Conjunctiva Mesenchymal Stem Cells Research Articles

The optimal electrical stimulation for neural differentiation of conjunctiva mesenchymal stem cells.

The combination of biomaterial conductive scaffolds and electrical stimulation (ES) dramatically promotes stem cell differentiation into electro-responsive cells like neural cells. In this study, we aimed to fabricate PCL/PPY nanofiber scaffolds through the electrospinning method and investigate the effect of ES duration on neural differentiation of Conjunctiva Mesenchymal Stem Cells (CJMSCs). The topography of the fabricated scaffold was characterized using SEM and TEM microscopy, and its mechanical and other properties were determined by tensile, TGA, FTIR, and Contact angle tests. CJMSCs were seeded on the scaffolds and then subjected to electrical current (115 V m-1 at 100 Hz) with durations of 1, 3, and 7 min for 3 days. Then the effect of nanofiber scaffold and electrical currents on cell viability and expression of neural marker genes (Nestin, β-tubulin, MAP-2) was investigated by MTT assay and qPCR analysis. Our results revealed the good biocompatibility of the PCL-PPy nanofiber scaffold, and according to q-PCR results, the electrical stimulation of 1 min day-1 for 3 days can induce neural differentiation of CJMSCs as indicated by the fold change of gene expression of Nestin (~127), B-tubulin (~30), and MAP-2 (~52). This study emphasizes that the utilization of an electrically conductive nanofibrous scaffold in conjunction with electrical current has potential applications in the field of neural tissue engineering.

Synergistic effect of miR-9 overexpression and electrical induction on differentiation of conjunctiva mesenchymal stem cells into photoreceptor-like cells.

A variety of genes and materials can induce the differentiation of stem cells. The purpose of this work was to investigate the effect of microRNA-9 (miR-9) overexpression and electrical induction on the photoreceptor differentiation of Conjunctiva Mesenchymal Stem Cells (CJMSCs). In this study, an electroconductive scaffold (silk fibroin polymer (SF) and reduced graphene oxide (rGo) nanoparticles) was fabricated by electrospinning method, and its characteristics such as diameter, graphene distribution, compound, conductivity, and toxicity were evaluated by scanning and transmission electron microscopy (SEM and TEM), FTIR, electrochemical impedance spectroscopy, and MTT assay. The cells were transduced by a lentiviral vector carrying miR-9, then electrical induction was implied on mir-9-CJMSCs, cultivated on the fabricated scaffold, and the expressions of neural and photoreceptor marker genes were evaluated by RT-qPCR. A uniform, smooth appearance with lower diameter, uniform distribution of rGo nanoparticles across the fibers, and lower resistance were shown in SF-rGo fibrous scaffold. After electrical stimulation, lower and higher expression of neural marker genes and photoreceptor marker genes (Rhodopsin, PKC) were documented, respectively. Finally, we proposed that the combinational approach of miR-9 overexpression and electrical induction leads CJMSCs to photoreceptor-like cells.

Surface modification of neurotrophin-3 loaded PCL/chitosan nanofiber/net by alginate hydrogel microlayer for enhanced biocompatibility in neural tissue engineering.

This study prepared a novel three-dimensional nanocomposite scaffold by the surface modification of PCL/chitosan nanofiber/net with alginate hydrogel microlayer, hoping to have the privilege of both nanofibers and hydrogels simultaneously. Bead free randomly oriented nanofiber/net (NFN) structure composed of chitosan and polycaprolactone (PCL) was fabricated by electrospinning method. The low surface roughness, good hydrophilicity, and high porosity were obtained from the NFN structure. Then, the PCL/chitosan nanofiber/net was coated with a microlayer of alginate containing neurotrophin-3 (NT-3) and conjunctiva mesenchymal stem cells (CJMSCs) as a new stem cell source. According to the cross-sectional FESEM, the scaffold shows a two-layer structure with interconnected pores in the range of 20 μm diameter. The finding revealed that the surface modification of nanofiber/net by alginate hydrogel microlayer caused lower inflammatory response and higher proliferation of CJMSCs than the unmodified scaffold. The initial burst release of NT-3 was 69% in 3 days which followed by a sustained release up to 21 days. The RT-PCR analysis showed that the expression of Nestin, MAP-2, and β-tubulin III genes were increased 6, 5.4, and 8.8-fold, respectively. The results revealed that the surface-modified biomimetic scaffold possesses enhanced biocompatibility and could successfully differentiate CJMSCs to the neuron-like cells.

The potency of hsa-miR-9-1 overexpression in photoreceptor differentiation of conjunctiva mesenchymal stem cells on a 3D nanofibrous scaffold

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.

In vitro differentiation of conjunctiva mesenchymal stem cells into insulin producing cells on natural and synthetic electrospun scaffolds

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-value ≤ 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 nanofibrous scaffolds reported herein could be used as a potential supportive matrix for islet tissue engineering.

Effect of let-7a overexpression on the differentiation of conjunctiva mesenchymal stem cells into photoreceptor-like cells.

Objective(s):MicroRNAs (miRNAs) could regulate many cellular processes such as proliferation and differentiation. let-7a miRNA is one of the key regulators in the developmental transition of retinal progenitor cells into differentiated cells. Current evidence suggests that mesenchymal stem cells (MSCs) can isolate from various tissues such as bone marrow and conjunctiva. In this study, we investigated the effect of let-7a overexpression on induced differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells.Materials and Methods:After isolation and characterization, CJMSCs were transduced with lentiviruses containing let-7a or empty vector. The effect of let-7a overexpression on expression of photoreceptor-specific markers was evaluated by quantitative real-time PCR (RT-qPCR) after 28 and 42 days of transduction.Results:The relative expression of rhodopsin and recoverin genes was evaluated by RT-qPCR in let-7a overexpressing cells, control vector transduced cells and untransduced CJMSCs (control cells). Our results indicated that following overexpression of let-7a, after 28 and 42 days of transduction, significant up-regulation in the expression of recoverin (574.7 and 43.9 folds) and rhodopsin (3334.7 and 53.1 folds) were observed, respectively.Conclusion:Our findings indicate that overexpression of let-7a microRNA can increase the expression of photoreceptor-specific genes in CJMSCs. Moreover, it is prospective that let-7a overexpression can use as an alternative protocol for the differentiation of mesenchymal stem cells into photoreceptors. It seems that the effect of let-7a on the differentiation of CJMSCs into photoreceptors is also time-dependent.

Conductive electrospun scaffolds with electrical stimulation for neural differentiation of conjunctiva mesenchymal stem cells.

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 1:115V/m, 100-Hz frequency and current 2:115v/m voltages, 0.1-Hz frequency) were applied for 7days. 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±20nm 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 β-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.

Differentiation of conjunctiva mesenchymal stem cells into secreting islet beta cells on plasma treated electrospun nanofibrous scaffold

Transplantation of stem cells using biocompatible nanofibrous scaffolds is a promising therapeutic method for treating Diabetic Mellitus. The aim of this study was to derive insulin-producing cells (IPCs) from conjunctiva-derived mesenchymal stem cell (CJMSCs) and to compare the functionality of differentiated IPCs in a three-dimensional (3D) culture with 2D. Furthermore, the effects of hydrophobicity of scaffold on IPCs differentiation were examined. Scanning electron microscopy (SEM), quantitative real times PCR (qPCR), Immunostaining and flow cytometry were used to analyze fabricated scaffold and the presence of IPCs. Functional maturity of differentiated cells was determined by measuring insulin release and the creation of IPCs was confirmed via gene and protein expression. In this study, the induced CJMSCs were morphologically similar to pancreatic islet-like cells. The expression of the islet-associated genes (glucagon, insulin and Pdx-1) and the insulin release (2.5-fold) in 3D-cultured cells was significantly higher than the 2D. The expression of IPCs genes was significantly higher in CJMSCs differentiated on plasma-treated nanofibers compared to those on untreated scaffolds. In conclusion, the results show that CJMSCs might be a new source for Diabetic Mellitus therapy and the nanofibrous scaffold could be used as a potential cell carrier for islet tissue engineering.

Core-shell fibrous scaffold as a vehicle for sustained release of retinal pigmented epithelium-derived factor (PEDF) for photoreceptor differentiation of conjunctiva mesenchymal stem cells.

Coaxial electrospinning technique was introduced as a flexible and promising technique for the fabrication of core-shell fibrous scaffold from poly ethylene glycol/poly caprolactone (PEG/PCL), where retinal pigmented epithelium-derived factor (PEDF) was encapsulated in the core, for photoreceptor differentiation of conjunctiva mesenchymal stem cells (CJMSCs) seed on scaffolds. The morphology and structure of fibers were characterized using SEM and TEM and photoreceptor differentiation was examined by quantitative real time PCR (qPCR). Release study showed that, a sustained release of PEDF from PEG/PCL scaffold was observed over 14 days. qPCR analysis demonstrated that rhodopsin (as a main photoreceptor gene) was significantly expressed in CJMSCs cultured on scaffold loaded with PEDF. According to the result, the core-shell scaffold loaded with PEDF (PEG + PEDF)/PCL) has superior control over factor release profile and has a potential for guiding photoreceptor differentiation of mesenchymal stem cells and promoting retinal regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3514-3519, 2017.

