs, but significantly reduced in those treated with IWP-2. Altogether, we declare first a higher neural potency of TM-MSCs compared to the more commonly used MSCs, BMSCs and ADSCs, and second that Wnt/beta-catenin activation directs the neurally induced TM-MSCs towards a DA fate.
Assuntos
Células-Tronco Mesenquimais , Via de Sinalização Wnt , Diferenciação Celular , Células Cultivadas , Células-Tronco Mesenquimais/metabolismo , Malha Trabecular/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND AND AIM: Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the destruction of the dopaminergic neurons in the nigrostriatal pathway, leading to motor-behavioral complications. Cell therapy has been proposed as a promising approach for PD treatment using various cellular sources. Despite a few disadvantages mesenchymal stem cells (MSCs) represent, they have more auspicious effects for PD cell therapy. The present study aimed to evaluate a new source of MSCs isolated from human Conjunctiva (CJ-MSCs) impact on PD complications for the first time. MATERIALS AND METHODS: Parkinson's was induced by stereotactic injection of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). An apomorphine-induced rotation test was used to confirm the model establishment. After PD model confirmation, green fluorescent protein (GFP) labeled CJ-MSCs and induced CJ-MSCs (microfluidic encapsulated and non-capsulated) were transplanted into the rats' right striatum. Then Rotation, Rotarod, and Open-field tests were performed to evaluate the behavioral assessment. Additionally, the immunohistochemistry technique was used for identifying tyrosine hydroxylase (TH). RESULTS: According to the obtained data, the cell transplantation caused a reduction in the rats' rotation number and improved locomotion compared to the control group. The previous results were also more pronounced in induced and microfluidic encapsulated cells compared to other cells. Rats recipient CJ-MSCs also have represented more TH-expressed GFP-labeled cell numbers in the striatum than the control group. CONCLUSION: It can be concluded that CJ-MSCs therapy can have protective effects against PD complications and nerve induction of cells due to their ability to express dopamine. On the other hand, CJ-MSCs microencapsulating leads to enhance even more protective effect of CJ-MSCs. However, confirmation of this hypothesis requires further studies and investigation of these cells' possible mechanisms of action.
Assuntos
Túnica Conjuntiva/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Doença de Parkinson/terapia , Animais , Túnica Conjuntiva/citologia , Corpo Estriado/patologia , Corpo Estriado/transplante , Modelos Animais de Doenças , Humanos , Técnicas Analíticas Microfluídicas , Oxidopamina/farmacologia , Doença de Parkinson/patologia , RatosRESUMO
The purpose of this study was to investigate miR-7 overexpression effects on neural differentiation of mesenchymal stem cells (MSCs) on both two-dimensional (2D) and three-dimensional (3D) culture systems. We upregulated miR-7 through lentiviral vector in trabecular meshwork MSCs (TMMSCs) and polymers of poly l-lactic acid/polycaprolactone fibrous scaffold were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). Neural markers expression was evaluated through quantitative-polymerase chain reaction (q-PCR) and immunostaining. The results showed that the high percentage of cell transduction (84.9%) and miR-7 expression (folds: 677.93 and 556.4) was detected in TMMSCs-miR-7(+). SEM and FTIR established the fabrication of the hybrid scaffold. q-PCR analysis showed that on days 14 and 21 of transduction, the expression level of Nestin and glial fibrillary acidic protein (GFAP) genes were significantly higher in the scaffold (3D) compared with tissue culture polystyrene (2D) culture. The expression of microtubule-associated protein-2 (MAP-2) and GFAP genes in TMMSCs-miR-7(+) cells were significantly higher than those miR-7(-) cells after 21 days of cell culture. Also, MAP-2 and Nestin proteins were detected in TMMSCs-miR-7(+) cells. Our results demonstrate that miR-7 is involved in neural differentiation of TMMSCs and scaffold can improve differentiate into glial and neural progenitor cells. These findings provided some information for future use of microRNAs and scaffold in tissue engineering and cell therapy for neurological diseases.
