Epidermal stem cell-based regenerative medicine has proven to be lifesaving for patients suffering from massive full-thickness burns or affected by genetic skin diseases, such as Junctional Epidermolysis Bullosa (EB). To ensure long-term epidermal restoration, epidermal cultures require an adequate number of stem cells (KSCs), detected as long-lived, self-renewing holoclone-forming cells. However, KSCs represent only a small proportion of clonogenic keratinocytes and cannot be prospectively isolated. These cells give rise to a population of clonogenic short-lived, transient amplifying progenitors (TACs), eventually generating terminally differentiated keratinocytes. While combined ex vivo cell and gene therapy has proven effective in treating recessive forms of EB, more than 50% of EB cases and other genodermatoses involve dominant inheritance, making gene addition unsuitable. Furthermore, due to the limited abundance of KSCs and challenges in their prospective identification and isolation, existing gene editing techniques lack the efficiency needed to ensure the genetic correction of the stem cell compartment. Recently, a distinctive molecular profile, called “holoclone signature”, has been identified by microarray analysis and further confirmed through single cell gene expression profiles. The expression level of this signature makes us able to distinguish stem cells from the other clonogenic progenitors, in retrospect. Starting from these assumptions, this thesis unfolds in two interconnected parts: 1. Reference dataset construction and KSCs markers identification: We curated a comprehensive dataset by integrating single cell RNA sequencing (scRNA-seq) data from keratinocyte cultures derived from 3 healthy donors. To enhance the robustness of our findings, we validated the identified clusters labels using additional keratinocyte cultures. Our differential expression analysis pinpointed three standout genes – NUSAP1, STMN1, and RRM2 – showcasing distinct expressions between KSCs and TACs. Their expression patterns were validated through both protein and RNA-based molecular assays, establishing these genes as novel markers useful for the unequivocal identification of holoclone-forming cells. 2. Bioinformatic prediction of reprogramming transcription factors (TFs) We conducted bioinformatic analyses on the same reference to pinpoint potential master regulators that can potentially reprogram TACs to KSCs. Employing the ARACNe-AP and the VIPER algorithms, we identified 7 putative master regulators, testing the differential enrichment of their regulons in different clonal types. A comparative analysis was carried out using the TransSynW tool for subpopulation conversion at the single-cell level. This software returned 7 TFs, 2 of which overlapped with the previously identified ones. The integration of these approaches resulted in the identification of 12 candidate genes that are being tested in intra-lineage reprogramming experiments. To promote the stochastic and combinatorial activation of these 12 genes, we are currently constructing a single-cell CRISPR activation (CRISPRa) screening strategy, using the CRISPRa-SAM (Synergistic Activation Mediator) system. The final aim of this approach is to identify the most effective combination to achieve a functional reprogramming.
La medicina rigenerativa basata sulle cellule staminali epidermiche si è dimostrata salvavita per pazienti affetti da gravi ustioni o da malattie cutanee genetiche, come l’Epidermolisi Bollosa Giunzionale (JEB). Per garantire il ripristino di una pelle funzionale a lungo termine, le colture epidermiche richiedono un adeguato numero di cellule staminali (KSC), definite come cellule auto-rinnovanti, con grande potenziale proliferativo e in grado di formare olocloni. Tuttavia, le KSC rappresentano solo una piccola proporzione di cheratinociti clonogenici e non possono essere isolate prospetticamente. Queste cellule danno origine a una popolazione di progenitori clonogenici a vita breve, che amplificano in maniera transiente (TAC), generando infine cheratinociti differenziati terminalmente. Anche se l’applicazione ex-vivo di una terapia cellulare-genetica combinata si è dimostrata efficace nel trattare le forme recessive di EB, oltre il 50% dei casi di EB e altre genodermatosi coinvolgono un'ereditarietà dominante, rendendo la gene-addition non adatta. Inoltre, a causa della limitata abbondanza delle staminali e delle sfide nella loro identificazione e isolamento prospettici, le tecniche esistenti di gene-editing mancano dell'efficienza necessaria per garantire la correzione genetica del compartimento delle cellule staminali. Recentemente, è stato identificato un profilo molecolare distintivo, chiamato "holoclone signature", mediante analisi microarray e successivamente confermato attraverso profili di espressione genica a singola cellula. Il livello di espressione di questa “firma” ci consente di distinguere le cellule staminali dagli altri progenitori clonogenici, a posteriori. Partendo da queste premesse, questa tesi si sviluppa in due parti interconnesse: 1. Costruzione del dataset di riferimento e identificazione dei marcatori delle staminali: Abbiamo curato un dataset completo integrando i dati di sequenziamento di RNA a singola cellula (scRNA-seq) dalle colture di cheratinociti derivate da 3 donatori sani. Per aumentare la robustezza delle nostre conclusioni, abbiamo convalidato le etichette dei cluster identificati utilizzando ulteriori colture di cheratinociti. La nostra analisi di espressione differenziale ha individuato tre geni - NUSAP1, STMN1 e RRM2 - che mostrano espressioni diverse tra KSC e TAC. I loro pattern di espressione sono stati convalidati attraverso test molecolari basati sia su proteine che su RNA, stabilendo questi geni come nuovi marcatori utili per l'identificazione inequivocabile delle staminali. 2. Predizione bioinformatica dei fattori di trascrizione (TFs) per la riprogrammazione: Abbiamo condotto un'analisi bioinformatica sullo stesso dataset per individuare potenziali regolatori che possano potenzialmente riprogrammare le TACs in KSCs. Utilizzando gli algoritmi ARACNe-AP - VIPER, abbiamo identificato 7 regolatori principali ipotetici, testando l'arricchimento differenziale dei loro reguloni nei diversi tipi clonali. Un'analisi comparativa è stata effettuata utilizzando il software TransSynW, sviluppato per la conversione tra sottopopolazioni partendo da dati di espressione a singola cellula. Questo software ha restituito 7 TF, 2 dei quali si sovrappongono con quelli precedentemente identificati. L'integrazione di questi approcci ha portato all'identificazione di 12 geni candidati che sono in fase di test in esperimenti di riprogrammazione intra-lineage. Per promuovere l'attivazione stocastica e combinatoria di questi 12 geni, stiamo attualmente costruendo una strategia di screening di attivazione basata sull’uso del sistema CRISPR, in particolare il CRISPRa-SAM (Synergistic Activation Mediator). L'obiettivo finale di questo approccio è identificare la combinazione più efficace per ottenere una riprogrammazione funzionale.
Definizione di marker molecolari delle cellule staminali epiteliali e strategie per il reprogramming dei cheratinociti / Grazia Marini , 2024 May 22. 36. ciclo, Anno Accademico 2022/2023.
Definizione di marker molecolari delle cellule staminali epiteliali e strategie per il reprogramming dei cheratinociti
MARINI, GRAZIA
2024
Abstract
Epidermal stem cell-based regenerative medicine has proven to be lifesaving for patients suffering from massive full-thickness burns or affected by genetic skin diseases, such as Junctional Epidermolysis Bullosa (EB). To ensure long-term epidermal restoration, epidermal cultures require an adequate number of stem cells (KSCs), detected as long-lived, self-renewing holoclone-forming cells. However, KSCs represent only a small proportion of clonogenic keratinocytes and cannot be prospectively isolated. These cells give rise to a population of clonogenic short-lived, transient amplifying progenitors (TACs), eventually generating terminally differentiated keratinocytes. While combined ex vivo cell and gene therapy has proven effective in treating recessive forms of EB, more than 50% of EB cases and other genodermatoses involve dominant inheritance, making gene addition unsuitable. Furthermore, due to the limited abundance of KSCs and challenges in their prospective identification and isolation, existing gene editing techniques lack the efficiency needed to ensure the genetic correction of the stem cell compartment. Recently, a distinctive molecular profile, called “holoclone signature”, has been identified by microarray analysis and further confirmed through single cell gene expression profiles. The expression level of this signature makes us able to distinguish stem cells from the other clonogenic progenitors, in retrospect. Starting from these assumptions, this thesis unfolds in two interconnected parts: 1. Reference dataset construction and KSCs markers identification: We curated a comprehensive dataset by integrating single cell RNA sequencing (scRNA-seq) data from keratinocyte cultures derived from 3 healthy donors. To enhance the robustness of our findings, we validated the identified clusters labels using additional keratinocyte cultures. Our differential expression analysis pinpointed three standout genes – NUSAP1, STMN1, and RRM2 – showcasing distinct expressions between KSCs and TACs. Their expression patterns were validated through both protein and RNA-based molecular assays, establishing these genes as novel markers useful for the unequivocal identification of holoclone-forming cells. 2. Bioinformatic prediction of reprogramming transcription factors (TFs) We conducted bioinformatic analyses on the same reference to pinpoint potential master regulators that can potentially reprogram TACs to KSCs. Employing the ARACNe-AP and the VIPER algorithms, we identified 7 putative master regulators, testing the differential enrichment of their regulons in different clonal types. A comparative analysis was carried out using the TransSynW tool for subpopulation conversion at the single-cell level. This software returned 7 TFs, 2 of which overlapped with the previously identified ones. The integration of these approaches resulted in the identification of 12 candidate genes that are being tested in intra-lineage reprogramming experiments. To promote the stochastic and combinatorial activation of these 12 genes, we are currently constructing a single-cell CRISPR activation (CRISPRa) screening strategy, using the CRISPRa-SAM (Synergistic Activation Mediator) system. The final aim of this approach is to identify the most effective combination to achieve a functional reprogramming.File | Dimensione | Formato | |
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