Respiratory diseases affect millions of people globally, regardless of age, group or socioeconomic status, emerging as one of the major causes of disability and death overall. Many diseases can alter the structure and function of the different tracts of the respiratory system, substantially affecting the patients' life. Even worst, the number of people affected by these disorders is dramatically increasing due to the ongoing COVID-19 pandemic. In the case of wide structural alterations, the available medical treatments and the standard surgical strategies are ineffective or totally inapplicable. Despite the surgical based approaches and the tissue-engineering (TE) strategies tested to face this urgent medical need, the positive results obtained have been very few and, to date, no strategy has become a well-established and routinely-applied clinical procedure. One of the major difficulties encountered in these clinical approaches is the regeneration of a stable/self-renewing epithelium onto the transplanted graft. This feature is imperative to preserve the respiratory function, avoid infections, granulation tissue formation and re-stenosis. In TE strategies, the adult airway epithelial cells would be the most appropriate cellular source for restoring the airway epithelium. However, many difficulties have been encountered in the in-vitro expansion of these cells. Indeed, they were described as effectively dividing for a very limited number of passages, beyond which they lose their differentiative potential and the ability to form a functional barrier. Therefore, it is reported that autologous airway epithelial cells do not meet the clinical regeneration needs and are unsuitable and unreliable cell sources for TE approaches. In the present study, we proved the ability of a culture system, largely used for the clinical expansion of different epithelial tissues, to safely and effectively maintain the long-term proliferative and differentiation potential of airway epithelial cells. Moreover, we established reproducible quality controls to be adopted from the biopsy collection up to the first steps of the generation of a TE construct. These quality controls include/verify 1) the tissue-regenerative properties of the cells extracted from the biopsy, 2) the expression of some critical markers identifying the cellular identity, the tissue integrity and the early epithelial differentiation of the cultures. These markers have to be verified during both the in-vitro cell expansion and during the TE process. 3) Crucial, we assessed the heterogeneity of the basal cells, identifying the stem cells of the airway epithelium. These cells can self-renew, withstanding even extreme differentiation conditions to regenerate the tissue, maintaining its physiological heterogeneity. Here, we hypothesized the use of some transcription factors as possible molecular markers to be adopted, alternatively to the clonal analysis, to evaluate the percentage of stem cells within an airway culture. Moreover, the multiple analyses conducted at the single-cell level allowed us to understand the mechanisms that underlie the cellular differentiation/specialization process. Therefore, here we propose a model showing how the airway epithelial renewal and differentiation process could occur. Finally, thanks to the knowledge acquired during the in-vitro cultures characterization and to the consistency of the results obtained from many human donors, we decided to start a pilot study envisaging the reconstruction of a pseudo-turbinate for the treatment of Empty Nose Syndrome’s patients. In this manuscript, we present our TE strategy and the data obtained by the initials steps on which this approach is based.
Le malattie respiratorie colpiscono milioni di persone in tutto il mondo, indipendentemente dalla fascia d'età o dallo status socioeconomico, emergendo come una delle principali cause di disabilità e morte. Molte di esse possono alterare la struttura e la funzione di diversi tratti respiratori, influenzando in modo sostanziale la vita dei pazienti. Purtroppo, il numero di persone affette da questi disturbi è drammaticamente in aumento a causa della pandemia da COVID-19 in corso. In caso di ampie alterazioni strutturali, i trattamenti medici disponibili e le strategie chirurgiche standard sono inefficaci o totalmente inapplicabili. Nonostante gli approcci chirurgici e le strategie di “tissue-engineering” (TE) proposti per affrontare questa urgente necessità medica, ad oggi nessuna strategia è diventata una procedura clinica ben consolidata e applicata di routine. Una delle maggiori difficoltà riscontrate in questi approcci clinici è la rigenerazione di un epitelio stabile sul costrutto trapiantato. Questa caratteristica è indispensabile per preservare la funzionalità respiratoria, evitare infezioni, formazione di tessuto di granulazione e re-stenosi. La fonte cellulare più appropriata per la rigenerazione dell’epitelio respiratorio in approcci di TE sarebbero le cellule epiteliali adulte del tessuto stesso. Tuttavia, la loro espansione in-vitro è resa difficile dal fatto è stato osservato siano capaci di dividersi efficientemente solo per pochissimi passaggi, oltre i quali perdono il loro potenziale di differenziazione e la capacità di formare una barriera funzionale. Pertanto si pensa che tali cellule siano inadatte e inaffidabili per approcci di TE. In questo studio abbiamo dimostrato la sicurezza e l’efficacia di un sistema di coltura, clinicamente validato per l’espansione di diversi tessuti epiteliali, nel mantenere il potenziale di proliferazione e differenziazione a lungo termine delle cellule epiteliali delle vie aeree. Inoltre, abbiamo stabilito dei controlli di qualità riproducibili da adottare nei diversi stadi di un approccio di TE. Tali controlli di qualità comprendono/verificano 1) le proprietà rigenerative delle cellule estratte dalla biopsia, 2) l'espressione di alcuni marcatori critici che identificano l'identità cellulare, l'integrità tessutale e il differenziamento epiteliale precoce delle colture. Questi marcatori devono essere verificati sia durante l'espansione cellulare in-vitro, sia durante il processo di TE. 3) Di cruciale importanza, abbiamo identificato le cellule staminali dell'epitelio delle vie aeree. Queste cellule sono in grado di auto-rinnovarsi, sopportando anche condizioni di differenziazione estreme per rigenerare il tessuto, mantenendo la sua eterogeneità fisiologica. Abbiamo ipotizzato l'uso di alcuni fattori di trascrizione, come possibili marcatori molecolari da adottare, in alternativa all'analisi clonale, per la valutazione della percentuale di cellule staminali all'interno di una coltura delle vie aeree. Inoltre, le molte analisi condotte a livello di singola cellula ci ha permesso di comprendere i meccanismi alla base del processo di specializzazione cellulare. Questo ci ha portato a proporre un modello che descrive come il rinnovamento epiteliale delle vie aeree e il processo di differenziazione potrebbero avvenire. Infine, grazie alle conoscenze acquisite durante la caratterizzazione delle colture in-vitro e alla coerenza dei risultati ottenuti da molti donatori umani, abbiamo deciso di avviare uno studio pilota che prevede la ricostruzione di uno pseudo-turbinato per il trattamento dei pazienti affetti da sindrome del naso vuoto. In questa tesi, presentiamo anche la nostra strategia di TE e i dati ottenuti dalle fasi iniziali su cui si basa questo approccio.
