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Mandoli, Amit (2012) Stem cells in molecular and regenerative medicine. [Ph.D. thesis]

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Abstract (english)

Stem Cells are rare cells with the crucial ability to self-renew and to generate mature cells of any tissue through differentiation. Adult stem cells hold great promise for regenerative medicine, tissue repair, and gene therapy. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, tenocytes, and haematopoiesis supporting stromal cells. Under normal condition these stem cells are tightly regulated by both intrinsic and extrinsic signals and malfunctioning in this balance can result in cancer. In this thesis we focused on two different aspects of stem cells: the leukemia stem/initiating cells in acute myeloid leukemia (AML) and the usage of stem cells in regenerative medicine.
In the first part we focused on the molecular mechanism of AML-ETO, a results from the t(8:21) translocation which has been associated with leukemic transformation. Acute myeloid leukaemia (AML) is defined as a heterogeneous group of clonal disorders caused by malignant transformation of a bone marrow-derived self-renewing stem or progenitor cell, which demonstrates an enhanced proliferation as well as aberrant differentiation resulting in haematopoietic insufficiency (i.e. granulocytopenia, thrombocytopenia or anaemia). These leukaemias are suggested to result from the acquisition of chromosomal rearrangements and multiple gene mutations in either a hematopoietic multipotent cell or a more differentiated, lineage-restricted progenitor cell that is transformed in a so-called leukaemic stem or initiating cell, which keeps the ability to self-renewal. AML is generally regarded as a stem cell disease and is commonly altered both at the epigenetic as well as the genetic level. AML is the most common acute leukemia affecting adults, and its incidence increases with age. Therapies based on the current knowledge target the bulk leukemic population and spare the leukemic stem cells. It is therefore critical to determine and characterize the exact molecular mechanism involved in leukemic transformation for the development of novel therapeutic targets. AML patients harboring the t(8:21) translocation has intermediate prognosis and the identification of genome wide events in this subset of AML is clinically relevant and would lead to the understanding of molecular mechanism of disease progression.
To this end we analyzed the DNA binding pattern of AML1-ETO in AML cell lines and in primary AML blasts. We demonstrate that AML1-ETO preferentially binds regions that contain RUNX1/AML1 and ETS core consensus sequences and that the AML1-ETO binding sites invariably consist of HEB and partially CBFβ, RUNX1/AML1 as well as of ETS factors such as ERG and FLI1. Subsequent analysis in t(8;21) and t(15;17) (another AML associated translocation) cells revealed cell type specific ETS factor binding and preferential AML1-ETO binding to the cell type specific ETS factor binding sites. In addition, we uncovered that binding of the ETS factor ERG correlates with the ‘active’ histone acetylation mark.
Together our results suggest that ETS factors demarcate hematopoietic regulatory sites that provide a target for (aberrant) epigenetic regulation by oncofusion proteins.
In the second part we attempted to evaluate the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of acellular esophageal matrix and Mesenchymal stem cells (MSCs).

Mesenchymal Stem Cells (MSCs) are multipotent precursors to many mesodermal cell lineages in vertebrate animals and are most often obtained from bone marrow. Certain attributes of MSCs, including migration toward sites of inflammation, ease of transduction, and lack of immunogenicity, suggest these cells may be potentially useful for regenerative medicine. Putative therapeutic uses include regeneration of damaged tissue, acting as a vessel for delivering a therapeutic transgene, support of other cell types for tissue repair, and modulating the immune reaction to co-transplanted cells or tissues. The use of MSCs in tissue engineering approaches avoids the moral and technical issues associated with the use of those from embryonic source and MSCs have already demonstrated their efficacy in preliminary tissue engineering application.
Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. In the present study we attempted to evaluate the adhesion of MSCs on acellular esophageal matrix for esophagus tissue engineering. MSCs were isolated from rabbit bone marrow, characterized, expanded in vitro, and seeded onto rabbit acellular esophageal matrix.
Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker. Moreover, they supported cell adhesion, and in as much as
just after 24 h from seeding, the scaffold appeared completely covered by MSCs in static as well as in bioreactor.
Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous MSCs may represent a promising tissue engineering approach for the repair of esophageal injuries

Abstract (italian)