Assuntos
Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Malha Trabecular/metabolismo , Diferenciação Celular , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Células HEK293 , Humanos , Lentivirus/metabolismo , Microscopia Eletrônica de Varredura , Nanofibras , Nestina/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Poliésteres/química , Poliestirenos/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
MiRNAs are small non-coding RNAs that are ordinarily involved in modulating mRNAs and stem cell differentiation. 3D nanofibrous scaffolds have an important role in the differentiation of stem cells due to their similarity to the extracellular matrix (ECM). In the present study, we tried to introduce a new approach to guiding the differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells by hsa-miR-9-1 delivery on both 2D and 3D substrates. First, the CJMSCs were transduced by a lentiviral vector carrying miR-9 (pCDH + hsa-miR-9-1) and then cell transduction efficacy verified by using fluorescent microscopy, flow cytometry, and qPCR analyses. Silk Fibroin-poly-L-lactic acid (SF-PLLA) scaffold was fabricated by the electrospinning technique while the scaffold characteristics including morphology, chemical properties, and biocompatibility were evaluated by SEM, FTIR, and MTT assays, respectively. Then, the miR-9-CJMSCs were seeded on both TCPS and the scaffold; photoreceptor gene and protein expressions were evaluated by RT-qPCR and immunostaining after 14 and 21 days of transduction. More than 80% of CJMSCs were transduced and miR-9 expression was significantly higher in miR-9-CJMSCs compared with empty vector (EV)-CJMSCs. SEM and FTIR confirmed the fabrication of the SF/PLLA hybrid structure. RT-qPCR and immunostaining analyses showed that the specific photoreceptor genes and proteins were expressed in miR-9 transduced CJMSCs. Mir-9 induced CJMSCs into photoreceptor-like cells in a time-dependent manneron on both TCPS and nanofibrous scaffold.We have proved that hsa-miR-9-1 has the potency to guide the photoreceptor differentiation of mesenchymal stem cells and promote retinal regeneration.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Nanofibras/química , Células Fotorreceptoras de Vertebrados/metabolismo , Alicerces Teciduais/química , Células Cultivadas , Túnica Conjuntiva/citologia , Fibroínas/química , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanofibras/ultraestrutura , Células Fotorreceptoras de Vertebrados/citologia , Poliésteres/química , Fatores de Tempo , Engenharia Tecidual/métodosRESUMO
Conductive nanofibrous scaffolds with that can conduct electrical current have a great potential in neural tissue engineering. The purpose of this study was to survey effects of electrical stimulation and polycaprolactone/polypyrrole/multiwall carbon nanotube (PCL/PPY/MWCNTs) fibrous scaffold on photoreceptor differentiation of trabecular meshwork mesenchymal stem cells (TM-MSCs). PCL/PPY/MWCNTs scaffold was made by electrospinning method. TM-MSCs were seeded on PCL/PPY/MWCNTs scaffold and stimulated with a potential of 115 V/m. Scanning electron microscopy, transmission electron microscopy, and FT-IR were used to evaluate the fabricated scaffold. Immunofluorescence and quantitative real-time polymerase chain reaction were used to examine differentiated cells. Scanning electron microscopy, transmitting electron microscopy, and FT-IR confirmed the creation of the composite structure of fibers. RT-qPCR analysis showed that the expression of rhodopsin and peripherin genes in electrically stimulated cells were significantly higher (5.7- and 6.23-fold, respectively; p ≤ 0.05) than those with no electrical stimulation. Collectively, it seems that the combination of PCL/PPY/MWCNTs scaffold, as a suitable conductive scaffold, and electrical stimulation could be an effective approach in the differentiation of stem cells in retinal tissue engineering.
RESUMO
Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.
