RESUMO
Mesenchymal stromal cells (MSC), with progenitor cell and immunological properties, have been cultivated from numerous vascularized tissues including bone marrow, adipose tissue and the corneal-limbus of the eye. After observing mesenchymal cells as contaminants in primary cultures of vascular endothelial cells derived from the choroidal tunic of the human eye, we investigated whether the choroid might also provide a source of cultured MSC. Moreover, we examined the effect of the choroidal stromal cells (Ch-SC) on the proliferation of freshly isolated choroidal vascular endothelial cells (ChVEC) in vitro. The phenotype of cultures established from five choroidal tissue donors was examined by flow cytometry and immunocytochemistry. The potential for mesenchymal cell differentiation was examined in parallel with MSC established from human bone marrow. Additional cultures were growth-arrested by treatment with mitomycin-C, before being tested as a potential feeder layer for ChVEC. The five unique cultures established from choroidal stroma displayed a phenotype consistent with the accepted definition for MSC (CD34-, CD45-, HLA-DR-, CD73+, CD90+, and CD105+), including the capacity for mesenchymal differentiation when cultivated under osteogenic, adipogenic and chondrogenic conditions. Growth-arrested Ch-SC inhibited the proliferation of ChVEC derived from five separate donors. Cultures of Ch-SC secreted approximately 40-fold higher concentrations of the anti-angiogenic factor pigment epithelium derived factor (PEDF/serpin F1) compared to the pro-angiogenic factor, vascular endothelial growth factor (VEGF), regardless of normal or growth-arrested state. Our results provide first evidence of a resident MSC cell type within the choroid and encourage investigation of new mechanisms for altering the growth of ChVEC.
Assuntos
Corioide/irrigação sanguínea , Células Endoteliais/citologia , Endotélio Vascular/citologia , Células-Tronco Mesenquimais/citologia , Células Estromais/citologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Corioide/citologia , Citometria de Fluxo , Humanos , Fenótipo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Silk fibroin membrane displays potential for ocular tissue reconstruction as demonstrated by its ability to support a functioning retinal pigment epithelium (RPE) in vitro. Nevertheless, translation of these findings to the clinic will require the use of membranes that can be readily handled and implanted into diseased retinas, with minimal impact on the surrounding healthy tissue. To this end, we optimized the physical properties of fibroin membranes to enable surgical handling during implantation into the retina, without compromising biocompatibility or permeability. Our central hypothesis is that optimal strength and permeability can be achieved by combining the porogenic properties of poly(ethylene glycol) (PEG) with the crosslinking properties of horseradish peroxidase (HRP). Our study reveals that PEG used in conjunction with HRP enables the production of fibroin membranes with superior handling properties to conventional fibroin membranes. More specifically, the modified membranes could be more easily implanted into the retinas of rats and displayed good evidence of biocompatibility. Moreover, the modified membranes retained the ability to support construction of functional RPE derived from pluripotent stem cells. These findings pave the way for preclinical studies of RPE-implantation using the optimized fibroin membranes.
Assuntos
Fibroínas/química , Membranas Artificiais , Próteses Visuais , Animais , Bombyx , Células-Tronco Embrionárias Humanas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Permeabilidade , Fagocitose , Ratos , Epitélio Pigmentado da Retina/citologia , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Resistência à TraçãoRESUMO
Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome-with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)-but very limited changes in the small RNA transcriptome-totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.
Assuntos
Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/parasitologia , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Ocular/genética , Toxoplasmose Ocular/imunologia , Idoso , Cadáver , Técnicas de Cultura de Células , Linhagem Celular , Separação Celular , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Fenômenos Imunogenéticos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RNA-Seq , Epitélio Pigmentado da Retina/citologia , Toxoplasmose Ocular/parasitologiaRESUMO
Herein, we review the safety, efficacy, regulatory standards and ethical implications of the use of stem cells in ocular disease. A literature review was conducted, registered clinical trials reviewed, and expert opinions sought. Guidelines and codes of conduct from international societies and professional bodies were also reviewed. Collated data is presented on current progress in the field of ocular regenerative medicine, future challenges, the clinical trial process and ethical considerations in stem cell therapy. A greater understanding of the function and location of ocular stem cells has led to rapid advances in possible therapeutic applications. However, in the context of significant technical challenges and potential long-term complications, it is imperative that stem cell practices operate within formal clinical trial frameworks. While there remains broad scope for innovation, ongoing evidence-based review of potential interventions and the development of standardized protocols are necessary to ensure patient safety and best practice in ophthalmic care.
