Regenerative approaches for congenital and acquired kidney disease have the potential to repair and restore critical cell populations, renal structures, and ultimately function of damaged kidneys. The overriding objective of the studies described in this dissertation was the development, characterization, and recellularization of a three-dimensional (3D) scaffold from rhesus monkey kidneys from different age groups. This was accomplished by optimization of decellularization methods for monkey kidney transverse sections followed by characterization of extracellular matrix (ECM) proteins and biomechanical properties. It was shown that decellularized kidney scaffolds retain expression of major ECM proteins, do not contain residual cell nuclei, and show alterations in biomechanical properties as a result of the decellularization process. Age-related differences in decellularization rate and recellularization capacity using kidney explant culture were assessed. It was found that scaffolds and renal cells from younger donors provided the best outcomes in terms of recellularization potential under the culture conditions described. Recellularization was further explored by utilizing fetal renal glomerular and tubular fractions, which demonstrated that the renal tubular fraction was more effective in recellularizing tubular structures than the glomerular fraction for recellularizing glomerular structures. Proteomics analysis of decellularized kidney scaffolds revealed the presence of several non-ECM proteins. The tissue specificity of kidney and lung scaffolds were compared and showed that the decellularized scaffolds have the ability to spatially organize cells into tissue-specific structures but do not drive tissue-specific gene expression. Lastly, recellularization of kidney scaffolds using NIH-approved human embryonic stem cells (hESC) and a dynamic culture system resulted in the generation of renal constructs that contained tubules with enzymatic activity. The research addressed in this dissertation provides key approaches to using decellularized kidney scaffolds to answer meaningful questions regarding age-related differences, tissue specificity, and tissue engineering strategies for generation of renal constructs for potential regenerative strategies.

The prevalence of end stage renal disease (ESRD) is dramatically increasing. It is estimated that the number of people in the United States undergoing treatment for chronic kidney disease will rise from 500,000 to two million by the year 2030. In infants and children, ESRD is commonly caused by developmental abnormalities including obstruction of the fetal urinary tract. Characterization of developing glomerular precursors including renal vesicles, C- and S-shaped bodies, and podocytes is necessary in order to determine effective ways to recapitulate ontogeny with the goal of repair. New strategies to repair damaged organs using decellularized biological scaffolds are being developed, with the hope of using cell therapy for tissue regeneration within a natural framework. Decellularized scaffolds must serve as a support for cellular interactions and not result in an immune response. This study characterizes fetal kidney development in a clinically relevant rhesus monkey model and assesses whether MHC Class I or Class II proteins remain in a decellularized kidney scaffold. This was addressed by staining fetal kidneys across gestation with hematoxylin and eosin (H&E), v and performing immunohistochemistry (IHC) and enzyme-linked immunosorbant assay (ELISA) on fresh kidneys and kidneys decellularized in 1% sodium dodecyl sulfate (SDS). Results show that renal development in the rhesus monkey mimics that in the human. Glomerular precursors are abundant in the cortex during the first and second trimesters, and by the third trimester, nearly all precursors have differentiated to mature glomeruli. Near term kidneys from the fetal rhesus monkey model of obstructive renal dysplasia show cystic glomeruli with cystic dilatation of Bowman0́9s space, and abnormal glomerular tuft development when compared with unobstructed kidneys. The podocyte marker, WT-1, is strongly expressed in the condensed mesenchyme and nephrogenic zone at the cortical border during the first and second trimesters. By the third trimester, WT-1 expression is confined to mature glomeruli. IHC and ELISA revealed that HLA-E and HLA-DR were removed from the scaffolds during decellularization, while modest amounts of HLA-DQ remained in juvenile decellularized kidney scaffolds. The results from this study add to the growing base of research concerning the use of decellularized scaffolds and will contribute to the ability to repair or replace damaged kidneys in vivo.

A comprehensive overview of the latest achievements, trends, and the current state of the art of this important and rapidly expanding field. Clearly and logically structured, the first part of the book explores the fundamentals of tissue engineering, providing a separate chapter on each of the basic topics, including biomaterials stem cells, biosensors and bioreactors. The second part then follows a more applied approach, discussing various applications of tissue engineering, such as the replacement or repairing of skins, cartilages, livers and blood vessels, to trachea, lungs and cardiac tissues, to musculoskeletal tissue engineering used for bones and ligaments as well as pancreas, kidney and neural tissue engineering for the brain. The book concludes with a look at future technological advances. An invaluable reading for entrants to the field in biomedical engineering as well as expert researchers and developers in industry.