Assuntos
Diferenciação Celular/fisiologia , Diabetes Mellitus Experimental/patologia , Células Secretoras de Insulina/fisiologia , Células-Tronco Mesenquimais/fisiologia , Malha Trabecular/fisiologia , Animais , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microfluídica/métodos , RNA Mensageiro/metabolismo , Ratos , Malha Trabecular/metabolismoRESUMO
Despite the advantages of transplantation of umbilical cord blood's (UCB's) hematopoietic stem cells (uHSCs) for hematologic malignancy treatment, there are two major challenges in using them: (a) Insufficient amount of uHSCs in a UCB unit; (b) a defect in uHSCs homing to bone marrow (BM) due to loose binding of their surface glycan ligands to BM's endothelium selectin receptors. To overcome these limitations, after poly l-lactic acid (PLLA) scaffold establishment and incubation of uHSCs with fucosyltransferase-VI and GDP-fucose, ex vivo expansion of these cells on selectin-coated scaffold was done. The characteristics of the cultured fucosylated and nonfucosylated cells on a two-dimensional culture system, PLLA, and a selectin-coated scaffold were evaluated by flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming unit (CFU) assay, and CXCR4 expression at the messenger RNA and protein levels. According to the findings of this study, optimized attachment to the scaffold in scanning electron microscopy micrograph, maximum count of CFU, and the highest 570 nm absorption were observed in fucosylated cells expanded on selectin-coated scaffolds. Furthermore, real-time polymerase chain reaction showed the highest expression of the CXCR4 gene, and immunocytochemistry data confirmed that the CXCR4 protein was functional in this group compared with the other groups. Considered together, the results showed that selectin-coated scaffold could be a supportive structure for fucosylated uHSC expansion and homing by nanotopography. Fucosylated cells placed on the selectin-coated scaffold serve as a basal surface for cell-cell interaction and more homing potential of uHSCs. Accordingly, this procedure can also be considered as a promising technique for the hematological disorder treatment and tissue engineering applications.
Assuntos
Sangue Fetal/citologia , Células-Tronco Hematopoéticas/fisiologia , Selectinas/química , Alicerces Teciduais/química , Linhagem Celular , Sobrevivência Celular , Fucose/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Nanoestruturas , Propriedades de Superfície , Sais de Tetrazólio , TiazóisRESUMO
An electrical stimulus is a new approach to neural differentiation of stem cells. In this work, the neural differentiation of conjunctiva mesenchymal stem cells (CJMSCs) on a new 3D conductive fibrous scaffold of silk fibroin (SF) and reduced graphene oxide (rGo) were examined. rGo (3.5% w/w) was dispersed in SF-acid formic solution (10% w/v) and conductive nanofibrous scaffold was fabricated using the electrospinning method. SEM and TEM microscopies were used for fibrous scaffold characterization. CJMSCs were cultured on the scaffold and 2 electrical impulse models (Current 1:115 V/m, 100-Hz frequency and current 2:115 v/m voltages, 0.1-Hz frequency) were applied for 7 days. Also, the effect of the fibrous scaffold and electrical impulses on cell viability and neural gene expression were examined using MTT assay and qPCR analysis. Fibrous scaffold with the 220 ± 20 nm diameter and good dispersion of graphene nanosheets at the surface of nanofibers were fabricated. The MTT result showed the viability of cells on the scaffold, with current 2 lower than current 1. qPCR analysis confirmed that the expression of ß-tubulin (2.4-fold P ≤ 0.026), MAP-2 (1.48-fold; P ≤ 0.03), and nestin (1.5-fold; P ≤ 0.03) genes were higher in CJMSCs on conductive scaffold with 100-Hz frequency compared to 0.1-Hz frequency. Collectively, we proposed that SF-rGo fibrous scaffolds, as a new conductive fibrous scaffold with electrical stimulation are good strategies for neural differentiation of stem cells and the type of electrical pulses has an influence on neural differentiation and proliferation of CJMSCs.
