Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.

Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.:Table of Contents LISTS OF FIGURES AND TABLES VI ACKNOWLEDGEMENTS..................................................................................VII ABSTRACT ............................................................................................................IX NOMENCLATURE ................................................................................................X 1 INTRODUCTION...................................................................................................1 2 FUNDAMENTALS..................................................................................................2 2.1 KIDNEY DEVELOPMENT AND REGENERATION ...............................................................................2 2.1.1 Function of the kidney............................................................................................2 2.1.2 Development of the metanephric kidney ................................................................2 2.1.3 Selfregenerative potential of the kidney.................................................................5 2.2 THE EXTRACELLULAR MATRIX AS BIOLOGICAL SCAFFOLD ...............................................................6 2.2.1 Molecular composition of the ECM........................................................................7 2.2.1.1 An overview of the main ECM components..................................................................................8 2.2.2 Cell/tissue-matrix interactions.............................................................................12 2.2.2.1 Biochemical signals....................................................................................................................13 2.2.2.2 Mechanical signals......................................................................................................................14 2.2.2.3 Structural signals........................................................................................................................15 2.3 TISSUE ENGINEERING FOR THERAPEUTIC PURPOSES .....................................................................15 2.3.1 An overview of tissue engineering and regenerative medicine.............................15 2.3.2 Biomaterials for tissue engineering and regenerative medicine...........................18 2.3.2.1 Decellularization approach as tool to extract natural matrices....................................................19 2.3.3 Tissue engineering and regenerative medicine in kidney treatment.....................19 2.4 ORGAN AND TISSUE CULTURE AS TOOL FOR TISSUE ENGINEERING...................................................22 2.4.1 Common organ culture systems............................................................................24 3 OBJECTIVES AND MOTIVATION...................................................................25 4 RESULTS AND DISCUSSION............................................................................27 4.1 A NOVEL, LOW-VOLUME METHOD FOR ORGAN CULTURE OF EMBRYONIC KIDNEYS THAT ALLOWS DEVELOPMENT OF CORTICO-MEDULLARY ANATOMICAL ORGANIZATION..............................................27 4.1.1 Additional evidences (to Appendix A) for stress reduction of kidney rudiments cultured in the novel system than those grown in conventional organ culture.....28 4.1.2 Additional evidences (to Appendix A) for corticomedullary zonation and improved development of kidney rudiments cultured in the novel system for a period of 12 days......................................................................................................................30 4.1.3 Additional evidences (to Appendix A) for the application of the glass based low volume culture system for other organs................................................................32 4.2 ECM MODULATED EARLY KIDNEY DEVELOPMENT IN ORGAN CULTURE ...........................................34 4.3 ESTABLISHING AND EVALUATING DECELLULARIZATION TECHNIQUES TO ISOLATE WHOLE KIDNEY ECMS FROM ADULT MURINE KIDNEYS................................................................................................37 4.4 THE ABILITY OF WHOLE DECELLULARIZED ECM CONSTRUCTS TO INFLUENCE MURINE EMBRYONIC STEM CELL DIFFERENTIATION AND RENAL TISSUE BEHAVIOUR IN A NEW STRAIGHT FORWARD APPROACH..........38 iv 5 SUMMARY AND OUTLOOK.............................................................................39 5.1 