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Migliorati, Angelo (2018) Advanced cell therapy strategies to correct corneal disorders. [Ph.D. thesis]

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Abstract (english)

Introduction
Epithelial homeostasis is guaranted by somatic stem cells wich, through different patways, such as p63 and Notch signaling, self-renew, differentiate and control tissue function and integrity. In vivo behavior of epithelial stem cells (ESCs) from different epithelia reflects differences in physiological role of the tissue. Limbal stem cells deficiency (LSCD) is characterized by conjunctival epithelial ingrowth, neovascularization, recurrent corneal erosion and persistent ulcers, as well as corneal scarring and ultimately leads to visual impairment and blindness. When the cornea is entirely covered with a fibro-vascular tissue, the chances of success of a traditional penetrating keratoplasty are virtually absent. Transplantation of autologous limbal epithelial stem cells (LESCs) or oral mucosa epithelial stem cells (OMESCs) cultured on fibrin glue has shown to be successful for unilateral or bilateral LSCD treatment, respectively. Despite that, fibrin presented several limitations including inability to repair or replace damaged corneal stroma.
Aim
The purpose of this work is to better understand the potentiality of somatic epithelial stem cells in order to identify possible approaches to exploit this potentiality. In the light of this, my research group has been able to identify and set up a cell therapy approach for a unique homozygous-heterozygous mosaicism of EEC Syndrome, demonstrating that epithelial stem cells have an intrinsic potential for regenerative medicine that can be exploited with a deeper characterization. We, also, aimed to test human keratoplasty lenticules (HKLs): particularly attractive, full-thickness scaffolds for corneal epithelial and stromal reconstruction that provides an interesting organotypic culture system for evaluation of growth, proliferation, and differentiation processes of epithelial stem cells (ESCs).
Results
During the three years of PhD I had the opportunity to collect and review cellular biology data of four diverse types of primary epithelial cells, derived from four different epithelia (skin, oral mucosa, limbus/cornea and conjunctiva) and, to better investigate the exhaustion of clonogenic potential and the self-renewal of epithelial stem cells we took in consideration also p63-defective oral mucosa primary cell lines obtained from three patients affected by EEC syndrome, already known to cause an acceleration in epithelial aging. Demonstrating that epithelial stem cells have an intrinsic potential for regenerative medicine that can be exploited with a deeper characterization, we have also been able to apply a cell therapy protocol to a patient affected by EEC syndrome in a rare form of mosaicism. We isolated the cell population with a mild phenotype from this patient, enriching these primary cells in vitro and producing well-organized and stratified epithelial sheets. The novelty and the importance of this case was related to the possibility to start a customized cell therapy approach for this unique case of EEC syndrome, based solely on epithelial stem cell manipulation.
Limbal stem cells (H-LESCs) expanded onto HKLs gave rise to a keratinized stratified squamous epithelium morphologically similar to that of normal corneas. To set up the cohort of animal patients, we proceded with the characterization of cell lines obtained from biopsies of the cornea (C-CESCs) and oral mucosa (C-OMESCs) of canine origin. Primary lines were serially propagated until exhaustion in order to get life span data to compare their behavior with human limbal stem cells. For each passage, we also performed colony forming efficiency assays (CFE) to estimate the proportion of clonogenic cells present in the culture. Results showed a trend of canine cell lines comparable to human limbal cells trend, with a similar decrease of clonogenic cells number, a similar percentage of aborted colonies during serial cultivation and a similar replicative senescence. Then, primary human and canine epithelial stem cells were seeded onto HKLs. The resulting epithelia was well organized and stratified into four to five cell layers with basal cuboidal cells differentiating upward to winged cells. The layer of basal cuboidal cells was firmly attached to the underlying ECM and to the basement membrane through integrinβ4. Maintenance of stemness potential and differentiation pathways were assessed checking the expression of the stem cells marker p63 and the terminally differentiated cells marker Involucrine. Importantly, expression of the different markers resembled that observed in normal epithelia, thus suggesting that HKLs are able to support the growth and maintain the differentiation pathways of epithelial stem cells.
Discussion and conclusions
Our findings demonstrate that primary epithelial cells have unique characteristics with an inimitable potential that makes them a malleable tool, able to adapt to different necessities. And so on, although these data are intriguing, further investigation could provide more and more useful data for their clinical application in regenerative medicine. As a proof-of-principle that epithelial stem cells have an intrinsic potential for regenerative medicine that can be exploited with a deeper characterization, my group have been able to apply, in vitro, a cell therapy protocol to a patient affected by a rare mosaic form of EEC syndrome.
HKLs appear to be particularly attractive, animal-free (feeder-free) and full-thickness scaffolds for corneal reconstruction. We have already started with the the recruitment of canine patients to assess the transplantability and functionality of these organotypic structures and, through the collaboration with different veterinary departments, we are creating a small cohort on which to start with first transplantation trials.