Assuntos
Túnica Conjuntiva/citologia , Células-Tronco Mesenquimais/citologia , Neurogênese , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Animais , Reatores Biológicos , Bombyx/química , Células Cultivadas , Condutividade Elétrica , Estimulação Elétrica/instrumentação , Desenho de Equipamento , Fibroínas/química , Humanos , Nanofibras/química , Nanofibras/ultraestruturaRESUMO
Polymers are used in tissue engineering as a scaffold. In this study the differentiation capability of conjunctiva mesenchymal stem cells (CJMSCs) on natural and synthetic nanofibrous electrospun scaffolds into insulin producing cells (IPCs) were studied. Natural Silk fibroin and synthetic PLLA polymers were used to fabricate electrospun scaffolds. These scaffolds are characterized by SEM and CJMSCs were differentiated into IPCs on these scaffolds. The differentiation efficiency was measured by analysis the expression of specific pancreatic markers by RT-qPCR and insulin release capacity via ELISA. Microscopy analysis showed the fabrication of uniform nanofibers and the formation of the islet-like clusters at the end of differentiation period. Significant differences in expression of Pdx-1 and glucagon were observed in PLLA scaffold compared to Silk scaffold (Fold: 1.625 and 1.434, respectively; P-valueâ¯≤â¯0.0001 for both). Furthermore, insulin secretion at high glucose concentration was significantly higher in cells differentiated on PLLA scaffold than those cultured on Silk scaffold (P-value: 0.012). The scaffolds can enhance the differentiation of IPCs from CJMSCs. In this way, PLLA synthetic scaffold was more efficient than Silk natural scaffold. We conclude that the nanoï¬brous scaffolds reported herein could be used as a potential supportive matrix for islet tissue engineering.
Assuntos
Diferenciação Celular , Túnica Conjuntiva/metabolismo , Fibroínas/química , Células Secretoras de Insulina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Nanofibras/química , Alicerces Teciduais/química , Células Cultivadas , Túnica Conjuntiva/citologia , Humanos , Células Secretoras de Insulina/citologia , Células-Tronco Mesenquimais/citologiaRESUMO
Stem cell transplantation is a new therapeutic strategy in the treatment of neurodegenerative disorders such as Parkinson's disease (PD). Therefore, in this study, the therapeutic effects of Trabecular Meshwork Mesenchymal Stem Cells (TM-MSCs) transplantation, as a new source of mesenchymal stem cells, were evaluated in the animal model of PD. After the development and confirmation of hemi-parkinsonian rats by administration of 6-hydroxy dopamine (6-OHDA) and apomorphine-induced rotation test, green fluorescent protein (GFP) labeled TM-MSCs (normal and induced cells) were transplanted in the striatum of rats. Next, the rotation test, rotarod test, open field, passive avoidance memory tests and immunohistochemistry for tyrosine hydroxylase (TH) were done. The results showed that the number of turns significantly decreased and the improvement of motor performance was achieved after cell transplantation. However, there was no significant difference in passive avoidance memory of animals documented by shuttle box test. The number of GFP- labeled cells expressing TH significantly is increased compared to the vehicle group. Collectively, it seems that TM-MSCs and induced TM-MSCs cell transplantation have positive effects on some aspects of the animal model of PD. Other studies may reveal the potentially positive aspects of these cells in the laboratory and clinical studies.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Atividade Motora , Doença de Parkinson Secundária , Malha Trabecular , Aloenxertos , Animais , Modelos Animais de Doenças , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Doença de Parkinson Secundária/metabolismo , Doença de Parkinson Secundária/patologia , Doença de Parkinson Secundária/terapia , Ratos , Ratos Wistar , Malha Trabecular/metabolismo , Malha Trabecular/patologiaRESUMO
The studies have been done on patient-specific human adipose-derived from mesenchymal stem cells (hADSCs) like a series of autologous growth factors and nanofibrous scaffolds (3D culture) will probably have many benefits for regenerative medicine in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a polyvinyl alcohol (PVA) scaffold and a differentiation protocol by adding platelet-rich plasma (PRP) that induces the hADSCs into insulin-producing cells (IPCs). The characteristics of the derived IPCs in 3D culture were compared with conventional culture (2D) groups evaluated at the mRNA and protein levels. The viability of induced pancreatic cells was 14 days. The in vitro studies showed that the treatment of hADSCs in the 3D culture resulted in differentiated cells with strong characteristics of IPCs including pancreatic-like cells, the expression of the islet-associated genes at the mRNA and protein levels in comparison of 2D culture group. Furthermore, the immunoassay tests showed that these differentiated cells in these two groups are functional and secreted C-peptide and insulin in a glucose stimulation challenge. The results of our study for the first time demonstrated that the PVA nanofibrous scaffolds along with the optimized differentiation protocol with PRP can enhance the differentiation of IPCs from hADSCs. In conclusion, this study provides a new approach to the future pancreatic tissue engineering and beta cell replacement therapies for T1DM.