Assuntos
Oftalmopatias/terapia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Humanos , Limbo da Córnea/citologia , Segurança do PacienteRESUMO
Purpose: RPE cell transplantation as a potential treatment for AMD has been extensively investigated; however, in AMD, ultrastructural damage affects both the RPE and its underlying matrix support, the Bruch's membrane (BrM). An RPE monolayer supported by a surrogate scaffold could thus provide a more effective approach to cell-based therapy for AMD. Toward this goal, we aimed to establish a functional human induced pluripotent stem cell-derived (hiPSC)-RPE monolayer on a Bombyx mori silk fibroin (BMSF) scaffold. Methods: RPE differentiated from five distinct hiPSC lines were cultured on BMSF membrane coated with extracellular matrix (ECM, COL1), and either regular tissue culture plastic or Transwell coated with ECM (LAM-TCP). Morphologic, gene and protein expression, and functional characteristics of the hiPSC-RPE cultured on different membranes were compared in longitudinal experiments spanning 1 day to ≥3 months. Results: The hiPSC-RPE monolayers on ECM-coated BMSF and TCP could be maintained in culture for ≥3 months and displayed RPE-characteristic morphology, pigmentation, polarity, and expression of RPE signature genes and proteins. Furthermore, hiPSC-RPE on both ECM-coated BMSF and TCP displayed robust expression and secretion of several basement membrane proteins. Importantly, hiPSC-RPE cells on COL1-BMSF and LAM-TCP showed similar efficacy in the phagocytosis and degradation of photoreceptor outer segments. Conclusions: A biomaterial scaffold manufactured from silk fibroin supports the maturation and long-term survival of a functional hiPSC-RPE monolayer. This has significant implications for both in vitro disease modeling and in vivo cell replacement therapy.
Assuntos
Lâmina Basilar da Corioide , Fibroínas/química , Células-Tronco Pluripotentes Induzidas/citologia , Membranas Artificiais , Epitélio Pigmentado da Retina/citologia , Western Blotting , Diferenciação Celular , Células Cultivadas , Materiais Revestidos Biocompatíveis , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/metabolismo , Alicerces TeciduaisRESUMO
The interactions of cells with the surface of materials is known to be influenced by a range of factors that include chemistry and roughness; however, it is often difficult to probe these factors individually without also changing the others. Here we investigate the role of roughness on cell adhesion while maintaining the same underlying chemistry. This was achieved by using a polymerization in mold technique to prepare poly(hydroxymethyl methacrylate) hydrogels with either a flat topography or a topography that replicated the microscale features of lotus leaves. These materials were then assessed for cell adhesion, and atomic force microscopy and contact angle analysis were then used to probe the physical reasons for the differing behavior in relation to cell adhesion.
Assuntos
Hidrogéis/química , Lotus/anatomia & histologia , Folhas de Planta/anatomia & histologia , Animais , Adesão Celular/efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Microscopia de Força Atômica , Poli-Hidroxietil Metacrilato/química , Poli-Hidroxietil Metacrilato/farmacologiaRESUMO
Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+ /K+ -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Antígenos de Diferenciação/biossíntese , Lâmina Basilar da Corioide , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica , Teste de Materiais , Membranas Artificiais , Epitélio Pigmentado da Retina/metabolismo , Linhagem Celular , Fibroínas , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologiaRESUMO
When used as scaffolds for cell therapies, biomaterials often present basic handling and logistical problems for scientists and surgeons alike. The quest for an appropriate mounting device for biomaterials is therefore a significant and common problem. In this review, we provide a detailed overview of the factors to consider when choosing an appropriate mounting device including those experienced during cell culture, quality assurance, and surgery. By way of example, we draw upon our combined experience in developing epithelial cell therapies for the treatment of eye diseases. We discuss commercially available options for achieving required goals and provide a detailed analysis of 4 experimental designs developed within our respective laboratories in Australia, the United Kingdom, and Belgium.
