ABSTRACT
The cell biological mechanism controlling the regeneration of renal tubules in renal failure after application of stem/progenitor cells is subject of actual research. Unsolved issues are the integration of stem/progenitor cells in a diseased organ environment, the differentiation into epithelial tissue and the formation of tubules in a spatial environment. Following this therapeutic strategy new biomaterials have to be found promoting spatial development of tubules. To obtain new information about the growth of tubules renal stem/progenitor cells from neonatal rabbit kidney were isolated and mounted in a tissue carrier between a selection of commercially available polyester fleeces. This procedure replaces coating by extracellular matrix proteins and creates an artificial interstitium supporting development of tubules. Perfusion culture was performed with chemically defined IMDM containing aldosterone as tubulogenic factor. Polyester fleeces were investigated by scanning electron microscopy. The spatial development of tubules was registered on whole-mount specimens and on cryosections labeled with SBA and antibodies indicating tubule differentiation. It is found that some polyester fleeces promote the spatial development of tubules between the fibers, whereat each of them produces its individual growth pattern.
Subject(s)
Kidney Tubules , Organ Culture Techniques/methods , Polyesters/chemistry , Regeneration/physiology , Stem Cells/physiology , Tissue Engineering/methods , Animals , Biocompatible Materials/chemistry , Cell Differentiation/physiology , Cells, Cultured , Kidney Tubules/cytology , Kidney Tubules/growth & development , Materials Testing , Organ Culture Techniques/instrumentation , Rabbits , Stem Cells/cytology , Tissue Engineering/instrumentationABSTRACT
In regenerative medicine, stem/progenitor cells are emerging as potential candidates for the treatment of renal failure. However, the mechanism of regeneration of renal tubules from stem/progenitor cells is not well-elucidated. In this study, a new method was developed for the generation of tubules replacing coating by extracellular matrix proteins. Renal stem/progenitor cells are mounted between layers of polyester fleece. This artificial interstitium supports spatial development of tubules within 13 days of perfusion culture in chemically defined Iscove's modified Dulbecco's medium (IMDM) containing aldosterone as the tubulogenic factor. Whole mount label by soybean agglutinin (SBA) showed that generated tubules exhibited a lumen and a continuously developed basal lamina. Immuno-labeling for cytokeratin Endo-A demonstrated the presence of isoprismatic epithelial cells, and laminin gamma1, occludin, and Na/K-ATPase alpha5 labeling revealed typical features of a polarized epithelium. To get first insight in the interface between tubules and polyester interstitium, transmission electron microscopy (TEM) was performed. The results showed that the generated tubules exhibited polar differentiation with a continuously developed basal lamina consisting of a lamina rara interna, lamina densa, and lamina rara externa. Collagen type III was found to be the linking molecule between the basal lamina and the surrounding polyester fibers by immuno labeling studies. Thus, the findings demonstrate that the spatial development involves the interface between the tubular basal lamina and the polyester interstitium of tubules and is not restricted to the epithelial portion.