Le cellule staminali sono una popolazione cellulare con la particolare capacità di moltiplicarsi indefinitamente autorinnovandosi e di differenziarsi in cellule mature di qualsiasi altro tessuto attraverso il processo di differenziazione. In particolare l'utilizzo delle cellule staminali adulte costituisce una promettente applicazione nel campo della medicina rigenerativa, la riparazione dei tessuti e la terapia genica. Le cellule staminali adulte da midollo osseo (BMCs) comprendono due popolazioni cellulari: le cellule staminali ematopoietiche (HSCs), dalle quali originano tutte le cellule mature del sangue, e le cellule staminali mesenchimali (MSCs) che possono differenziare in osteoblasti, condrociti, adipociti, miociti, tenociti e cellule stromali di supporto per l'ematopoiesi. In condizioni normali l'autorinnovamento della popolazione staminale è strettamente regolato sia da segnali estrinseci che intrinseci ed un'alterazione di questo equilibrio può portare all'instaurarsi di un cancro.
In questa tesi abbiamo analizzato due differenti aspetti delle cellule staminali: le cellule staminali che danno origine a leucemia nella leucemia mieloide acuta (AML) e l'utilizzo delle cellule staminali nella medicina rigenerativa.
Nella prima parte del lavoro abbiamo approfondito il meccanismo molecolare dell' AML-ETO, risultato della traslocazione genica t(8:21) che viene associata alla trasformazione leucemica. La leucemia mieloide acuta (AML) è definita come un gruppo eterogeneo di disordini clonali causati dalla trasformazione maligna di cellule staminali o progenitori staminali di derivazione midollare, che mostrano un aumento della capacità proliferativa così come un differenziamento aberrante che porta ad una insufficienza ematopoietica (per esempio: granulocitopenia, trombocitopenia o anemia). Questi tipi di leucemia sembrano essere il risultato dell'acquisizione di riarrangiamenti cromosomici e mutazioni geniche multiple da parte delle cellule ematopoietiche multipotenti o di progenitori cellulari più differenziati e indirizzati verso una linea cellulare specifica, che risultano così trasformati in cellule staminali leucemiche o cellule inizianti la leucemia, che mantengono la capacità di autorinnovamento. L' AML è solitamente considerata una malattia delle cellule staminali e comunemente presenta alterazioni sia a livello genetico che epigenetico. L' AML è la forma più comune di leucemia acuta che colpisce soprattutto la popolazione adulta e la sua incidenza aumenta con l'età. Gli attuali approcci terapeutici hanno come target le cellule staminali leucemiche e la popolazione leucemica per intero. E' quindi di cruciale importanza riuscire a determinare e caratterizzare l'esatto meccanismo molecolare coinvolto nella trasformazione leucemica per lo sviluppo di nuovi bersagli terapeutici. I pazienti affetti da AML che manifestano la traslocazione t(8:21) hanno una prognosi intermedia e l'identificazione di ampi eventi genici in questo subset delle AML è clinicamente rilevante in quanto potrebbe portare alla comprensione dei meccanismi molecolari della progressione della malattia.
A questo scopo sono stati analizzati i pattern di legame al DNA di AML1-ETO nelle cellule di linea AML e nei blasti di AML. Abbiamo dimostrato che AML1-ETO lega preferenzialmente le regioni che contengono le sequenze di consenso RUNX1/AML1 e ETS e che i siti di legame di AML1-ETO si sovrappongono invariabilmente a quelli di HEB e parzialmente a quelli di CBFβ, RUNX1/AML1 così come accade per i fattori ETS, quali ERG e FLI1. Le successive analisi sulle cellule t(8;21) e t(15;17) (un'altra traslocazione associata con l' AML) hanno evidenziato il legame di fattori ETS specifici per questi tipi cellulari e il legame preferenziale di AML1-ETO ai siti di legame per i fattori ETS specifici per il tipo cellulare. Inoltre è stato anche scoperto che il legame di un fattore ETS, ERG, correla con un segnale di acetilazione istonica "attiva".
Presi insieme questi risultati suggeriscono che i fattori ETS demarcano i siti regolatori ematopoietici che forniscono un target per la regolazione epigenetica (aberrante) da parte delle proteine di oncofusione.

Nella seconda parte di questa tesi è stata testata la possibilità di ottenere in vitro un esofago ingegnerizzato composto da matrice acellulare esofagea e cellule staminali mesenchimali (MSCs)
che potesse essere impiantato in vivo.
Le cellule staminali mesenchimali (MSCs) nei vertebrati sono precursori multipotenti di molte linee cellulari di origine mesodermica e vengono ottenute per la maggior parte dal midollo osseo. Alcune caratteristiche delle MSCs, inclusa la capacità di migrare verso i siti di infiammazione, la facilità di trasduzione e la perdita di immunogenicità, suggeriscono che queste cellule possano essere potenzialmente utilizzabili nella medicina rigenerativa. I probabili usi terapeutici includono la possibilità di rigenerare un tessuto danneggiato, agendo come veicolo per il trasporto di transgeni terapeutici, di supportare altri tipi cellulari per il riparo tessutale, e di modulare la reazione immunitaria dell'ospite nei confronti delle cellule o dei tessuti co-trapiantati. L'uso delle MSCs permette di evitare i problemi di natura etica e morale associati all'utilizzo delle cellule staminali di origine embrionale; inoltre le MSCs hanno già dimostrato la loro efficacia in studi preliminari che prevedevano la loro applicazione in ingegneria tessutale.
I materiali artificiali e i tessuti autologhi utilizzati per la ricostruzione dell'esofago spesso comportano complicazioni come stenosi e rottura dell'impianto nei follow-up a lungo termine. Nel presente studio è stata valutata l'adesione delle MSCs ad una matrice acellulare di esofago per la costruzione di un tessuto esofageo ingegnerizzato. Le MSCs sono state isolate da midollo osseo di coniglio, caratterizzate, espanse in vitro e seminate su una matrice esofagea di coniglio.
Le matrici acellulari ottenute attraverso un metodo detergente-enzimatico non presentavano marker per il complesso maggiore di istocompatibilità. Inoltre supportavano l'adesione cellulare e in non più di 24 ore dalla semina lo scaffold appariva completamente coperto dalle MSCs sia in condizione statica che in bioreattore.
Complessivamente questi risultati suggeriscono che i tessuti ingegnerizzati composti da matrice acellulare omologa e MSCs autologhe possono rappresentare un promettente approccio per il riparo di danni all'esofago

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EPrint type:Ph.D. thesis
Tutor:Parnigotto, Pier Paolo
Supervisor: Martens, Joost
Data di deposito della tesi:19 January 2012
Anno di Pubblicazione:19 January 2012
Key Words:Stem cells, regenerative medicine and cancer stem cells
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/16 Anatomia umana
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Farmaceutiche
Codice ID:4396
Depositato il:29 Oct 2012 10:40
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