Fibrin gel as a scaffold for photoreceptor cells differentiation from conjunctiva mesenchymal stem cells in retina tissue engineering

Stem cell-based therapies are attraction approaches for regenerative medicine for treating retinal diseases. One of the limitations in cell therapy is cell death following post-injection whit preventing functional integration with retinal tissue. Fibrin gel, a bio-polymeric material with excellent biocompatibility, provides numerous advantages as a tissue engineering scaffold and a stem cell carrier. Therefore, current research is focusing on developing fibrin hydrogel scaffolds to protect stem cells during delivery and to stimulate endogenous regeneration through interactions of transplanted stem cells and retinal tissue. In this study fibrin gel was used as hydrogel scaffold for immobilization of cells. The structural characteristics of fibrin gel scaffold were examined with SEM. Rheological properties of fibrin gel were measured by rheometer and biodegradation rate of fibrin were assayed for 2 weeks. After isolation of stem cells CJMSCs, the cells were differentiated into photoreceptor-like cells by exposing with taurin for 14 days in tissue culture plate (TCP group) and fibrin hydrogel (3 D group). The attachment of cells was analyzed with SEM and MTT. The expression of rhodopsin, PKC, CRX, recoverin, peripherin, nestin and RPE65 as photoreceptor-like cell markers was evaluated by immunocytochemistry and quantitative real-time PCR (RT-PCR) in TCP and 3 D groups. The results of SEM analysis showed CJMSCs were well attached in fibrin gels and there were good integrity between cells and scaffold. The elastic modulus and constant degradation of the gel contributes to the growth and proliferation of cells. There was no toxicity effect of fibrin hydrogel on cells and the viability of cultured cells was higher in 3 D fibrin gels in comparison with TCP groups. After 2 weeks, the expression of rhodopsin, PKC, CRX, peripherin, recoverin, nestin and RPE65 as special markers of photoreceptor cells were detected by Real time PCR and immunofluorescence that these expressions in 3 D groups were higher than TCP groups. In conclusion, our findings showed that application of readily available sources of adult stem cells like human conjunctiva stem cells encapsulated in fibrin gel could be interesting strategy to enhance photoreceptor progenitor cell numbers for repair and regeneration of retina disease such as photoreceptor injury.

Conjunctiva derived mesenchymal stem cell (CJMSCs) as a potential platform for differentiation into corneal epithelial cells on bioengineered electrospun scaffolds.

With the aid of tissue engineering, the capability of nanofibrous scaffolds was examined for corneal epithelial differentiation of Conjunctiva mesenchymal stem cells (CJMSCs) as a new source. This study tried to provide a synthetic corneal sheet which could be applied in corneal regeneration. The hybrid arrangement of fine Silk fibers with the diameter of 93.65 nm and the polyurethane (PU) fibers with an average diameter of 575.5 makes a mat with sufficient mechanical properties and interconnected pores that could accelerate nutrient diffusion. After methanol and plasma treatment, the highest biocompatibility of Silk-PU hybrid nanofibrous scaffolds based on cell attachment and proliferation were proven using MTT assay. The differentiation morphology of cells was observed by SEM micrographs of cells seeded on the scaffold after 12 days treatment through the differentiation medium. Despite up-regulation of Cytokeratin (CK)3, CK8, CK12, Desmocollin (DSC)1, and Desmoglein (DSG)1 in the treated PU and silk hybrid nanofibrous scaffolds, the relative expression of CK3, CK8, and CK12 genes were highest among TCP, treated PU and silk nanofibrous scaffolds. Immunocytochemistry (ICC) analysis was used to characterize the protein expression of CK3 in CJ-MSCs seeded on different groups. Although, For both TCP and hybrid group, CJ-MSCs were positive for corneal epithelial-specific marker CK3, ICC data demonstrated that CJ-MSCs seeded on the hybrid group likewise PU or Silk pure nanofibrous scaffolds, could differentiate into corneal epithelial-like cells. Finally, it was inferred that the hybrid PU and silk nanofibrous scaffold has the potential for the treatment of corneal epithelium. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2703-2711, 2017.