Assuntos
Peptídeo C/genética , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Adipócitos/metabolismo , Adulto , Doadores de Sangue , Peptídeo C/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Regulação da Expressão Gênica/genética , Glucose/metabolismo , Humanos , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Plasma Rico em Plaquetas/metabolismo , Álcool de Polivinil/química , Álcool de Polivinil/metabolismo , Alicerces Teciduais/químicaRESUMO
Coculture systems are widely used in tissue engineering to mimic cell-cell interactions between different populations. This study aimed to find an improved and convenient system for the corneal epithelial differentiation of conjunctiva derived mesenchymal stem cells (CJMSCs). Thus, the cells were used to reconstruct corneal epithelial cells. Obtained by flow cytometry data, 51.9% of isolated CJMSCs were immune reactive for SSEA4+ antibody which are more potent to differentiate into corneal epithelial cells. A differential medium in a single culture plate was applied and compared to a Coculture with a SHEM medium and a Coculture with a commercial medium. It was found that CJMSCs can be induced to corneal epithelial cells through in vitro co-culturing in a SHEM medium; this was confirmed with immunostaining results. Moreover, relative gene expression results showed that a Coculture system with a SHEM medium can provide a more favorable microenvironment for cells to differentiate into epithelial cells than a single culture or a Coculture with a CnT-Prime commercial medium. Finally, as platforms for cell differentiation, CJMSCs can differentiate into epithelial lineages. This was proven using immunofluorescence staining.
Assuntos
Diferenciação Celular , Túnica Conjuntiva/metabolismo , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual , Técnicas de Cocultura , Túnica Conjuntiva/citologia , Células Epiteliais/citologia , Epitélio Corneano/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Nicho de Células-TroncoRESUMO
Studies on patient-specific human-induced pluripotent stem cells (hiPSCs) as well as a series of autologous growth factors presumably will reveal their many benefits for cell base replacement therapy in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a multistep protocol by adding platelet-rich plasma (PRP) that induce the hiPSCs into insulin-producing cells (IPCs). We present here a differentiation protocol consisting of five stages in two groups including protocol with PRP and without PRP. Charac-teristics of derived IPCs in both groups were evaluated at the mRNA and protein levels, cell cycle and viability in the end stage of cell differentiation. The in vitro studies indicated the treatment of hiPSCs in the protocol with PRP resulting in differentiated cells with strong characteristics of IPCs including islet-like cells, the expression of mature and functional pancreatic beta cell specific marker genes. In addition to these pancreatic specific markers were detected by immunochemistry and Western blot. Our differentiated cells in two groups secreted insulin and C-peptide in a glucose stimulation test by ELISA showing in vitro functional. The results of the cell cycle assay confirmed that differentiation has been done. We reported for the first time that PRP might be ideal additive in the culture medium to induce pancreatic differentiation in the hiPSCs. This study provides a new approach to investigate the role of PRP in pancreatic differentiation protocols and enhance the feasibility of using patient-specific iPSCs and autologous PRP for future beta cells replacement therapies for T1DM. J. Cell. Physiol. 232: 2878-2886, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Plasma Rico em Plaquetas/metabolismo , Western Blotting , Peptídeo C/metabolismo , Técnicas de Cultura de Células , Ciclo Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Forma Celular , Sobrevivência Celular , Meios de Cultura/metabolismo , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Glucose/metabolismo , Humanos , Secreção de Insulina , Fenótipo , Fatores de TempoRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder of the brain which is produced by the damage to dopaminergic neurons. Stem cell transplantation with a nanofibrous scaffold is one of the encouraging strategies for Parkinson's disease therapy. In this study, human mesenchymal stem cells (MSCs) from eye trabecular meshwork (TM) were differentiated into dopaminergic neurons on nanofibrous scaffold. After Trabecular meshwork biopsy, MSCs were isolated, cultivated on Poly-l-Lactide Acid (PLLA) nanofibrous scaffold (fabricated by electrospinning methods) and treated with medium containing DMEM supplemented with RA, IBMX and forskolin for 7 days. Scanning electron microscopy imaging, qPCR and immunostaining were used to analyze differentiated TM-MSCs on scaffold and their expression of dopaminergic-specific markers such as TH and Nurr-1. qPCR analysis revealed the expression of dopaminergic neuron genes such as TH, Nurr-1 on fibrous scaffold as well as TCPS. Immunostaining revealed that the differentiated TM-MSCs on TCPS and Scaffold not only express TH and Nurr-1 genes, but also express TH protein. In conclusion, the results indicate that TM-MSCs might be a suitable source for cell transplantation therapy. In addition the nanofibrous scaffold reported herein could be used as a potential cell carrier for the central system diseases such as PD.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Mesenquimais/citologia , Malha Trabecular/citologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Nanofibras/química , Nanofibras/ultraestrutura , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poliésteres/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Malha Trabecular/metabolismo , Malha Trabecular/ultraestruturaRESUMO
Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1.
