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Simoni, Silvia/SS (2012) Estrazione di cellule staminali da cordone ombelicale e loro differenziamento in colangiociti. [Ph.D. thesis]

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Abstract (english)

All acquired or congenital cholestatic diseases (primary biliary cirrhosis, sclerosing cholangitis, pediatric biliary atresia) represent one of major indications for liver transplantation. In the last 20 years new and innovative cell-based therapeutic strategies using post-natal multipotent stem cells have been characterised and proposed for hepatic surgery.
This work develops a research program aimed to identify and isolate adult stem cells from an easy and ethic approved source like Wharton jelly (CCO) for liver applications in particular for cholangiopathies. In perspective, CCO cells have been in vitro
stimulated with specific inducers in order to test their ability to differentiate into cholangiocyte-like cells.
By cytometrical analysis, the isolated fibroblastic populations have showed to be positive for stem cell markers like CD105, CD90, CD133 and negative for CD45, cKit, CD44 and HLA-DR. Although cell immunophenotype revealed to keep stable all over
subculturing, after 15 passages a significative percentage increase of CD133, CD44 and cKit positive cells has been observed. Similarly to stem cells defined as “migrating”, CCO populations expressed mRNAs for matrix metalloproteinases such as MMP2, MMP3. Moreover, they showed to respond to differentiative adipogenic, osteogenic and chondrogenic stimula until XV culture passage, as demonstrated by cytoplasmic lipid
droplets and expression of specific markers as perlecan, Runx-2, osteocalcin and osteopontin.
In order to evaluate the cholangiocyte differentiative potential of CCOs, it has been set on: a) 2D culture system based on unconditionated plates; b) 3D culture system for studying the in vitro tubulogenesis using a scaffold constituted of collagen type 1 and MatrigelTM (MATCO); c) MATCO coating-based culture system. After 7 days of stimulation, by immunofluorescence and cytometrical analysis, cells cultured in 2D system demonstrated the expression of typical cholangiocite markers as CK19 and
GGT-1. The MATCO matrix prepared as a coating layer showed to sustain the differentiaton of CCOs into cholangiocyte-like cells. Indeed, by RT-PCR study, the encapsulated cells into MATCO demonstrated to express only GGT-1 marker and, by
morphogenesis study, resulted not to be organized into tubular like structures. When induced and cultured on MATCO coating, CCOs showed similarities to cholangiocytes as demonstrated by the expression of mRNAs for GGT-1, CK19, MMP1, MMP2 and
aquaporin 1. No expression of ALB, INTβ4, HNF1B.
So, the present work demonstrated that, using a standadized procedure, it is possible to isolate from Wharton jelly a multipotent stem cell population, characterized by stable immunophenotype, long-term growth and responsivity to cholangiocyte differentiative stimulus on unconditioned plates or on matrix prepared with collagen type 1 and MatrigelTM. The in vivo study using a biliary damage model will permit to evaluate the real differentiative potential of CCO populations.

Abstract (italian)

Le malattie colestatiche croniche congenite o acquisite (cirrosi biliare primitiva, colangite sclerosante, atresia pediatrica delle vie biliari) rappresentano nel loro insieme una delle principali indicazioni al trapianto di fegato. Nell’ultimo ventennio, nuove ed
innovative strategie terapeutiche basate sull’impiego di cellule staminali adulte, ovvero cellule staminali multipotenti identificate in tessuti diversi dell’organismo in età postnatale, sono state caratterizzate e proposte in chirurgia epatica.
Tale ricerca si inserisce in un programma di studio che in campo epatologico mira all’identificazione di popolazioni cellulari staminali adulte (CCO), che, isolate da una fonte di facile accesso ed eticamente approvata quale la gelatina di Wharton., possono rappresentare candidati ideali per la terapia cellulare delle colangiopatie. In tale prospettiva, le popolazioni cellulari CCO sono state stimolate in vitro con fattori induttivi per testare la loro capacità di differenziare in cellule simil colangiociti.
All’analisi di citofluorimetria, le popolazioni fibroblastoidi ottenute hanno mostrato positività all’espressione di marcatori di staminalità quali CD105, CD90, CD133 e negatività alla presenza di CD45, cKit, CD44 e HLA-DR. Sebbene l’immunofenotipo si sia mantenuto stabile nel corso delle subcolture, è stato osservato, dopo 15 passaggi, un significativo aumento percentuale di cellule positive per i marcatori CD133, CD44 e cKit. Similmente alle popolazioni cellulari staminali definite “migranti”, le popolazioni CCO presentano mRNA specifici per le metalloproteinasi di matrice quali MMP2 e MMP3 e rispondono agli stimoli differenziativi in senso adipogenico, osteogenico e condrogenico fino alla subcoltura XV, come dimostrato dai tipici accumuli lipidici citoplasmatici e dall’espressione di specifici marcatori quali il perlecano, Runx-2,
osteocalcina ed osteopontina.
Per valutare il grado di potenzialità differenziativa colangiocitaria sono stati allestiti a) un sistema di coltura bidimensionale per semina su piastre di polistirene non condizionate, b) un sistema di coltura tridimensionale per lo studio della tubulogenesi in vitro mediante incapsulazione delle cellule in una matrice di collagene 1 e Matrigel (MATCO), c) un sistema di coltura su coating di matrice MATCO. Dopo 7 giorni di stimolazione, le cellule coltivate nel sistema bidimensionale hanno dimostrato, all’analisi di immunofluorescenza e di citofluorimetria, l’espressione di marcatori tipici di linea colangiocitaria quali CK19 e GGT-1. La matrice MATCO si è dimostrata adatta a sostenere il differenziamento delle cellule CCO in senso simil-colangiocitario solo nella forma di substrato. Infatti, all’analisi d’espressione mediante RT-PCR le cellule incapsulate in MATCO hanno dimostrato dopo trattamento induttivo la sola espressione di RNA messaggero per il marcatore GGT-1 e, all’analisi di morfogenesi, non hanno evidenziato alcuna organizzazione simil tubulare. Le cellule CCO differenziate su coating MATCO hanno acquisito caratteristiche simili a quelle dei colangiociti come dimostrato dall’espressione di mRNA per i marcatori GGT-1, CK19, MMP1, MMP2 e acquaporina 1. Non è stata osservata l’espressione di marcatori quali ALB, INTb4 e fattore HNF1B.
Dal presente studio è quindi emerso che, mediante una procedura standardizzata, è possibile isolare da gelatina di Wharton una popolazione cellulare staminale multipotente, dotata di caratteristiche immunofenotipiche stabili, potenziale di crescita a lungo termine in vitro e capace di rispondere allo stimolo differenziativo colangiocitario su piastre di polistirene condizionate o meno con una matrice di collagene 1 e matrigel.
L’impianto in vivo in un modello animale di danno biliare consentirà la valutazione del reale potenziale differenziativo delle popolazioni CCO.

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EPrint type:Ph.D. thesis
Tutor:Floreani, Annarosa/FA
Supervisor:Di Liddo, Rosa/DLR
Ph.D. course:Ciclo 24 > Scuole 24 > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > SCIENZE EPATOLOGICHE E GASTROENTEROLOGICHE
Data di deposito della tesi:30 January 2012
Anno di Pubblicazione:30 January 2012
Key Words:Cellule staminali/Stem cells, gelatina di Wharton/Wharton's jelly, rigenerazione dei dotti bilairi/biliary duct regeneration
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/12 Gastroenterologia
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Chirurgiche Gastroenterologiche "Pier Giuseppe Cevese"
Codice ID:4512
Depositato il:07 Nov 2012 10:00
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