The notion of being able to engineer complete organs has inspired an entire generation of researchers. While recent years have brought significant progress in regenerative medicine and tissue engineering, the immense challenges encountered when trying to engineer an entire organ have to be acknowledged. Despite a good understanding of cell phenotypes, cellular niches and cell-to-biomaterial interactions, the formation of tissues composed of multiple cells remains highly challenging. Only a step-by-step approach will allow the future production of a living tissue construct ready for implantation and to augment organ function. In this book, expert authors present the current state of this approach. It offers a concise overview and serves as a great starting point for anyone interested in the application of tissue engineering or regenerative medicine for organ engineering. Each chapter contains a short overview including physiological and pathological changes as well as the current clinical need. The potential cell sources and suitable biomaterials for each organ type are discussed and possibilities to produce organ-like structures are illustrated. The ultimate goal is for the generated small tissues to unfold their full potential in vivo and to serve as a native tissue equivalent. By integrating and evolving, these implants will form functional tissue in-vivo. This book discusses the desired outcome by focusing on well-defined functional readouts. Each chapter addresses the status of clinical translations and closes with the discussion of current bottlenecks and an outlook for the coming years. A successful regenerative medicine approach could solve organ shortage by providing biological substitutes for clinical use - clearly, this merits a collaborative effort.

Murine embryonic stem cells were seeded into the decellularized scaffolds and cultured up to 14 days with one of three protocols: whole scaffold, perfused scaffold, and sectioned scaffold culture. Cells delivered into the renal artery showed initial glomerular localization with subsequent expansion into adjacent vasculature, interstitium, and tubules. Over time, histological analysis revealed distinct cell morphologies, patterns of cell division and apoptosis, and cell arrangements with Pax-2 and cytokeratin positivity. Cytokeratin is an epithelial cell marker and Pax-2 is necessary for kidney development. The evidence of cellular change of mouse stem cells in response to a rat scaffold is a promising step toward establishment of a xenogenic scaffold source for engineered kidneys.

This multidisciplinary book provides up-to-date information on clinical approaches that combine stem or progenitor cells, biomaterials and scaffolds, growth factors, and other bioactive agents in order to offer improved treatment of urologic disorders including lower urinary tract dysfunction, urinary incontinence, neurogenic bladder, and erectile dysfunction. In providing clinicians and researchers with a broad perspective on the development of regenerative medicine technologies, it will assist in the dissemination of both regenerative medicine principles and a variety of exciting therapeutic options. After an opening section addressing current developments and future perspectives in tissue engineering and regenerative medicine, fundamentals such as cell technologies, biomaterials, bioreactors, bioprinting, and decellularization are covered in detail. The remainder of the book is devoted to the description and evaluation of a range of cell and tissue applications, with individual chapters focusing on the kidney, bladder, urethra, urethral sphincter, and penis and testis.

This contributed volume is the first of a series that introduces safe, feasible, and practical decellularization and recellularization techniques for tissue and organ reconstruction. We have put special emphasis on the research areas most likely to develop well-engineered scaffolds for tissue and organ engineering, while presenting easily applicable bench-to-bedside approaches highlighting the latest technical innovations in the field. This book includes both a fundamental discussion for a broad understanding of the basis of tissue repair and substitution, as well as chapters written by world renowned specialists from 20 countries providing deeper discussions and analysis of related sub disciplines. Within these pages, the reader will find state-of-the-art protocols and current clinical challenges in cell and tissue biology, including accurate and comprehensive information on extracellular matrices, natural biomaterials, tissue dynamics, morphogenesis, stem cells, cellular fate progressions, cell and tissue properties for in-vitro and in-vivo applications. This comprehensive and carefully organized treatise provides a clear framework for graduate students and postdoctoral researchers new to the field, but also for researchers and practitioners looking to expand their knowledge on tissue and organ reconstruction.