Assuntos
Transdiferenciação Celular/fisiologia , Túnica Conjuntiva/citologia , Endoderma/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Ativinas/genética , Ativinas/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/fisiologia , Túnica Conjuntiva/metabolismo , Endoderma/citologia , Células HEK293 , Humanos , Camundongos , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMO
Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.
Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fibroblastos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Tretinoína/administração & dosagemRESUMO
Wounds refer to physical injuries in which the integrity of the skin or other body organs is disturbed. Wound care includes proper management and treatment of the injuries to promote healing while avoiding infection. Here, a core-shell scaffold is developed comprising polyethylene glycol/silk fibroin-chitosan nanoparticles loaded with curcumin. Chitosan nanoparticles and PEG/Silk fibrous scaffold were synthesized by a microfluidic system and electrospinning technique, respectively. TEM, DLS, and FTIR techniques were used to examine the nanoparticles; whereas nanofibers were characterized by SEM, TEM, and FTIR. Drug loading and release from nanoparticles and scaffolds were assessed by optical spectroscopy. MTT assay and hemolysis test were performed to examine the toxicity of the scaffolds. The hydrophobicity or hydrophilicity of nanofibers was explored by the contact angle test. Scaffolds were examined on the full-thickness wound created on Wistar rats, followed by histological analyses and coagulation tests. The results of FTIR, TEM, and SEM indicated the proper distribution of nanoparticles and core-shell scaffold. The drug loading was about 3 %. About 80 % of the drug was released in the first 7 days. Scaffolds showed hydrophobic properties (114.63° ± 3.6) with no cytotoxicity. The proposed scaffold was able to close 94 % of the wound era after 14 days in the animal model and positively affected re-epithelization and angiogenesis. Moreover, nanofibers containing chitosan nanoparticles exhibited a proper blood coagulation ability in the tail cut model. Finally, it was found that this scaffold, in addition to a biological dressing, can be considered as a drug delivery, and according to the results obtained, this dressing has hydrophobic properties and has also shown good performance against superficial bleeding coagulation. And it has not shown any cytotoxicity for red blood cells and mesenchymal stem cells.
