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In JoVE (1)
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Articles by Basak E. Uygun in JoVE
JoVE Bioengineering
وDecellularization Recellularization الأكباد من الجامعة
decellularization التروية هو تقنية جديدة لإنتاج السقالات الكبد كله أن يحتفظ تكوين الجهاز في المصفوفة خارج الخلية والمعمارية المصغرة. هنا ، وصفت طريقة إعداد السقالات الجهاز بأكمله باستخدام decellularization نضح وإعادة تعمير لاحقة مع خلايا الكبد. يمكن أن تتولد ترقيع الكبد الوظيفية وزرع باستخدام هذه التقنية.
Other articles by Basak E. Uygun on PubMed
Differentiating Stem Cells into Liver
Research involving differentiated embryonic stem (ES) cells may revolutionize the study of liver disease, improve the drug discovery process, and assist in the development of stem-cell-based clinical therapies. Generation of ES cell-derived hepatic tissue has benefited from an understanding of the cytokines, growth factors and biochemical compounds that are essential in liver development, and this knowledge has been used to mimic some aspects of embryonic development in vitro. Although great progress has been made in differentiating human ES cells into liver cells, current protocols have not yet produced cells with the phenotype of a mature hepatocyte. There is a significant need to formally establish criteria that would define what constitutes a functional human stem cell-derived hepatocyte. Here, we explore current challenges and future opportunities in development and use of ES cell-derived liver cells. ES-derived hepatocytes could be used to better understand liver biology, begin the process of "personalizing" health care, and to treat some forms of liver disease.
Retinal Pigment Epithelium Differentiation of Stem Cells: Current Status and Challenges
Degeneration and loss of retinal pigment epithelium (RPE) is the cause of a number of degenerative retinal diseases, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, leading to blindness that affects three million Americans as of now. Transplantation of RPE aims to restore retinal structure and the interaction between the RPE and photoreceptors, which is fundamental to sight. Although a significant amount of progress has been made in the past 20 years in autologous RPE transplantation, sources for RPE cells are limited. Recent advances in stem cell culture and differentiation techniques have allowed the generation of RPE cells from pluripotent stem cells. In this review, we discuss strategies for generating functional RPE cells from human embryonic stem cells and induced pluripotent stem cells, and summarize transplantation studies of these derived RPEs. We conclude with challenges in cell-replacement therapies using human embryonic and induced pluripotent stem cell-derived RPEs.
Effects of Immobilized Glycosaminoglycans on the Proliferation and Differentiation of Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are adult stem cells with potential for multilineage differentiation. They represent an attractive cell source alternative to embryonic stem cells for therapeutic applications. Optimal utilization of MSCs for tissue engineering requires improved biomaterials that can enhance their growth and direct differentiation. The biological activity of glycosaminoglycans (GAGs) has been previously exploited for use in tissue engineering applications. In this study, MSC proliferation and differentiation was studied on GAG-derivatized chitosan membranes. The GAGs included heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid. The covalent GAG immobilization method and amount of immobilized GAG were varied. It was found that MSC growth increased as much as fivefold on GAG-immobilized surfaces compared to tissue culture plastic and chitosan-only controls. The MSC growth rates increased significantly with increasing GAG density on the culture surfaces. The MSC proliferation rates on heparin, heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate exhibited nonlinear increases with the level of fibronectin binding on these surfaces. In contrast, MSC proliferation on hyaluronic acid and chondroitin 4-sulfate was found to be independent of fibronectin or vitronectin binding on the surfaces, suggesting that these GAGs influenced MSC proliferation through different mechanisms. In conclusion, the results indicate that GAG immobilization on chitosan scaffolds provides an effective means of manipulating MSC proliferation and has promising potential for directing MSC differentiation in tissue engineering applications employing chitosan.
High Throughput Single Cell Bioinformatics
Advances in systems biology and bioinformatics have highlighted that no cell population is truly uniform and that stochastic behavior is an inherent property of many biological systems. As a result, bulk measurements can be misleading even when particular care has been taken to isolate a single cell type, and measurements averaged over multiple cell populations in a tissue can be as misleading as the average height at an elementary school. There is a growing need for experimental techniques that can provide a combination of single cell resolution, large cell populations, and the ability to track cells over multiple time points. In this article, a microwell array cytometry platform was developed to meet this need and investigate the heterogeneity and stochasticity of cell behavior on a single cell basis. The platform consisted of a microfabricated device with high-density arrays of cell-sized microwells and custom software for automated image processing and data analysis. As a model experimental system, we used primary hepatocytes labeled with fluorescent probes sensitive to mitochondrial membrane potential and free radical generation. The cells were exposed to oxidative stress and the responses were dynamically monitored for each cell. The resulting data was then analyzed using bioinformatics techniques such as hierarchical and k-means clustering to visualize the data and identify interesting features. The results showed that clustering of the dynamic data not only enhanced comparisons between the treatment groups but also revealed a number of distinct response patterns within each treatment group. Heatmaps with hierarchical clustering also provided a data-rich complement to survival curves in a dose response experiment. The microwell array cytometry platform was shown to be powerful, easy to use, and able to provide a detailed picture of the heterogeneity present in cell responses to oxidative stress. We believe that our microwell array cytometry platform will have general utility for a wide range of questions related to cell population heterogeneity, biological stochasticity, and cell behavior under stress conditions.
Membrane Thickness is an Important Variable in Membrane Scaffolds: Influence of Chitosan Membrane Structure on the Behavior of Cells
Cell and tissue responses to polymeric materials are orchestrated in part by the conformations of adsorbed plasma proteins. Thus, the chemical properties of a polymer membrane that govern protein adsorption behavior can play an important role in determining the biological properties of tissue engineered scaffolds derived from that polymer. In this study, we explored the role of membrane thickness as a factor influencing cell adhesion and proliferation on chitosan membranes with and without covalently attached glycosaminoglycans. Rat mesenchymal stem cells (MSCs) cultured on chitosan membranes of various thicknesses demonstrated significantly improved cell adhesion, spreading and proliferation as membrane thickness was increased. Rat hepatocytes displayed increased spreading on the substrate with increasing membrane thickness, similar to MSCs. Increased thickness reduced the overall crystallinity of the membrane, and the data indicate that the improved cellular responses were likely due to enhanced adsorption of serum vitronectin, presumably due to reduced membrane crystallinity. These results demonstrate that membrane thickness is an important design variable that can be manipulated in chitosan-based scaffolds to achieve enhanced cell spreading, proliferation and function.
Cell Delivery: from Cell Transplantation to Organ Engineering
Cell populations derived from adult tissue and stem cells possess a great expectation for the treatment of several diseases. Great efforts have been made to generate cells with therapeutic impact from stem cells. However, it is clear that the development of systems to deliver such cells to induce efficient engraftment, growth, and function is a real necessity. Biologic and artificial scaffolds have received significant attention for their potential therapeutic application when use to form tissues in vitro and facilitate engraftment in vivo. Ultimately more sophisticated methods for decellularization of organs have been successfully used in tissue engineering and regenerative medicine applications. These decellularized tissues and organs appear to provide bioactive molecules and bioinductive properties to induce homing, differentiation, and proliferation of cells. The combination of decellularized organs and stem cells may dramatically improve the survival, engraftment, and fate control of transplanted stem cells and their ultimate clinical utility, opening the doors to a new era of organ engineering.
Organ Reengineering Through Development of a Transplantable Recellularized Liver Graft Using Decellularized Liver Matrix
Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.
