The mechanism underlying the recovery of renal cell injury is still a matter of debate that concerns the involvement of fully differentiated cells, or the existence of quiescent scattered multipotent stem cells. By sphere forming assay and sorting, our group isolated a population of PKH26 most fluorescent cells with characteristics of adult renal stem-like cells (PKHhigh cells). We previously assessed the ability of PKHhigh cells to differentiate in vitro along epithelial, podocytic and endothelial lineages. We also demonstrated that PKHhigh population is heterogeneous in composition and within nephrospheres the cells with stem capacities are PKHhigh/CD133+/CD24- (RSC). We recently published that our nephrosphere cells, comprising PKHhigh cells and their PKHlow/neg progeny, are able to repopulate human decellularized renal scaffolds. With this research, we now aim: I) to prove the regenerative capabilities of PKHhigh stem-like cells, even in absence of their PKHlow/neg progeny. II) to find the molecular signature of RSC that among PKHhigh cells are those with the wider stem capacities. To reach these aims, we cultured isolated PKHhigh cells on acellular scaffolds for 30 days and the cells of the repopulated structures were characterized by sequential immunofluorescence using specific markers of differentiation. Some structures indicated a specific lineage differentiation into proximal and distal tubular epithelium and endothelium. Only few structures coexpressed some or all markers tested, indicating still immature phenotypes. For the disclosure of RSC molecular signature, transcriptomic analysis of RSC, of their PKHlow/neg progeny and of terminally differentiated primary cell cultures (PCC) was performed and differentially expressed genes (DEG) were evidenced. Bioinformatic Gene Set Enrichment Analysis suggested a renal immature status of our RSC, but different from embryonic stem cells. Crossing DEG lists potential markers were obtained and some of them were selected and validated. In conclusion, we highlighted the proximal and distal tubular epithelial and endothelial differentiative and regenerative abilities of PKHhigh cells. Moreover, we showed that PKHhigh cells, completely lacking any endothelial marker, were able to give rise to endothelial-like structures. The occasional coexpression of epithelial and endothelial markers in repopulated structures may indicate a transitional early status toward cell differentiation. The eventual role of the PKHlow/neg progeny of PKHhigh cells in speeding up the complete PKHhigh differentiation will be clarified. Finally, the obtained RSC signature would open the possibility for the direct isolation of adult renal stem-like cells from normal kidney tissue.
Il meccanismo alla base della riparazione di un danno a cellule renali è ancora oggi oggetto di dibattito e potrebbe coinvolgere sia cellule terminalmente differenziate che l'esistenza di cellule staminali multipotenti quiescenti. Sfruttando il saggio di formazione di sfere e la tecnica del FACS sorting, il nostro gruppo ha isolato una popolazione di cellule marcate col clorante PKH26 che avessero caratteristiche di cellule staminali renali adulte (PKHhigh). In precedenza abbiamo valutato la capacità di queste cellule di differenziare in vitro in lineage epiteliale, podocitico ed endoteliale. Abbiamo anche dimostrato che la popolazione PKHhigh è eterogenea nella sua composizione e all'interno delle nefrosfere le cellule con capacità simil-staminali sono PKHhigh/CD133+/CD24- (RSC). Abbiamo recentemente pubblicato che le nostre cellule delle nefrosfere, che comprendono cellule PKHhigh e la loro progenie PKHlow/neg, sono in grado di ripopolare scaffold renali umani decellularizzati. Con questa ricerca, ora abbiamo come scopo: i) di dimostrare le capacità rigenerative delle cellule staminali PKHhigh, anche in assenza della loro progenie PKHlow/neg. ii) di trovare la signature molecolare delle RSC che, tra PKHhigh, sono le cellule con capacità staminali più ampie. Per raggiungere questi obiettivi, abbiamo messo in coltura cellule PKHhigh su scaffold acellulari per 30 giorni e le cellule delle strutture ripopolate sono state caratterizzate mediante immunofluorescenza sequenziale, utilizzando specifici marcatori di differenziamento. Alcune strutture hanno indicato un differenziamento terminale in lineage epiteliale tubulare prossimale o distale o in quello endoteliale. Solo poche strutture mostravano invece la coespressione di alcuni o di tutti i marcatori testati, suggerendo un fenotipo ancora immaturo. Per la comprensione della signature molecolare delle RSC, è stata eseguita l'analisi trascrittomica delle RSC stesse, della loro progenie PKHlow/neg e di colture primarie terminalmente differenziate e sono stati evidenziati geni differenzialmente espressi (DEG). L’analisi bioinfomatica mediante Gene Set Enrichment Analysis ha suggerito uno stato immaturo delle nostre RSC, ma comunque diverso da cellule staminali embrionali. Infine, incrociando le liste di DEG sono stati ottenuti potenziali marcatori e alcuni di essi sono stati selezionati e validati. In conclusione, abbiamo evidenziato le capacità differenziative in senso epiteliale sia prossimale che distale ed endoteliale delle cellule PKHhigh. Inoltre, abbiamo dimostrato che le cellule PKHhigh, completamente prive di qualsiasi marcatore endoteliale, sono in grado di dare origine a strutture simil-endoteliali. La coespressione occasionale di marcatori epiteliali ed endoteliali in strutture ripopolate potrebbe indicare uno stato precoce di transizione verso un differenziamento terminale. Il possibile ruolo della progenie PKHlow / neg nel velocizzare il differenziamento terminale delle cellule PKHhigh dovrà essere chiarito. Infine, la signature delle RSC ottenuta potrà aprire la possibilità di isolamento diretto di cellule staminali renali adulte da tessuto renale normale.
STUDY OF MULTIPOTENT RENAL PKHHIGH STEM-LIKE CELLS, ISOLATED FROM HUMAN NEPHROSPHERES: REGENERATIVE ABILITIES AND TRANSCRIPTOMIC PROFILE
MEREGALLI, CHIARA
2018
Abstract
The mechanism underlying the recovery of renal cell injury is still a matter of debate that concerns the involvement of fully differentiated cells, or the existence of quiescent scattered multipotent stem cells. By sphere forming assay and sorting, our group isolated a population of PKH26 most fluorescent cells with characteristics of adult renal stem-like cells (PKHhigh cells). We previously assessed the ability of PKHhigh cells to differentiate in vitro along epithelial, podocytic and endothelial lineages. We also demonstrated that PKHhigh population is heterogeneous in composition and within nephrospheres the cells with stem capacities are PKHhigh/CD133+/CD24- (RSC). We recently published that our nephrosphere cells, comprising PKHhigh cells and their PKHlow/neg progeny, are able to repopulate human decellularized renal scaffolds. With this research, we now aim: I) to prove the regenerative capabilities of PKHhigh stem-like cells, even in absence of their PKHlow/neg progeny. II) to find the molecular signature of RSC that among PKHhigh cells are those with the wider stem capacities. To reach these aims, we cultured isolated PKHhigh cells on acellular scaffolds for 30 days and the cells of the repopulated structures were characterized by sequential immunofluorescence using specific markers of differentiation. Some structures indicated a specific lineage differentiation into proximal and distal tubular epithelium and endothelium. Only few structures coexpressed some or all markers tested, indicating still immature phenotypes. For the disclosure of RSC molecular signature, transcriptomic analysis of RSC, of their PKHlow/neg progeny and of terminally differentiated primary cell cultures (PCC) was performed and differentially expressed genes (DEG) were evidenced. Bioinformatic Gene Set Enrichment Analysis suggested a renal immature status of our RSC, but different from embryonic stem cells. Crossing DEG lists potential markers were obtained and some of them were selected and validated. In conclusion, we highlighted the proximal and distal tubular epithelial and endothelial differentiative and regenerative abilities of PKHhigh cells. Moreover, we showed that PKHhigh cells, completely lacking any endothelial marker, were able to give rise to endothelial-like structures. The occasional coexpression of epithelial and endothelial markers in repopulated structures may indicate a transitional early status toward cell differentiation. The eventual role of the PKHlow/neg progeny of PKHhigh cells in speeding up the complete PKHhigh differentiation will be clarified. Finally, the obtained RSC signature would open the possibility for the direct isolation of adult renal stem-like cells from normal kidney tissue.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/76671
URN:NBN:IT:UNIMIB-76671