Abstract (italian)

Introduzione
L'omeostasi epiteliale è garantita da cellule staminali somatiche che, attraverso diversi pathways, come quello di p63 o di Notch, si auto-rinnovano, differenziano e controllano la funzione e l'integrità del tessuto. Il comportamento in vivo delle cellule staminali epiteliali (ESC) di diversi epiteli riflette le differenze nel ruolo fisiologico che queste cellule hanno nel tessuto di origine. Il deficit di cellule staminali limbali (LSCD) è caratterizzato da crescita epiteliale congiuntivale, neovascolarizzazione, erosione corneale ricorrente e ulcere persistenti, nonché cicatrici corneali, e porta infine a deficit visivo e cecità. Quando la cornea è interamente coperta da un tessuto fibro-vascolare, le probabilità di successo di una cheratoplastica penetrante tradizionale sono praticamente assenti. Il trapianto di cellule staminali epiteliali autologhe del limbus (LESCs) o di cellule staminali epiteliali della mucosa orale (OMESCs) coltivate su scaffold di fibrina ha dimostrato di avere successo per il trattamento unilaterale o bilaterale di LSCD, rispettivamente. Nonostante ciò, la fibrina presentava numerose limitazioni tra cui l'incapacità di riparare o sostituire lo stroma corneale danneggiato.
Scopo
Lo scopo di questo lavoro è quello di comprendere meglio la potenzialità delle cellule staminali epiteliali somatiche al fine di identificare possibili approcci per sfruttare questa potenzialità. Alla luce di ciò, il mio gruppo di ricerca è stato in grado di identificare e impostare un approccio di terapia cellulare per un caso unico di mosaicismo omozigote-eterozigote della Sindrome EEC, dimostrando che le cellule staminali epiteliali hanno un potenziale intrinseco per la medicina rigenerativa che può essere sfruttata con una caratterizzazione più profonda. Un secondo scopo della tesi è stato quello di testare gli Human Keratoplasty Lenticules (HKL): scaffolds particolarmente attraenti per la ricostruzione a tutto spessore della superficie anteriore dell’occhio che fornisce, anche, un interessante sistema di coltura organotipica per la valutazione dei processi di crescita, proliferazione e differenziazione delle cellule staminali epiteliali (ESCs ).
Risultati
Durante i tre anni di dottorato ho avuto l'opportunità di raccogliere e rivedere i dati di biologia cellulare di quattro diversi tipi di cellule epiteliali primarie, derivate da quattro diversi epiteli (pelle, mucosa orale, limbus/cornea e congiuntiva) e, per studiare meglio l'esaurimento del potenziale clonogenico e dell'auto-rinnovamento delle cellule staminali epiteliali abbiamo preso in considerazione anche delle linee cellulari primarie di mucosa orale con difetti genetici del gene p63 già noti per causare un'accelerazione nell'invecchiamento epiteliale, ottenute da tre pazienti affetti da sindrome EEC. Dimostrando che le cellule staminali epiteliali hanno un potenziale intrinseco per la medicina rigenerativa che può essere sfruttato con una caratterizzazione più profonda, siamo stati anche in grado di applicare un protocollo di terapia cellulare a un paziente affetto da sindrome EEC in una rara forma di mosaicismo. Abbiamo isolato la popolazione cellulare con un fenotipo lieve da questo paziente, arricchendo queste cellule primarie in vitro e producendo foglietti epiteliali ben organizzati e stratificati. La novità e l'importanza di questo caso è legata alla possibilità di iniziare un approccio di terapia cellulare personalizzata per questo caso unico di sindrome EEC, basata esclusivamente sulla manipolazione delle cellule staminali epiteliali.
