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Da Sacco, Stefano (2009) Amniotic Fluid: Stem Cells and Progenitor Cells for Kidney Regeneration. [Tesi di dottorato]

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Abstract (inglese)

End Stage Renal Disease is a condition of kidney failure with loss of functionality in all the primary renal functions. The major cause of ESRD is Chronic Kidney Disease (CKD) characterized by a slow progression of kidney injury fibrosis and glomerular sclerosis. Many are the causes for Chronic Kidney Disease and treatments are directed to slow down the progress of the renal functional deterioration before the necessity for dialysis and/or transplant.
Alport Syndrome (AS) is a well known model for CKD. AS is a hereditary disease caused by mutations in the genes that codify for collagen IV alpha chains. Collagen IV is the main constituent of the glomerular basal membrane (GBM), essential for a proper filtration activity of the nephron. No definitive treatments are available for Alport Syndrome and CKD. The use of stem cells has been a breakthrough for Regenerative Medicine in the last years. In this study we are investigating potential of stem cells and progenitor cells from Amniotic Fluid for renal regeneration, in particular for CKD.
Amniotic fluid fills the amniotic cavity, providing an environment in which floats the developing embryo and late the fetus. The volume and composition of the amniotic fluid changes during pregnancy and reflects the physiology of the fetus. Amniotic Fluid is principally composed by fetal urine and fetal lung liquid, with minor contribution from the amnion. In 2007, the presence of a pluripotent population of Amniotic Fluid Stem Cells (AFSC) was reported.
Beside AFSC that constitutes about the 0.8-1% of the total population, mesenchymal and hematopoietic cells have been shown to be present within Amniotic Fluid but its composition is still poorly understood.
Two different approaches were investigated in this study for possible use in of stem cells in renal regeneration. Since Amniotic Fluid by 8 weeks of gestation is mainly derived by fetal urination, it could represent a source for kidney derived cells. In order to better understand Amniotic Fluid cell composition a wide characterization has been performed for cells derived from the three germ layers(ectoderm, mesoderm, endoderm), pluripotent cells and organ-specific progenitor cells, focusing mainly on renal precursors.
The expression of markers from endoderm and mesoderm was seen to be higher in samples of earlier gestational age, while ectodermal markers showed constant rates of expression across gestational ages. A population of Metanephric Mesenchyme derived cells was isolated from human Amniotic Fluid.
Further immunoseparations were performed using specific markers to select cells committed to four of the renal cell types: mesangial cells, podocytes, stromal cells and Mesenchymal To Epithelial Transition cells.
In our second approach, we injected AFSC into Alport Syndrome mice. We investigated life span, levels of haematic creatinine and nitrogens, urinary albumin and collagen IV alpha 1 deposition in the glomerular compartment
AFSC injected AS mice where compared with non treated AS mice and Wild Type mice from the same background.
We have shown a decrease in haematic creatinine and nitrogens, decrease of urinary albumin, decrease in deposition of Collagen IV alpha 1 and prolongation of life in injected Alport Syndrome mice. Evaluation of the results obtained, with allows us to speculate that AFSC can be useful for CKD treatment.
In summary, Amniotic Fluid is a repository for many cells that could be used for kidney regeneration. Specific progenitor cells are residing within Amniotic Fluid and may be tested for kidney differentiation in in vitro and in vivo models.
In addition, AFSC are able to integrate within the glomerular structure and participate to the restoration of the homeostasis. Investigation about AFSC mechanism of action and evaluation with multiple injections and different time points could give us better information about their potential.

Abstract (italiano)

Negli ultimi anni l’incidenza di malattie renali croniche ha visto un netto incremento. L’allungamento dell’aspettativa di vita e il numero crescente di pazienti con malattie quali diabete e ipertensione, che provocano danno renale e possono portare allo sviluppo di malattia renale cronica, sono considerate causa principale di questo aumento.
I trattamenti farmacologici disponibili sono scarsi e si limitano a rallentare la progressione della malattia e la dialisi presenta importanti effetti collaterali. La mancanza di organi rende la pratica del trapianto scarsamente accessibile all’elevato numero di pazienti nelle liste d’attesa.
La Sindrome di Alport (AS) rappresenta un ben noto modello per la malattia renale di tipo cronico. AS e’ una malattia ereditaria provocata da una mutazione nel gene codificante il Collagene IV, costituente principale della menbrana basale glomerulare. In particolare, le cellule staminali da liquido amniotico (AFSC), si sono dimostrate, in studi precedenti, capaci di integrarsi e differenziare in reni embrionici, provando la loro potenzialita’ per la rigenerazione renale. L’assenza di teratomi, la facilita’ di coltura e l’accessibilita’ alla fonte rendono le AFSC candidati ideali per la loro applicazione clinica.
Nel presente studio abbiamo scelto due diversi approcci per la cura della malattia renale cronica: 1. Identificazione di specifici progenitori renali all’interno del liquido amniotico per un eventuale utilizzo in medicina rigenerativa 2. Valutazione in vivo della capacita’ di AFSC nel supportare la rigenerazione renale in un modello murino di Aport Syndrome.

Nel primo esperimento sono state studiate ed identificate le varie popolazioni cellulari presenti nel liquido amniotico a diverse settimane di gestazione. I campioni, di eta’ compresa tra le 15 e le 20 settimane di gestazione, sono stati ottenuti tramite amniocentesi, tecnica usata per studiare il cariotipo del feto durante lo sviluppo.
L’analisi e la caratterizzazione della popolazione totale presente nel liquido amniotico e’ stata effettuata utilizzando RT-PCR, Real Time PCR e Western Blotting, analizzando l’espressione specifica di geni che sono coinvolti nel mantenimento della pluripotenzialita’, geni che identificano specificatamente i tre foglietti embrionali ed infine geni che identificano progenitori organo-specifici. Sono state inoltre identificate popolazioni specifiche renali, tramite immunoseparazione con biglie magnetiche (MASC). L’espressione di marcatori per i foglietti embrionali endoderma e mesoderma e’ piu’ alta in campioni piu’ giovani rispetto a campioni con tempo di gestazione maggiore mentre, per l’ectoderma, rimane pressoche’ invariata nel tempo.
La presenza di cellule pluripotenti e’ costante cosi’ come le cellule staminali mesenchimali mentre le cellule progenitrici ematopoietiche, investigate tramite CD34, fanno la loro comparsa successivamente alle 17 settimane di gestazione.
La presenza di progenitori tessuto specifici già “committed” e’ evidente nei campioni di gestazione più avanzata sia per quantitita’ che per specificità dell’organo preso in esame.
E’ stata approfondita l’analisi di cellule progenitrici renali, utilizzando un ampio pannello di marcatori che identificano sia la componente tubulare che quella glomerulare del nefrone. E’ stata identificata e studiata una popolazione derivante dal mesenchima metanefrico da cui ha origine il nefrone ed e’ una delle due strutture embrionali fondamentali per lo sviluppo del rene. Da questa popolazione principale sono state ottenute 4 nuove sottopopolazioni che identificano sottocompartimenti del glomerulo, come per esempio le cellule corticali stromogeniche, i podociti, le cellule del mesangio e le cellule in transizione mesenchima-epitelio. Tramite PCR e Real Time PCR e’ stata dimostrata la forte specificita’ di ogni singola linea cellulare.
Parallelamente uno studio e’ stato effettuato in vivo, con l’utilizzo di un modello murino affetto da Sindrome di Alport. AFSC sono state iniettate intracardialmente e i progressi sono stati seguiti comparando il progresso della malattia con quelli in topi AS non iniettati e topi Wild Type. I risultati preliminari ottenuti hanno evidenziato un allungamento dell’aspettativa di vita nei topi AS trattati con parallela diminuzione dei livelli di proteinuria, valutati con misurazione di livelli ematici di nitrogeni e creatinina e di albumina nelle urine. Analisi istologiche hanno evidenziato una diminuzione di produzione di collagene fetale IV alpha 1 nella capsula di Bowman’s e nella membrana basale glomerulare degli animali trattati. E’ necessario uno studio approfondito che preved per le AKPC differenziazioni in vitro, utilizzando diversi fattori di crescita nefro-specifici in modo tale da confermare la loro completa differenziazione in cellule renali mature.
Un approfondimento sul meccanismo d’azione e sulle migliori tempisitiche di somministrazione, sono i punti fondamentali da chiarire per comprendere il meccanismo d’azione delle AFSC in vivo. Questo ricerca puo’ essere una base fondamentale per future applicazioni cliniche in pazienti che soffrono di nefropatie.

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Tipo di EPrint:Tesi di dottorato
Relatore:Conconi, MariaTeresa
Correlatore:Perin, Laura - De Filippo, Roger
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:27 Gennaio 2009
Anno di Pubblicazione:27 Gennaio 2009
Parole chiave (italiano / inglese):amniotic fluid Alport Syndrome Stem cells Progenitors Podocytes
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:1497
Depositato il:27 Gen 2009
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