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Ferraro, Silvia (2009) Studi di riprogrammazione su cellule estratte da tessuto adiposo. [Tesi di dottorato]

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Abstract (inglese)

Introduction. In the late few years reprogramming studies were undergone on somatic cells to get insight into the epigenetic mechanisms of cell differentiation and developmental biology. DNA methylation was identified as having crucial role on the expression of tissue-specific proteins and in gene silencing of embryonic transcriptional factors. On the other hand, demethylation treatments exhibited to be successful in restoring hTERT and OCT4 in animal models and stabilized cell lines. The aim of this study was to investigate the effects of demethylation treatment on somatic cells isolated from human adipose tissue.
Materials and methods. In this work, tissue samples from liposuction or abdominoplastic surgery were processed for cell isolation. Optical microscopy and immunofluorescence, together with cytofluorimetric analysis and further western blotting indages were performed to collect informations regarding morphological and hymmunophenotipical features. Preliminary studies were carried out on mitochondrial dehydrogenases activity and proliferative capacity to estimate the cytotoxicity of the demethylation treatment.
Results. The out come of cytotoxic analysis was a reduction of mitochondrial activity in treated cells, compared to untreated cells. Furthermore, we obtained indications of working concentration of the demethylation agent.
Cell suffering was observed during clonal expansion in populations treated with demethylation agent. Differences on the harvested cell number resulted between treated and control sample, since lower number was registerd for the demethylated population
On the other hand, both treated end untreated samples showed comparable proliferative capacity, during and after the incubation time with demethylation agent. Taken together, the last two results give rise the hypothesis that the demethylation treatment may reduce cell adhesion in monolayer culture after clonal expansion, more than affecting the proliferative capacity.
No differences between treated and untreated cell samples were observed with phase contrast optical microscopy regarding cell complexity and distribution and with electronic scansion microscopy concenring dimension and cell spreading.
Cells isolated from adipose tissue are able of multipotency. Thus, adipogenic and osteogenic differentiation studies were carried out to evaluate wether 5-Azacytidine could affect the differentiation ability of adipose-derived cells, but no differences were observed between treated differentiated and untrated differentiated.
The expression of the adipose tissue-specific transcriptional factor SREBP, was evaluated with fluorescence microscopy and western blotting. SREBP signals were registered in treated cells as well as in the papulation of origin. The research of the transcriptional factors NANOG and OCT4, responsible of stem cell self-renewing in pluripotent cells, didn’t get positive results, even in those populations long cultured and expanded after the treatment.
Characterization studies with immunofluorescence techniques were undergone on freshly isolated from adipose tissue. Cytometric data revealed the presence of diverse cell linages that were lost during the cell expansion. By contrast an enrichment of the cell component, described elsewhere as bone marrow stem population, CD105+/CD90+/ CD29+, was observed in the III and IV generations.
In long-cultured adipose tissue-derived cells was registered lost of adipose tissue-specific marker ADRP as well as the depletion of cytoplasmatic adipose vescicles. The investigation with confocal microscopy and western blotting revealed no modifications in the expression of ADRP and CD105 markers in 5-Azacytidine treated cells, while new signal of STRO1 related to the bone marrow-derived stem cells, was detected in long cultured adipose-derived cells, in treated as well untreated sample.
Conclusions. No positive results were obtained through demethylation treatment of human adipose-derived cells with 5-Azacytidine, in contrast to the experiences with animal cell models. Would be extremely interesting to get insight into the methylation level of DNA for defined factors in the long-cultured adipose-derived cells CD105+/ CD90+/STRO1+/ADRP-. Moreover, new approach with association of a variety of demethylation agents in association with stem growth factors may give interesting results.

Abstract (italiano)