Le cellule staminali limbari (H-LESC) coltivate su HKL hanno dato origine a un epitelio squamoso stratificato cheratinizzato morfologicamente simile a quello delle cornee normali. Per costituire la coorte di pazienti animali, abbiamo proceduto alla caratterizzazione di linee cellulari ottenute da biopsie della cornea (C-CESCs) e mucosa orale (C-OMESCs) di origine canina. Le linee primarie sono state propagate in serie fino ad esaurimento al fine di ottenere dati di life span per confrontare il loro comportamento con le cellule staminali limbari umane. Per ogni passaggio, abbiamo anche eseguito analisi di Colony Forming Efficiency (CFE) per stimare la proporzione di cellule clonogeniche presenti nella coltura. I risultati hanno mostrato una tendenza delle linee cellulari canine comparabile all'andamento delle cellule limbari umane, con una diminuzione simile del numero di cellule clonogeniche, una percentuale simile di colonie abortive durante i passaggi in coltura e una simile senescenza replicativa. Quindi, cellule staminali epiteliali primarie umane e canine sono state seminate su HKL. L'epitelio risultante era ben organizzato e stratificato in quattro o cinque strati cellulari con cellule cuboidali basali che differenziano le cellule verso l'alto e quelle alate. Lo strato di cellule cuboidali basali era saldamente attaccato alla matrice extracellulare sottostante e alla membrana basale attraverso l'integrina-4. Sono stati valutati il mantenimento del pool di cellule staminali e i corretti processi di differenziamento controllando l'espressione del marcatore di cellule staminali, p63, e del marcatore di cellule terminalmente differenziate, Involucrina. È importante sottolineare che l'espressione dei diversi marcatori assomiglia a quella osservata negli epiteli normali, suggerendo quindi che gli HKL sono in grado di supportare la crescita e il mantenimento di tessuti epiteliali ricostruiti.
Discussione e Conclusioni
I nostri risultati dimostrano che le cellule epiteliali primarie hanno caratteristiche uniche con un potenziale inimitabile che le rende uno strumento malleabile, in grado di adattarsi alle diverse necessità. Sebbene questi dati siano intriganti, ulteriori indagini potrebbero fornire dati sempre più utili per la loro applicazione clinica nella medicina rigenerativa. Come prova di principio che le cellule staminali epiteliali hanno un potenziale intrinseco per la medicina rigenerativa che può essere sfruttato con una caratterizzazione più profonda, il mio gruppo è stato in grado di applicare, in vitro, un protocollo di terapia cellulare a un paziente affetto da un raro mosaicismo di sindrome EEC. Le HKL sembrano essere scaffold particolarmente attraenti (animal-free) per la ricostruzione a tutto spessore della cornea. Abbiamo già iniziato con il reclutamento di pazienti canini per valutare la trapiantabilità e la funzionalità di queste strutture organotipiche e, attraverso la collaborazione con diversi dipartimenti veterinari, stiamo creando una piccola coorte sulla quale iniziare con le prime prove di trapianto.

EPrint type:Ph.D. thesis
Tutor:Palù, Giorgio
Supervisor:Di Iorio, Enzo
Ph.D. course:Ciclo 30 > Corsi 30 > MEDICINA MOLECOLARE
Data di deposito della tesi:14 January 2018
Anno di Pubblicazione:14 January 2018
Key Words:Epithelial Stem Cells, Cell Therapy, Tissue Engineering, Cornea, EEC Syndrome
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/03 Genetica medica
Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Area 06 - Scienze mediche > MED/30 Malattie apparato visivo
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:10792
Depositato il:16 Nov 2018 09:11
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