The development of the nephron is piloted by interactions between epithelial and surrounding mesenchymal stem/progenitor cells. Data show that an astonishingly wide interstitial space separates both kinds of stem/progenitor cells. A simple contrasting procedure was applied to visualize features that keep renal epithelial and mesenchymal stem/progenitor cells in distance. The kidney of neonatal rabbits was fixed in solutions containing glutaraldehyde (GA) in combination with alcian blue, lanthanum, ruthenium red, or tannic acid. To obtain a comparable view to the renal stem/progenitor cell niche, the tissue was exactly orientated along the axis of collecting ducts. Fixation with GA or in combination with alcian blue or lanthanum revealed an inconspicuous interstitial space. In contrast, fixation with GA containing ruthenium red exhibits strands of extracellular matrix lining from epithelial stem/progenitor cells through the interstitium up to the surface of mesenchymal stem/progenitor cells. Fixation with GA containing tannic acid shows that the basal lamina of epithelial stem/progenitor cells, the adjacent interstitial space and also the surface of mesenchymal stem/progenitor cells are connected over a net of extracellular matrix. The applied technique appears to be a suitable method to illuminate the interstitium in stem/progenitor cell niches of specialized tissues, the microenvironment of tumors and extension of degeneration.

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Abstract-An increasing number of investigations is dealing with the repair of acute and chronic renal failure by the application of stem/progenitor cells. However, accurate data concerning the cell biological mechanisms controlling the process of regeneration are scarce. For that reason new implantation techniques, advanced biomaterials and morphogens supporting regeneration of renal parenchyma are under research. Special focus is directed to structural and functional features of the interface between generating tubules and the surrounding interstitial space. The aim of the present experiments was to investigate structural features of the interstitium during generation of tubules. Stem/ progenitor cells were isolated from neonatal rabbit kidney and mounted between layers of a polyester fleece to create an artificial interstitium. Perfusion culture was performed for 13 days in chemically defined Iscove's Modified Dulbecco's Medium containing aldosterone (1 9 10 À7 M) as tubulogenic factor. Recordings of the artificial interstitium in comparison to the developing kidney were performed by morphometric analysis, scanning and transmission electron microscopy. The degree of differentiation was registered by immunohistochemistry. The data reveal that generated tubules are embedded in a complex network of fibers consisting of newly synthesized extracellular matrix proteins. Morphometric analysis further shows that the majority of tubules within the artificial interstitium develops in a surprisingly close distance between 5 and 25 lm to each other. The abundance of synthesized extracellular matrix acts obviously as a spacer keeping generated tubules in distance. For comparison, the same principle of construction is found in the developing parenchyma of the neonatal kidney. Most astonishingly, scanning electron microscopy reveals that the composition of interstitial matrix is not homogeneous but differs along a cortico-medullary axis of proceeding tubule development.Keywords-Tissue engineering, Perfusion culture, Kidney, Tubule, Artificial interstitium, Collagen type III. INTRODUCTIONRecovery from renal failure requires the replacement of injured tissue with new cells that restore epithelial integrity and functionality within tubules. An increasing number of papers is therefore dealing with strategies for repair of parenchyma by the help of stem/progenitor cells. 5,12 However, recent data show that an effective therapy is still far away from a widespread clinic application. Unsolved issues in renal tissue engineering are the concentration of stem/progenitor cells at the site of damage, their integration in a diseased environment, the process of differentiation into nephron-specific cells, and the spatial development of tubules within the kidney. 32 Part of actual research is focusing on cell biological mechanisms involved in the formation of tubules during regeneration. 4 Due to the spatial microarchitecture of the kidney experiments are frequently performed applying three-dimensional culture experiments in combination wi...

In regenerative medicine much attention is given to stem/progenitor cells for a future therapy of acute and chronic renal failure. However, up to date sound cell biological knowledge about nephron renewal within the diseased kidney is lacking. Of special interest are steroid hormones occurring in the interstitial fluid. It is unknown, if they have promoting or hampering effects on the development of stem/progenitor cells. To investigate the influence of steroid hormones on regenerating tubules a realistic culture system is of great importance. An advanced technique creates an artificial interstitium promoting renal stem/progenitor cells to develop tubules between layers of polyester fleece. During perfusion culture with chemically defined Iscove's Modified Dulbecco's Medium (IMDM) individual steroid hormones can be administered. After a culture period of 13 days the effect on tubule development is analyzed by histochemical methods. Present experiments reveal that substances such as cholesterol and 17-α-hydroxyprogesterone do not exhibit a visible effect on renal stem/progenitor cells. However, atypical aggregation of few SBA-labeled cells in form of islets is found after treatment with progesterone, pregnenolone, 11-deoxycorticosterone and dihydrotestosterone. Numerous SBA-labeled cells within extended clusters are observed after application of testosterone, 17-β-estradiol, corticosterone and dexamethasone. In contrast, application of aldosterone exclusively leads to the formation of numerous intact tubules. In consequence, it is shown that distinct steroid hormones induce a shift in development from intact tubules to atypical cell islets and clusters. Such harmful effects have to be considered, when stem/progenitor cells are exposed after implantation to the interstitial fluid within diseased renal parenchyma.