Tannic acid label indicates abnormal cell development coinciding with regeneration of renal tubules

Abstract

BackgroundStem/progenitor cells are in the focus of research as a future therapeutic option to stimulate regeneration in diseased renal parenchyma. However, current data indicate that successful seeding of implanted stem/progenitor cells is prevented by harmful interstitial fluid and altered extracellular matrix. To find out possible parameters for cell adaptation, the present investigation was performed.MethodsRenal stem/progenitor cells were mounted in an artificial interstitium for perfusion culture. Exposure to chemically defined but CO2-independent culture media was tested during 13 days. Cell biological features were then analyzed by histochemistry, while structural details were investigated by transmission electron microscopy after conventional and improved fixation of specimens.ResultsCulture of renal stem/progenitor cells as well in Leibovitz’s L-15 Medium as CO2 Independent Medium shows in fluorescence microscopy spatial development of numerous tubules. Specimens of both media fixed by conventional glutaraldehyde exhibit in electron microscopy a homogeneous cell population in developed tubules. In contrast, fixation by glutaraldehyde including tannic acid illuminates that dispersed dark marked cells of unknown function are present. The screening further demonstrates that the dark cell type does not comply with cells found in embryonic, maturing or matured renal parenchyma.ConclusionsThe actual data show that development of abnormal cell features must be taken into account, when regeneration of renal tubules is simulated under in vitro conditions.

Detection of Abnormal Extracellular Matrix in the Interstitium of Regenerating Renal Tubules

“…In contrast to previous series ( Figure 2 a,b, Figure 3 a,b and Figure 4 a,b), the epithelium shows after fixation in GA solution containing tannic acid a heterogeneously composed cell population as it was earlier described [ 13 ]. The one type of cells exhibits a bright cytoplasm, while the other type shows a dark-grey cytoplasm.…”

“…For first orientation, semithin sections of specimens were performed as it was earlier described [ 13 ].…”

“…The focus was directed to morphological features arising in generated tubules. The actual data show that fixation of specimens in glutaraldehyde solution containing cupromeronic blue, ruthenium red or tannic acid for transmission electron microscopy unmasks previously not visible structures [ 13 ]. Based on these observations, for the first time causes for abnormal extracellular matrix in the interstitium of generated tubules can be systematically investigated.…”

Atypical Features in Regenerating Tubules Point to a Risk for Implantation of Renal Stem/Progenitor Cells

“…However, neither the extended basal lamina nor the heterogenously composed epithelium consisting of bright and dark cells is observed in intact renal parenchyma. Since in none of the cases a coincidence is found, unveiled morphological details in generated tubules have to be ascribed to atypical cell development as it was recently described [35,36].…”

“…In this coherence it makes no difference, whether atypical cell development is caused by here presented culture conditions or is evoked by harsh environment of diseased parenchyma after implantation. Irrespectively of whether it may be induced by culture conditions or evoked by the environment after implantation, it is shown that in renal stem/ progenitor cells principally both intact formation of tubules and a sleeping capacity for development of atypical cell and extracellular matrix is contained [35,36].…”