Academic literature on the topic 'Nephrosphere'

The mechanism upon which human kidneys undergo regeneration is debated, though different lineage-tracing mouse models have tried to explain the cellular types and the mechanisms involved. Different sources of human renal progenitors have been proposed, but it is difficult to argue whether these populations have the same capacities that have been described in mice. Using the nephrosphere (NS) model, we isolated the quiescent population of adult human renal stem-like PKHhigh/CD133+/CD24− cells (RSC). The aim of this study was to deepen the RSC in vitro multipotency capacity. RSC, not expressing endothelial markers, generated secondary nephrospheres containing CD31+/vWf+ cells and cytokeratin positive cells, indicating the coexistence of endothelial and epithelial commitment. RSC cultured on decellularized human renal scaffolds generated endothelial structures together with the proximal and distal tubular structures. CD31+ endothelial committed progenitors sorted from nephrospheres generated spheroids with endothelial-like sprouts in Matrigel. We also demonstrated the double commitment toward endothelial and epithelial lineages of single RSC. The ability of the plastic RSC population to recapitulate the development of tubular epithelial and endothelial renal lineages makes these cells a good tool for the creation of organoids with translational relevance for studying the parenchymal and endothelial cell interactions and developing new therapeutic strategies.

In recent years, numerous cancers have been described as having a "cancer stem cell" (CSC) population also known as "cancer initiating cells". CSCs refer to a subset of tumour cells that has the ability to self-renew and generate the diverse cells that comprise the tumor. Their name derives from their "stem-like" properties and ability to continually sustain tumorigenesis. CSCs have the same properties that define a normal tissue adult stem cell, even if they are aberrant: self-renewal and differentiation. Renal cell carcinoma (RCC) accounts for about 3% of adult cancers and is among the 10 most common malignancies in Europe. RCC has several morphological subtypes and clear cell RCC accounts for ~80% of cases. RCC has a late diagnosis and is therapy resistant. In renal pathologies there are situation in which the presence and function of adult renal stem cells may have clinical relevance. In the last years the existence of different sources of renal stem cells has been proposed even if the phenotype of a resident stem cell of the kidney has not been exhaustively described. Even in RCC the definition of a kidney cancer stem cell may have a role for better understanding renal cell carcinoma biology. A number of approaches based on the exploitation of renal stem cell markers have allowed the prospective isolation of human renal stem cells, even if the relative promiscuity of these markers limits their usefulness when highly purified stem cells are needed. So we decided to use a functional approach for the isolation of normal and cancer stem cells of the kidney by culturing the cells in suspension, non-adherent conditions, at low density with specific growth factors. In these conditions only a little percentage of cells survives growing as spherical aggregates in suspension. We called them "nephrospheres". Using fluorescent lipophilic dyes PKH, we demonstrated the clonal origin of the spheres and the presence of a heterogeneous population inside the spheres. In fact the dye dilutes in active replicating cells while is retained in quiescent cells; we can observe in normal and cancer nephrospheres some most fluorescent cells, the quiescent stem cells, and some less fluorescent or not fluorescent cells, that are the active replicating progenitors. We performed a characterization of the nephrospheres by immunofluorescence after cytospin or FACS evaluating the expression of some epithelial and stem cell markers. By Real Time PCR we found that some genes related with stemness or involved in the maintenance of pluripotency are overexpressed in normal and cancer nephrospheres if compared with the corresponding differentiated primary cell cultures. We then evaluated the differentiative abilities of the cells derived from normal nephrospheres by culturing the cells in specific media and semisolid substrates; the cells are able to differentiate into epithelial and neuronal-like phenotype and to form tubular/glomerular-like tridimensional structures. We the isolated the stem cell population form normal nephrospheres on the basis of the PKH fluorescence. We identified 3 PKH populations: PKHhigh population, with a high level of fluorescence, PKHlow population, with a low level of fluorescence, and PKHneg population, negative for PKH. The populations were separated with cell sorting and cultivated to form spheres. Only PKHhigh cells were able to generate new spheres, demostrating that the PKHhigh cells represent the stem cell population inside the nephrospheres. Normal and cancer PKHhigh cells will be deeply characterized in the future.

