RESUMEN
Inherited junctional epidermolysis bullosa is a severe genetic skin disease that leads to epidermal loss caused by structural and mechanical fragility of the integuments. There is no established cure for junctional epidermolysis bullosa. We previously reported that genetically corrected autologous epidermal cultures regenerated almost an entire, fully functional epidermis on a child who had a devastating form of junctional epidermolysis bullosa. We now report long-term clinical outcomes in this patient. (Funded by POR FESR 2014-2020 - Regione Emilia-Romagna and others.).
Asunto(s)
Epidermis/trasplante , Epidermólisis Ampollosa de la Unión/terapia , Queratinocitos/trasplante , Transducción Genética , Transgenes , Autorrenovación de las Células , Células Cultivadas/trasplante , Niño , Células Clonales , Epidermis/patología , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/patología , Estudios de Seguimiento , Enfermedades Genéticas Congénitas/patología , Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Vectores Genéticos , Humanos , Queratinocitos/citología , Queratinocitos/fisiología , Masculino , Regeneración , Células Madre/fisiología , Trasplante AutólogoRESUMEN
Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.
Asunto(s)
Células Epidérmicas/citología , Proteína Forkhead Box M1/metabolismo , Queratinocitos/citología , Análisis de la Célula Individual/métodos , Células Madre/citología , Transcriptoma/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Adhesión Celular/genética , Línea Celular , Autorrenovación de las Células/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Células Epidérmicas/metabolismo , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/metabolismo , Proteína Forkhead Box M1/genética , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Queratinocitos/metabolismo , Ratones , Análisis por Micromatrices , Familia de Multigenes , RNA-Seq , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.
Asunto(s)
Terapia Genética/métodos , Enfermedades de la Piel/genética , Enfermedades de la Piel/terapia , Animales , Sistemas CRISPR-Cas , Epidermis/metabolismo , Epidermólisis Ampollosa/terapia , Epidermólisis Ampollosa Distrófica/terapia , Epidermólisis Ampollosa de la Unión/terapia , Genes Dominantes , Genes Recesivos , Vectores Genéticos , Humanos , Lentivirus/genética , Síndrome de Netherton/terapia , Retroviridae/genética , Simplexvirus , Piel/metabolismo , Células Madre/citologíaRESUMEN
Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and α6ß4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular , Epidermis/metabolismo , Epidermólisis Ampollosa , Terapia Genética , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Epidermis/patología , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/metabolismo , Epidermólisis Ampollosa/patología , Epidermólisis Ampollosa/terapia , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Células Madre/patología , Factores de Transcripción/genética , Proteínas Señalizadoras YAP , KalininaRESUMEN
Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.