Assuntos
Materiais Biocompatíveis/química , Terapia Baseada em Transplante de Células e Tecidos/métodos , Âmnio/citologia , Humanos , OftalmologiaRESUMO
Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.
RESUMO
A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.
RESUMO
A monomer that contained the RGD ligand motif was synthesized and copolymerized with 2-hydroxyethyl methacrylate using polymerization-induced phase separation methods to form poly(2-hydroxyethyl methacrylate)-based hydrogel sponges. The sponges had morphologies of aggregated polymer droplets and interconnected pores, the pores having dimensions in the order of 10 µm typical of PHEMA sponges. RGD-containing moieties appeared to be evenly distributed through the polymer droplets. Compared to PHEMA sponges that were not functionalized with RGD, the new sponges containing RGD allowed greater invasion by human corneal epithelial cells, by advancing the attachment of cells to the surface of the polymer droplets.
Assuntos
Materiais Biocompatíveis/química , Oligopeptídeos/química , Poli-Hidroxietil Metacrilato/química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Ligantes , Microscopia Eletrônica de TransmissãoRESUMO
We have compared the effects of different sterilization techniques on the properties of Bombyx mori silk fibroin thin films with the view to subsequent use for corneal tissue engineering. The transparency, tensile properties, corneal epithelial cell attachment and degradation of the films were used to evaluate the suitability of certain sterilization techniques including gamma-irradiation (in air or nitrogen), steam treatment and immersion in aqueous ethanol. The investigations showed that gamma-irradiation, performed either in air or in a nitrogen atmosphere, did not significantly alter the properties of films. The films sterilized by gamma-irradiation or by immersion in ethanol had a transparency greater than 98% and tensile properties comparable to human cornea and amniotic membrane, the materials of choice in the reconstruction of ocular surface. Although steam-sterilization produced stronger, stiffer films, they were less transparent, and cell attachment was affected by the variable topography of these films. It was concluded that gamma-irradiation should be considered to be the most suitable method for the sterilization of silk fibroin films, however, the treatment with ethanol is also an acceptable method.
Assuntos
Materiais Biocompatíveis/química , Bombyx , Técnicas de Cultura de Células/métodos , Fibroínas/química , Animais , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Fibroínas/farmacologia , Fibroínas/fisiologia , Humanos , Esterilização , Propriedades de Superfície , Resistência à Tração/fisiologia , Engenharia TecidualRESUMO
We have presently evaluated membranes prepared from Bombyx mori silk fibroin (BMSF), for their potential use as a prosthetic Bruch's membrane and carrier substrate for human retinal pigment epithelial (RPE) cell transplantation. Porous BMSF membranes measuring 3 µm in thickness were prepared from aqueous solutions (3% w/v) containing poly(ethylene oxide) (0.09%). The permeability coefficient for membranes was between 3 and 9 × 10(-5) cm/s by using Allura red or 70 kDa FITC-dextran respectively. Average pore size (±sd) was 4.9 ± 2.3 µm and 2.9 ± 1.5 µm for upper and lower membrane surfaces respectively. Optimal attachment of ARPE-19 cells to BMSF membrane was achieved by pre-coating with vitronectin (1 µg/mL). ARPE-19 cultures maintained in low serum on BMSF membranes for approximately 8 weeks, developed a cobble-stoned morphology accompanied by a cortical distribution of F-actin and ZO-1. Similar results were obtained using primary cultures of human RPE cells, but cultures took noticeably longer to establish on BMSF compared with tissue culture plastic. These findings encourage further studies of BMSF as a substrate for RPE cell transplantation.