RESUMO
Transplantation of stem cells using biodegradable and biocompatible nanofibrous scaffolds is a promising therapeutic approach for treating inherited retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. In this study, conjunctiva mesenchymal stem cells (CJMSCs) were seeded onto poly-L-lactic acid (PLLA) nanofibrous scaffolds and were induced to differentiate toward photoreceptor cell lineages. Furthermore, the effects of orientation of scaffold on photoreceptor differentiation were examined. Scanning electron microscopy (SEM) imaging, quantitative real time RT-PCR (qPCR) and immunocytochemistry were used to analyze differentiated cells and their expression of photoreceptor-specific genes. Our observations demonstrated the differentiation of CJMSCs to photoreceptor cells on nanofibrous scaffolds and suggested their potential application in retinal regeneration. SEM imaging showed that CJMSCs were spindle shaped and well oriented on the aligned nanofiber scaffolds. The expression of rod photoreceptor-specific genes was significantly higher in CJMSCs differentiated on randomly-oriented nanofibers compared to those on aligned nanofibers. According to our results we may conclude that the nanofibrous PLLA scaffold reported herein could be used as a potential cell carrier for retinal tissue engineering and a combination of electrospun nanofiber scaffolds and MSC-derived conjunctiva stromal cells may have potential application in retinal regenerative therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem da Célula , Túnica Conjuntiva/citologia , Células-Tronco Mesenquimais/citologia , Nanofibras/química , Células Fotorreceptoras de Vertebrados/citologia , Alicerces Teciduais/química , Adolescente , Adulto , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Ácido Láctico/farmacologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Nanofibras/ultraestrutura , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Poliésteres , Polímeros/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Engenharia Tecidual , Adulto JovemRESUMO
Neural differentiation of the CD133+/CD34+ subpopulation of human umbilical cord blood stem cells was investigated, and neuro-miR (mir-9 and mir-124) expression was examined. An efficient induction protocol for neural differentiation of hematopoietic stem cells together with the exclusion of retinoic acid in this process was also studied. Transcription of some neural markers such as microtubule-associated protein-2, beta-tubulin III, and neuron-specific enolase was evaluated by real-time PCR, immunocytochemistry, and western blotting. Increased expression of neural indicators in the treated cells confirmed the appropriate neural differentiation, which supported the high efficiency of our defined neuronal induction protocol. Verified high expression of neuro-miRNAs along with neuronal specific proteins not only strengthens the regulatory role of miRNAs in determining stem cell fate but also introduces these miRNAs as novel indicators of neural differentiation. These data highlight the prominent therapeutic potential of hematopoietic stem cells for use in cell therapy of neurodegenerative diseases.
Assuntos
Antígenos CD34/biossíntese , Antígenos CD/biossíntese , Diferenciação Celular/genética , Sangue Fetal/metabolismo , Glicoproteínas/biossíntese , MicroRNAs/metabolismo , Neurônios/metabolismo , Antígeno AC133 , Antígenos CD/sangue , Antígenos CD/isolamento & purificação , Antígenos CD34/sangue , Antígenos CD34/isolamento & purificação , Células Cultivadas , Feminino , Sangue Fetal/citologia , Glicoproteínas/sangue , Glicoproteínas/isolamento & purificação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Neurônios/citologia , Peptídeos/sangue , Peptídeos/isolamento & purificaçãoRESUMO
Manipulation of stem cells and microencapsulation through microfluidic chips has shown more promising results in treating complex conditions, such as spinal cord injury (SCI), than traditional treatments. This study aimed to investigate the potency of neural differentiation and its therapeutic role in SCI animal model of trabecular meshwork mesenchymal stem/stromal cells (TMMSCs) via miR-7 overexpression and microchip-encapsulated. TMMSCs are transduced with miR-7 via a lentiviral vector (TMMSCs-miR-7[+]) and encapsulated in alginate-reduced graphene oxide (alginate-rGO) hydrogel via a microfluidic chip. Neuronal differentiation of transduced cells in hydrogel (3D) and tissue cultures plate (2D) was assessed by expressing specific mRNAs and proteins. Further evaluation is being carried out through 3D and 2D TMMSCs-miR-7(+ and -) transplantation into the rat contusion SCI model. TMMSCs-miR-7(+) encapsulated in the microfluidic chip (miR-7-3D) increased nestin, ß-tubulin III, and MAP-2 expression compared with 2D culture. Moreover, miR-7-3D could improve locomotor behavior in contusion SCI rats, decrease cavity size, and increase myelination. Our results revealed that miR-7 and alginate-rGO hydrogel were involved in the neuronal differentiation of TMMSCs in a time-dependent manner. In addition, the microfluidic-encapsulated miR-7 overexpression TMMSCs represented a better survival and integration of the transplanted cells and the repair of SCI. Collectively, the combination of miR-7 overexpression and encapsulation of TMMSCs in hydrogels may represent a promising new treatment for SCI.