Introduzione. L’ottenimento di cellule dotate di caratteristiche differenziative di tipo pluripotente a partire da cellule somatiche adulte è reso possibile mediante manipolazione dello status epigenetico. Il processo di metilazione del DNA è responsabile dell’espressione di proteine tessuto-specifiche e del silenziamento genico di fattori di trascrizione tipici della cellula germinale o tumorale. Al contrario, è stato dimostrato che il trattamento con agenti demetilanti è in grado di indurre in cellule animali multipotenti e in cellule stabilizzate l’espressione di proteine e fattori di trascrizione presenti nella cellula germinale (hTERT, OCT4). In questo studio è stato valutato l’effetto di un trattamento demetilante in vitro su cellule umane isolate da tessuto adiposo adulto (PLA cells).
Materiali e metodi. Lo studio ha richiesto l’estrazione di cellule da tessuto adiposo omentale ottenuto mediante addominoplastica o liposuzione e l’analisi delle caratteristiche morfologiche e fenotipiche delle PLA cells mediante tecniche di microscopia ottica e a fluorescenza, citofluorimetria e Western blotting a seguito del trattamento con l’agente demetilante 5-Azacitidina. Sono stati condotti studi preliminari sulla citotossicità del trattamento demetilante con saggi sull’attività delle deidrogenasi mitocondriali e sulla capacità proliferativa.
Risultati. Negli studi di citotossicità dell’agente demetilante 5-Azacitidina, si è registrato una riduzione dell’attività metabolica mitocondriale nella popolazione trattata rispetto al controllo. Lo studio ha anche fornito le conoscenze per definire la concentrazione di agente demetilante rispetto al range riportato in letteratura.
Le popolazioni trattate con l’agente demetilante hanno mostrato sofferenza durante l’espansione generazionale; la popolazione cellulare riseminata dopo distacco enzimatico presenta un numero di cellule significativamente inferiore rispetto al campione di controllo. D’altro lato, la popolazione presenta capacità proliferativa paragonabile a quella dei campioni non trattati, durante e dopo il trattamento demetilante. I due risultati potrebbero indicare che il trattamento demetilante non influisca sulla proprietà replicativa della cellula, ma ne riduca la capacità di adesione nella coltura in monostrato dopo il cambio generazionale.
Poiché le PLA cells sono in grado di differenziare in altre tipologie cellulari, sono stati condotti studi di differenziamento in senso adipogenico, osteogenico e miogenico su popolazioni tratte in precedenza con l’agente demetilante. Il confronto con la risposta agli stimoli differenziativi di cellule non trattate ha dimostrato che l’agente demetilante non ha modificato la capacità differenziativa.
Le indagini di microscopia a fluorescenza e Western blotting hanno mostrato che il fattore di trascrizione tessuto-specifico SREBP, presente nelle popolazioni di origine, è ugualmente espresso dopo il trattamento con 5-Azacitidina. La ricerca dei fattori di trascrizione NANOG e OCT4, responsabili della capacità di self-renewing nelle cellule pluripotenti non ha dato risultati positivi. Le cellule trattate con 5-Azacitidina sono state mantenute in coltura ed espanse, per valutare un eventuale rimodellamento proteomico nelle cellule replicate, ma non è stata registrata espressione dei due marcatori nucleari.
Gli studi di caratterizzazione condotti con immunofluorescenza confocale e analisi citometriche hanno dimostrato che la popolazione cellulare isolata da tessuto adiposo è eterogenea. E’ stato osservato che, l’espansione generazionale determina un arricchimento nella componente cellulare CD90+/CD105+/CD29+, e cioè una popolazione con forti analogie fenotipiche con le cellule staminali stromali del midollo osseo, a scapito delle cellule endoteliali ed ematopoietiche presenti in origine. Inoltre, le cellule mantenute in coltura perdono il segnale dell’ADRP, proteina associata al commitment adipogenico, ed esibiscono una deplezione delle vescicole adipose citoplasmatiche, mentre è stato osservato nelle cellule di terza e quarta generazione la comparsa del marcatore di staminalità mesenchimale STRO1, espresso unicamente nella frazione stromale delle cellule staminali estratte da midollo osseo.
Conclusioni. PLA cells di origine umana non si comportano in vitro dopo trattamento demetilante come le corrispondenti cellule di origine animale e cioè non sembrano essere in grado, nelle condizioni sperimentali usate in questo studio, di essere riprogrammate ad un fenotipo diverso da quello di origine. Questo risultato pone il problema dello stato di metilazione del DNA nelle cellule isolate da tessuto adiposo che mantenute a lungo in vitro assumono il fenotipo CD105+/ CD90+/STRO1+/ADRP-, e se un trattamento che preveda l’associazione di diversi agenti demetilanti/deacetilanti in combinazione con fattori di crescita possa realizzare in queste cellule una reale riprogrammazione funzionale in vitro.

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Tipo di EPrint:Tesi di dottorato
Relatore:Grandi , Claudio
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:01 Febbraio 2009
Anno di Pubblicazione:01 Febbraio 2009
Parole chiave (italiano / inglese):PLA cells, demetilazione; marcatori tessuto specifici SREBP e di staminalità mesenchimale STRO1, CD105, CD90.
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:1863
Depositato il:01 Feb 2009
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