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Stem/progenitor cells are seen as a therapeutic option for repair of diseased renal parenchyma. However, actual data show that survival of implanted stem/progenitor cells is impeded by harmful interstitial environment.To learn about parameters for cell adaptation, renal stem/progenitor cells were mounted in a polyester (POSI-4) interstitium during perfusion culture. Controlled fluid environment was maintained by chemically defined CO 2 -independent culture media for 13 days. Cell biological features were then analyzed by immunohistochemistry, while structural details were investigated by advanced fixation of specimens for microscopy. When stem/progenitor cells are kept in Leibovitz's L-15 Medium or CO 2 Independent Medium, spatial development of numerous tubules is observed. Immunolabel for TROMA-III, cingulin and laminin ɣ1 depicts that a homogenous cell population is contained. Semithin sections of specimens fixed in traditional glutaraldehyde (GA) solution reflect an unobtrusive morphology. In contrast, fixation by GA solution including ruthenium red unveils in tubules a thickened basal lamina. Fixation by GA solution including tannic acid illustrates atypical development of a heterogeneous epithelium consisting of bright and dark cells.Thus, advanced fixation of specimens makes pathological features visible, when regeneration is investigated by renal stem/ progenitor cells. To what extent a comparable risk lurks behind an implantation, has to be elaborated.
Stem/progenitor cells are seen as a therapeutic option for repair of diseased renal parenchyma. However, actual data show that survival of implanted stem/progenitor cells is impeded by harmful interstitial environment.To learn about parameters for cell adaptation, renal stem/progenitor cells were mounted in a polyester (POSI-4) interstitium during perfusion culture. Controlled fluid environment was maintained by chemically defined CO 2 -independent culture media for 13 days. Cell biological features were then analyzed by immunohistochemistry, while structural details were investigated by advanced fixation of specimens for microscopy. When stem/progenitor cells are kept in Leibovitz's L-15 Medium or CO 2 Independent Medium, spatial development of numerous tubules is observed. Immunolabel for TROMA-III, cingulin and laminin ɣ1 depicts that a homogenous cell population is contained. Semithin sections of specimens fixed in traditional glutaraldehyde (GA) solution reflect an unobtrusive morphology. In contrast, fixation by GA solution including ruthenium red unveils in tubules a thickened basal lamina. Fixation by GA solution including tannic acid illustrates atypical development of a heterogeneous epithelium consisting of bright and dark cells.Thus, advanced fixation of specimens makes pathological features visible, when regeneration is investigated by renal stem/ progenitor cells. To what extent a comparable risk lurks behind an implantation, has to be elaborated.
Stem/progenitor cells are in the focus of research as a therapeutic perspective to cure acute and chronic renal failure. Following this innovative strategy one has to consider that stem/progenitor cells are first kept in the beneficial atmosphere of a CO 2 -dependent culture medium, while after implantation they are exposed to unbalanced interstitial fluid of diseased parenchyma. However, this abrupt transition has consequences, since it limits survival of implanted stem/progenitor cells. To offer a constant environment during isolation, culture, surgical processing and initial seeding within diseased parenchyma, an improved concept is under current work. In present experiments stabilization of fluid environment was investigated by isolating renal stem/progenitor cells in chemically defined and CO 2 -stabilized culture media. Interstitial influences on spatial development of tubules were tested by mounting stem/progenitor cells in a polyester fleece as a substitute for extracellular matrix and by transporting always fresh medium in perfusion culture for 13 days. Finally, morphological quality of raised constructs was analyzed by histochemistry, light and transmission electron microscopy. The present results demonstrate that three different culture media can be short-listed for protecting stem/progenitor cells during the process of implantation. Earlier approved Iscove´s Modified Dulbecco´s Medium buffered by HEPES revealed formation of numerous tubules. Also application of Leibovitz's L-15 Medium and CO 2 Independent Medium demonstrated unexpected promoting effects on the generation of tubules. In all series typical features of transporting tubule cells were registered. Formation of an excess of vacuoles as an indicator for cytotoxicity was not observed. In conclusion, although very different in chemical composition the tested culture media reflect an advantageous microenvironment for isolation, implantation and development of stem/ progenitor cells.
The renal stem/progenitor cell niche is found during development at the inner side of the organ capsule. A reciprocal exchange of morphogenetic factors between epithelial and mesenchymal cells leads here to a successive anlage of nephrons. It is generally supposed that a close contact exists between involved cells and that transmission of signals occurs via diffusion. However, morphological analysis of specimens fixed in glutaraldehyde (GA) solution reveals that both types of cells are separated by an extended interface. To investigate this special cell arrangement in detail, kidneys of neonatal rabbits were fixed by GA containing lanthanum, alcian blue, cupromeronic blue, ruthenium red or tannic acid. To obtain a comparable view to the niche, parenchyma was cut along the axis of lining collecting ducts for analysis by transmission electron microscopy. Present data illustrate that fixation of specimens by GA including alcian blue, cupromeronic blue, ruthenium red or tannic acid unmasks four different textures of filigree extracellular matrix within the interface. Applied contrasting illuminates a supplementary pattern. Further on, projections of mesenchymal cells lining to epithelial cells are specifically integrated in detected matrix. Experiments are under work to elucidate, whether detected cell to cell contacts via tunneling nanotubes are involved in transmission of morphogenetic signals.
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