Cellule staminali dell’epitelio respiratorio: proprietà rigenerative, potenziale differenziativo e loro applicazione in ingegneria dei tessuti per la ricostruzione delle vie aeree umane / Davide Adamo , 2021 Nov 18. 33. ciclo, Anno Accademico 2019/2020.
Cellule staminali dell’epitelio respiratorio: proprietà rigenerative, potenziale differenziativo e loro applicazione in ingegneria dei tessuti per la ricostruzione delle vie aeree umane.
ADAMO, DAVIDE
2021
Abstract
Respiratory diseases affect millions of people globally, regardless of age, group or socioeconomic status, emerging as one of the major causes of disability and death overall. Many diseases can alter the structure and function of the different tracts of the respiratory system, substantially affecting the patients' life. Even worst, the number of people affected by these disorders is dramatically increasing due to the ongoing COVID-19 pandemic. In the case of wide structural alterations, the available medical treatments and the standard surgical strategies are ineffective or totally inapplicable. Despite the surgical based approaches and the tissue-engineering (TE) strategies tested to face this urgent medical need, the positive results obtained have been very few and, to date, no strategy has become a well-established and routinely-applied clinical procedure. One of the major difficulties encountered in these clinical approaches is the regeneration of a stable/self-renewing epithelium onto the transplanted graft. This feature is imperative to preserve the respiratory function, avoid infections, granulation tissue formation and re-stenosis. In TE strategies, the adult airway epithelial cells would be the most appropriate cellular source for restoring the airway epithelium. However, many difficulties have been encountered in the in-vitro expansion of these cells. Indeed, they were described as effectively dividing for a very limited number of passages, beyond which they lose their differentiative potential and the ability to form a functional barrier. Therefore, it is reported that autologous airway epithelial cells do not meet the clinical regeneration needs and are unsuitable and unreliable cell sources for TE approaches. In the present study, we proved the ability of a culture system, largely used for the clinical expansion of different epithelial tissues, to safely and effectively maintain the long-term proliferative and differentiation potential of airway epithelial cells. Moreover, we established reproducible quality controls to be adopted from the biopsy collection up to the first steps of the generation of a TE construct. These quality controls include/verify 1) the tissue-regenerative properties of the cells extracted from the biopsy, 2) the expression of some critical markers identifying the cellular identity, the tissue integrity and the early epithelial differentiation of the cultures. These markers have to be verified during both the in-vitro cell expansion and during the TE process. 3) Crucial, we assessed the heterogeneity of the basal cells, identifying the stem cells of the airway epithelium. These cells can self-renew, withstanding even extreme differentiation conditions to regenerate the tissue, maintaining its physiological heterogeneity. Here, we hypothesized the use of some transcription factors as possible molecular markers to be adopted, alternatively to the clonal analysis, to evaluate the percentage of stem cells within an airway culture. Moreover, the multiple analyses conducted at the single-cell level allowed us to understand the mechanisms that underlie the cellular differentiation/specialization process. Therefore, here we propose a model showing how the airway epithelial renewal and differentiation process could occur. Finally, thanks to the knowledge acquired during the in-vitro cultures characterization and to the consistency of the results obtained from many human donors, we decided to start a pilot study envisaging the reconstruction of a pseudo-turbinate for the treatment of Empty Nose Syndrome’s patients. In this manuscript, we present our TE strategy and the data obtained by the initials steps on which this approach is based.File | Dimensione | Formato | |
---|---|---|---|
TESI DOTTORATO DAVIDE ADAMO.pdf
Open Access dal 18/11/2024
Descrizione: TESI DOTTORATO DAVIDE ADAMO
Tipologia:
Tesi di dottorato
Dimensione
8.86 MB
Formato
Adobe PDF
|
8.86 MB | Adobe PDF | Visualizza/Apri |
Pubblicazioni consigliate
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris