Abstract
To highlight and define the intricate pathways governing epithelial stem cells, we propose to use a multi-omics approach to evaluate.
Results achieved
In line with the project's evolution from proposal to commencement, the activities included in Task 3.2 have been modified, as agreed with the Principal Investigator. In particular, the exploratory efficacy experiment envisaged in the project proposal (evaluating the impact of Col7 RDEB-corrected keratinocyte application on skin repair using the full skin excision model) has been replaced with a safety experiment. The safety of Col7 RDEB-corrected keratinocytes has been tested using a tumourigenicity test following intradermal injection (in accordance with OECD guidelines 451 and 453). The safety of Col7 RDEB-corrected keratinocytes has been established using a study design and protocols that comply with regulatory requirements for procedures (OECD 451 and 453), as well as a number of animals. The outcome is a report on the tumourigenic risk of applying Col7 RDEB-corrected keratinocytes to the skin, as well as data suitable for scientific publication. All the experiments have been carried out according to ARRIVE+ principles, under the authorization N° 880/2024-PR. BALB/c Nude N male mice (CAnN.Cg-Foxn1nu/Crl, Charles River), 8-10 ws old were use, including a total number of 35 animals. In order to test tumorigenicity of mutated Col7 RDEB corrected keratinocytes, mice were intradermally injected (upper back, bilaterally, at dermal-epidermal junction, under gaseous anaesthesia) with the cell suspension in matrigel, 2x10^6cells, 75 microl each side. The following experimentsl groups were prepared: A431 Tumor cell line- positive control; RDEB cell – control; RDEB cell – SIN-gammaRV (Col7A1); K82 cell – control; K82 cell SIN-gammaRV (eGFP); K86 cell – control; K86 cell SIN-gammaRV (eGFP); K88 cell – control; K88 cell SIN-gammaRV (eGFP); RDEB 23 cell – control; RDEB 23- SIN-gammaRV (Col7A1). After injection, animals were monitored every second day for general health conditions, body weight, and tumor onset up to 35 post-injection days. At sacrifice, a sample of skin was taken at the injection site, including the tumor mass, if present, and fixed in 10% NBF (neutral buffered formalin) for histological and immunohistochemical analysis at PI lab, and a sample was quickly frozen (N2) for molecular analysis. In parallel, RU3 conducted experiments to investigate the efficacy of human cell derivatives in improving wound healing quality. This study examined the impact of secretome derived from various human cell types (adult and foetal mesenchymal stromal cells) on a skin lesion model (full skin excision), supported by a ring stent for cell deposition. The experiment was carried out using genetically diabetic male mice (C57BL/KsJ-m+/+Leprdb - db/db, Jackson Laboratories), aged 11–12 weeks (authorisation no. 695-2024-PR). Secretome was applied weekly, and animals were sacrificed at two time points, corresponding to re-epithelisation and wound remodelling. Skin biopsies were analysed by histology and immunohistochemistry for re-epithelisation (epidermal thickness), re-innervation (PGP9.5 and small fibre immunoreactivity), angiogenesis (PECAM/laminin immunoreactivity) and fibrosis (α-SMA and myofibroblasts). mRNA expression analysis (RT-PCR) including 80 genes involved in fibrosis and ECM deposition was carried out to discover pathways and predict protein–protein interactions (web software STRING 10 and Gene Ontology databases). These results will inform therapies based on Col7 RDEB-corrected keratinocytes with regard to fibrosis the QUALITY and SAFETY of normal and gene-corrected KSC for cell and gene therapy. We will accurately define a fully comprehensive atlas mapping both the transcriptional and proteomic profile of KSC and TA in cell cultures derived from healthy donors and EB patients, before and after gene correction. Data deriving from the following analysis will define a KSC-specific and EB signature. Thanks to the classification of exclusive molecular patterns, in vitro and in vivo bio-safety assays will be developed to demonstrate the safety of γRV for gene therapy. A strong scientific rationale and an integrated network of the expertise shared between UNIMORE, UNITO, UNIBO, will lead to innovative improvement in the translational application of stem cells. The project can be intended as a proof of concept.Dettagli del progetto
Responsabile scientifico: Laura Calzà
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
Università degli studi di Modena e Reggio Emilia - UNIMORE(Italy)
Contributo totale Unibo: Euro (EUR) 53.867,00
Durata del progetto in mesi: 24
Data di inizio
28/09/2023
Data di fine:
28/02/2026