La fragilità è una sindrome geriatrica definita da un progressivo declino età-correlato di diverse facoltà fisiologiche, che si traduce in una ridotta funzionalità d’organo e in un’aumentata vulnerabilità in condizioni di stress. Fried e coll. propongono una definizione operativa delle fragilità. La letteratura riporta un’alta prevalenza di fragilità in individui affetti da insufficienza renale cronica. Tra questi soggetti, il rischio di sviluppare fragilità è aumentato di due/tre volte rispetto ai soggetti sani. Ad oggi, la definizione e la valutazione della fragilità in questi pazienti è ancora controversa. Tuttavia quello che si sa è che l’invecchiamento è un processo strettamente correlato alla ridotta capacità delle cellule staminali di auto-rinnovarsi e differenziarsi. Quest’alterazione delle funzioni delle cellule staminali può svolgere un ruolo chiave nella fisiopatologia delle malattie associate all’invecchiamento, inclusa la disfunzione renale. Il nostro gruppo ha identificato e isolato le cellule staminali renali adulte a partire da colture clonali di nefrosfere umane. All’interno di queste sono presenti cellule a diversa differenziazione e maturazione, tra queste anche le staminali identificate come PKHhigh/CD133+/CD24-, multipotenti e in grado di ripopolare scaffold renali decellularizzati. Date queste premesse l'obiettivo del progetto è valutare gli effetti di condizioni biologiche legate alla fragilità sul comportamento delle cellule staminali renali adulte umane e capire se tali condizioni sono in grado di esaurire il pool di cellule staminali e alterarne la funzione. In primo luogo abbiamo arruolato soggetti fragili, pre-fragili e non-fragili e giovani sani come controllo e raccolto i rispettivi plasmi e cellule mononucleate dal sangue periferico (PBMC). Sia nelle PBMC che nelle cellule staminali/progenitrici ematopoietiche circolanti abbiamo valutato il danno al DNA osservando una percentuale di cellule positive al danno statisticamente più alta nei pazienti fragili rispetto agli altri gruppi. Per valutare il reale effetto delle condizioni biologiche legate alla fragilità sulle proprietà delle RSC, le colture di NS, ottenute da nefrectomie, sono state trattate con il plasma dei soggetti arruolati. Abbiamo valutato dapprima le capacità di autorinnovamento delle cellule trattate e osserviamo una significativa diminuzione dell'efficienza di formazione della sfera, indice di autorinnovamento, nei soggetti fragili rispetto ai non fragili e ai giovani. Successivamente, abbiamo valutato il danno al DNA, i ROS intracellulari, la proliferazione e la vitalità nelle cellule staminali/progenitrici renali ottenute dopo la dissociazione delle NS. Non sono state evidenziate differenze nella vitalità e nella proliferazione cellulare tra i gruppi, mentre il danno al DNA e i ROS intracellulari sono aumentati nelle cellule delle NS trattate con plasma di anziani fragili rispetto a quelle trattate con gli altri plasmi. Ciò potrebbe indicare che la diminuzione della capacità di autorinnovamento nelle cellule trattate con il plasma di pazienti fragili e un aumento del danno al DNA e dei ROS intracellulari non sono correlati con la morte o la proliferazione cellulare, ma con un'elevata presenza di mediatori infiammatori e ROS nel plasma dei pazienti fragili. Per confermare questi dati abbiamo analizzato lo stress ossidativo e il profilo di 40 citochine infiammatorie nel plasma dei soggetti arruolati. Si ha un aumento dello stress ossidativo nel plasma dei soggetti fragili rispetto agli altri gruppi, così come un’aumentata presenza o l’esclusività di alcune citochine infiammatorie. Questi dati preliminari suggeriscono che esiste una combinazione di stress ossidativo e citochine pro-infiammatorie nel plasma di pazienti fragili che contribuiscono ad aumentare il danno al DNA e i ROS intracellulari alterando conseguentemente le caratteristiche di staminalità delle cellule delle NS.
Frailty is a geriatric syndrome that can be defined as an age-related progressive impairment of multiple physiological systems, resulting in a significantly reduced capacity to compensate for external stressors. Fried and colleagues proposed a phenotype characterization of frailty through five physical criteria, so this can be possible only after the onset of clinical manifestations without the possibility of a precocious diagnosis. Several studies report a high prevalence of frailty in both old and young individuals with kidney dysfunction, and this further increases with advancing age and progressive decline of renal function. Elderly individuals with chronic kidney disease (CKD) are two to three times more likely to be frail than those with normal renal function. However, the relationship between CKD and frailty is still unclear. The aging process can have adverse effects on stem cells; their self-renewal ability declines and their differentiation potential into the various cell types is altered. Aging-induced exhaustion and deterioration of stem cell pool and functions may play a key role in the pathophysiology of aging-associated diseases, including kidney dysfunction. Our group isolated a pure population of multipotent renal stem-like cells by a functional approach, taking advantage from the ability of renal stem cells (RSC) to grow as nephrospheres (NS). Investigating the expression of renal progenitor markers described in literature, our group identified in NS a homogeneous PKHhigh/CD133+/CD24- cell population displaying in vitro stem-cell properties, able to repopulate human decellularized renal scaffold and exhibiting multipotency. In this scenario, we tested whether in the organism of elderly and frail people there are biological conditions able to alter RSC behavior, justifying the high prevalence of chronic kidney dysfunction in the frail status and its severity. First, we recruited frail, pre-frail and non-frail subjects, and young subjects as controls and we obtained whole blood that was separated into plasma and PBMC. We studied DNA damage in both PBMC and circulating hematopoietic progenitor/stem cells (cHPSC) and we observed a statistically higher percentage of cells positive for DNA damage in frail patients compared to all the other groups. To assess the real effect of biological conditions related to frailty on adult RSC properties, NS cultures, obtained from nephrectomies, were treated with 10% plasma of enrolled frail and non-frail subjects and healthy young. We first evaluated the self-renewal abilities of treated cells and we observe a significant decrease in sphere forming efficiency, indication of self-renewal, in frail subjects compared to both non-frail and young people. Subsequently, we evaluated DNA damage, intracellular ROS, proliferation and viability in renal stem/progenitor cells obtained after NS dissociation after plasma treatment. We find no differences in viability and proliferation between groups, while DNA damage and intracellular ROS increased in NS cells treated with plasma of frail seniors compared to those treated with the other plasmas. This might indicate that the decrease of self-renewal ability in cell treated with plasma of frail patients and an increase of DNA damage and intracellular ROS are not correlated to cell death or proliferation, but with a high presence of inflammatory mediators and ROS in the plasma of frail patients. To confirm these data we analyzed the oxidative stress and the profile of 40 inflammatory cytokines on plasma of enrolled subjects. We observed an increase in oxidative stress and osome inflammatory cytokines in frail plasma compared to other plasmas. These preliminary data suggested that there is a combination of oxidative stress and pro-inflammatory cytokines in plasma of frail patients that contribute to increase DNA damage and intracellular ROS and consequently alter stem characteristics of NS cells.

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.
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.