SUMMARY..........................................................................................................................39 5.2 OUTLOOK...........................................................................................................................42 6 BIBLIOGRAPHY.................................................................................................49 7 APPENDICES..........................................................................................................I 7.1 APPENDIX A: A NOVEL, LOW-VOLUME METHOD FOR ORGAN CULTURE OF EMBRYONIC KIDNEYS THAT ALLOWS DEVELOPMENT OF CORTICO-MEDULLARY ANATOMICAL ORGANIZATION......................I 7.2 APPENDIX B: ECM MODULATED EARLY KIDNEY DEVELOPMENT IN EMBRYONIC ORGAN CULTURE ....XIX 7.3 APPENDIX C: THE DEWAXED ECM: AN EASY METHOD TO ANALYZE CELL BEHAVIOUR ON DECELLULARIZED EXTRACELLULAR MATRICES.......................................................................XLIV 7.4 PUBLICATIONS AND SCIENTIFIC CONTRIBUTIONS......................................................................LXV 7.5 SELBSTSTÄNDIGKEITSERKLÄRUNG......................................................................................LXIX

Organoide aus adulten Mäusen sind vielversprechende Modelle für die Nierenforschung. Ihre Charakterisierung wurde jedoch nicht auf ein zufriedenstellendes Niveau gebracht. Hier habe ich ein langfristiges 3D-Maus-Organoid (Tubuloid)-Modell etabliert und charakterisiert, das die Erneuerung und die Reparatur sowie die Architektur und die Funktionalität der adulten tubulären Epithelien rekapituliert. In der Zukunft wird das Modell detaillierte Untersuchungen der Trajektorien selbsterneuernder Zellen sowohl zur teilweisen Wiederherstellung der Niere als auch zur malignen Transformation der Niere ermöglichen. Das klarzellige Nierenzellkarzinom (ccRCC) ist der häufigste und aggressivste Nierenkrebs. Die Inaktivierung des Tumorsuppressorgens Von Hippel-Lindau (VHL) ist der Haupttreiber des ccRCCs. Zuvor hatten wir die Hochregulation der Wnt- und Notch-Signalübertragung in den CXCR4+MET+CD44+-Krebsstammzellen (CSC) aus primären humanen ccRCC-Tumoren identifiziert. Das Blockieren von Wnt und Notch in von Patienten stammenden Xenotransplantaten, Organoiden und nicht-anhaftenden Sphären unter Verwendung von niedermolekularen Inhibitoren beeinträchtigte die Selbsterneuerung der CSC und das Tumorwachstum. Um CSC-gesteuertes humanes ccRCC in genetischen Mausmodellen nachzuahmen, begann ich mit der Erzeugung von zwei Doppelmausmutanten; β-Catenin-GOF; Notch-GOF und Vhl-LOF; β-Catenin-GOF. Sowohl die β-Catenin-GOF; Notch-GOF Mausmutante als auch die Vhl-LOF; β-Catenin-GOF Mausmutante entwickelten innerhalb einiger Monate schwere Krankheitssymptome. Überraschenderweise beobachtete ich weder Tumore oder Tumorvorläuferläsionen noch höhere Zellproliferationsraten in den mutierten Nieren. Weitere Analysen ergaben, dass die Mausmutanten Merkmale chronischer Nierenerkrankung (CKD) aufwiesen. / Adult mouse organoids are promising models for kidney research. However, their characterization has not been pushed forward to a satisfying level. Here, I have generated and characterized a long-term 3D mouse organoid (tubuloid) model, which recapitulates renewal and repair, and the architecture and functionality of the adult tubular epithelia. In the future, the model will allow detailed investigations of trajectories of self-renewing cells towards both the partial recreation and malignant transformation of the kidney. Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive kidney cancer. Inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene is the major driver of ccRCC. Earlier, we identified the upregulation of Wnt and Notch signaling in CXCR4+MET+CD44+ cancer stem cells (CSCs) from primary human ccRCCs. Blocking Wnt and Notch in patient-derived xenografts, organoids and non-adherent spheres using small-molecule inhibitors impaired self-renewal of CSCs and tumor growth. To mimic CSC-governed human ccRCC in genetic mouse models, I started from the generation of two double mouse mutants; β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF. Surprizingly, I observed neither tumors or tumor precursor lesions nor higher cell proliferation rates in the mutant kidneys. Further analyses revealed that the mutant mice displayed features of chronic kidney disease (CKD). Thus, β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF mouse mutants did not develop kidney tumors under the given experimental conditions.