Characterization of PKHhigh renal stem cells isolated by a functional approach

Introduction and Aims In renal pathologies the regeneration of injured kidney portions may rely on the ability of resident stem/progenitor cells to proliferate and replace the damaged tissue, although the intrinsic capacity for tissue repair and regeneration is limited in mammalian kidney. However, the existence and identity of renal stem/progenitor cells with nephrogenic potential and their possible use in renal tissue repair is largely debated. Aim of this work is to evaluate the possibility to obtain adult renal stem cells from cultures of clonal human nephrospheres. Methods Nephrospheres were obtained from adult renal cells and sphere forming efficiency (SFE) was calculated. To identify and isolate stem cells from nephrospheres the PKH dye was used and CD133 and CD24 markers permitted to better characterize them. The ability of stem cells to differentiate into multiple lineages was evaluated using specific media. The regenerative abilities were evaluated transplanting the cells under the renal capsule of nude mice and by culturing nephrosphere and PKHhigh cells on decellularized slices of human renal tissue, obtained by using different detergents. Results Nephrospheres were obtained after 10 days in culture with a SFE of 0.8%. On the basis of PKHhigh status, a purified stem cell population able to self-renew and generate a differentiated epithelial, endothelial and podocytic progeny was identified in the nephrospheres. PKHhigh cells were able to survive in vivo maintaining an undifferentiated status. A PKHhigh subpopulation, characterized by CD133+/CD24- phenotype, identified a homogeneous cell population displaying strong in vitro self-renewal and multipotency capacity. Moreover nephrosphere cells were able to repopulate decellularized slices of human renal tissue and also PKHhigh stem cells seem to have the same ability as shown by our preliminary data. Conclusions The sphere-forming functional approach coupled with the PKHhigh status, that still has to be disclosed, permitted the isolation of a purified and homogeneous population of resident renal stem cells. The demonstrated abilities of these cells to survive in vivo and to repopulate decellularized slices of renal tissues make them attractive candidates for nephron regeneration and application in regenerative medicine.