Retina tissue engineering by conjunctiva mesenchymal stem cells encapsulated in fibrin gel: Hypotheses on novel approach to retinal diseases treatment

BackgroundRetinitis pigmentosa (RP) and age related macular degeneration (AMD) are two retinal diseases that progress by photoreceptor cells death. In retinal transplantation studies, stem and progenitor cells inject into the sub retinal space or vitreous and then these cells can be migrate to the site of retinal degeneration and locate in the host retina and restitute vision. Presentation of the hypothesisOur hypothesis suggests that using human conjunctiva stem cells (as the source for increasing the number of human stem cells progenitor cells in retina dysfunction diseases) with fibrin gel and also assessing its relating in vitro (cellular and molecular processes) and in vivo (vision tests and pathology) could be a promising strategy for treatment of AMD and RP disorders. Testing the hypothesisIn this idea, we describe a novel approach for retina tissue engineering with differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells in fibrin gel with induction medium contain taurine. For assessment of differentiation, immunocytochemistry and real time PCR are used for the expression of Rhodopsin, RPE65, Nestin as differentiated photoreceptor cell markers in 2D and 3D culture. The results show that fibrin gel will offer a proper 3D scaffold for CJMSCs derived photoreceptor cell-like cells. Implications of the hypothesisApplication of immune-privileged, readily available sources of adult stem cells like human conjunctiva stem cells with fibrin gel would be a promising strategy to increase the number of photoreceptor progenitor cells and promote involuntary angiogenesis needed in retina layer repair and regeneration.

Effect of parameters on the quality of core-shell fibrous scaffold for retinal differentiation of conjunctiva mesenchymal stem cells.

This article describes the coaxial electrospinning to generate core-shell fibers from polycaprolactone (PCL) and polyethylene glycol (PEG) for differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells by delivery of taurine. Also, the effects of many parameters such as polymer concentration, nozzle collector distance, applied voltage, and outer solution flow rate on creation of the core-shell structure were examined. The morphology and structure of fibers were characterized using scanning, transmission electron microscopy, and fourier transform infrared spectroscopy and then retinal differentiation was examined by quantitative real time PCR (qPCR). Significant variations between 25% and 35% PEG concentration groups for fiber diameter were documented. As faster flowing rates from the outer nozzle (PCL fluid) were applied, the creation possibility of fibrous scaffold was increased. The lowest diameter and the best quality alignment of core-shell fibrous scaffold were achieved in 22 Kv and 24 Kv. As rising distancing were applied, the fibrous diameter increased and spraying was observed. qPCR analysis demonstrated the differentiation of CJMSCs to photoreceptor like cells on PEG/PCL scaffolds. According to the result, we have proved successful in the creation of core/shell fibrous scaffold of PEG/PCL by coaxial electrospinning for retinal tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 189-197, 2017.

Isolation and Expansion of Mesenchymal Stem Cells from Human Conjunctival Tissue.

Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells (MSCs) from conjunctiva stromal tissue, with applications to stem cell biology and regenerative medicine. This protocol is based on an explant culture protocol for the adhesion and migration of MSCs from tissue biopsy in primary culture and expansion of the MSC population by passaging cells on the surface of plastic culture dishes. Conjunctiva mesenchymal stem cells (CJMSCs) are generally isolated from small biopsies of human conjunctival tissue. Epithelial sheets are carefully loosened and removed by enzyme treatment after 18 hr and placed in fresh medium supplemented with growth factors. When the cells migrating from tissue segments in primary cultures become nearly confluent, the culture is treated with trypsin. A purified population of CJMSCs that retain proliferation and differentiation potential for many passages can be obtained 2 weeks after the initiation of culture.

Erratum to: High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds

Erratum to: High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds

High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds

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.

Neurogenic differentiation of human conjunctiva mesenchymal stem cells on a nanofibrous scaffold

The selection of a good quality scaffold is an essential strategy for tissue engineering. Ideally, the scaffold should be a functional and structural biomimetic of the native extracellular matrix and support multiple tissue morphogenesis. However, investigators have previously shown that three-dimensional nanofibrous scaffolds are capable of influencing cellular behavior. In this study, we experimented with a three-dimensional nanofibrous scaffold fabricated from aligned-poly L-lactic acid (PLLA) for its ability to support neurogenic and hinder dopaminergic differentiation of conjunctiva mesenchymal stem cells (CJMSCs) in vitro. In this work, CJMSCs were seeded onto nanofibrous scaffolds, and were induced to differentiate along neurogenic lineages by culturing in specific differentiation media. Scanning electron microscopy imaging, RT-PCR and immunocytochemistry were used to analyze cultivated CJMSCs on scaffold and their expression of neurogenic-specific markers. We found a lack of expression of dopaminergic genes in CJMSCs seeded on align PLLA scaffold, while neurocyte-cell markers including Nestin, NSE, MAP-2 and beta-Tubulin III were expressed in these cells. On the basis of these experimental results, we conclude that the nanofibrous PLLA scaffold reported herein could be used as a potential cell carrier in neural tissue engineering and that these scaffolds could be useful for the partial inhibition of the dopaminergic differentiation of CJMSCs.