Grisafi, Davide (2008) Possibile impiego delle cellule staminali del fluido amniotico per la riparazione del danno polmonare in un modello animale per broncodiplasia: valutazione in vitro e in vivo. [Tesi di dottorato]
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Since our knowledge on the replication and differentiating capability of the respiratory tract is still limited, the therapeutic potential of this system is quite unexplored. The respiratory disease in pediatric age concerns several pathologies among which broncopulmonary dysplasia (BPD) in neonate and premature newborns, asthma and cistyc fibrosys are the most studied because of their diffusion among children and their infaust prognosis.. In the last few years some studies have shown the possibility of deriving progenitors with various potential from the amniotic fluid. Amniocentesis is a widely accepted method for prenatal diagnosis; it is associated with low risk both for the mother and the fetus and overcomes the ethical problems commonly associated to other sources. Recently, it has been described that amniotic fluid stem (hAFS) cells, for their ability to differentiate to various lineages, could represent a good candidate for therapeutic applications. The recent characterization of hAFS and the consolidation of the techniques for intratracheal transplantation have shown new perspectives for gene and cell therapy applications. In particular, for these purposes hAFS cells should be genetically modified with a therapeutic gene and delivered systematically or injected directly into the tissue of interest.
Materials and methods
The in vitro phase has evaluated for the first time the possibility to infect hAFS with first generation E1-deleted adenoviral vectors, and the mantainence of the stemness and differentiating capability even after transduction with foreign gene sequences.
In the in vivo phase of the project we verified the pulmonary homing and the eventual engraftment of hAFS cells, after intratracheal administration, in a 60% O2 rat model presenting a respiratory disease similar to the one observed in human patients affected by broncopulmonary dysplasia (BPD) and cystic fibrosis. The symptoms were reproduced by using OXYCYCLER, with two pressurized rooms in which animals are exposed at controlled percentages of O2 and CO2. In this trial, hAFS cells have been infected AdHCMVsp1LacZ, a first generation E1 deleted viral vector transducing LacZ, the β-gal specific gene used as marker.
At first, we investigated the feasibility of transducing hAFS cells with adenoviral vectors and to determine whether transduced stem cells retain the ability to differentiate into different lineages. Herein, we showed that hAFS cells could be efficiently infected by first generation adenovirus vectors. In addition, we demonstrated that infection and expression of two different marker genes, LacZ and EGFP, have no effect on cells phenotype and differentiation potential. In particular, on undifferentiated status, hAFS cells continued to express both the transgenes and stemness cell markers OCT4 and SSEA4 (stage-specific embryonic antigen 4). When cultured under mesenchymal conditions, infected cells could still differentiate into osteocytes and adipocytes expressing lineage specific genes.
Differently to what observed in embryonic stem cells, the amniotic fluid stem cells easily infect very efficiently. This could represent an excellent starting point for gene therapy studies in which a transient expression would be a necessary condition to the therapeutic approach.
In the in vivo phase we transplanted hAFS cells with an intratracheal administration in a rat model generated exposing newborns at 60% O2 for two weeks, reproducing in this way the chronic damage that can be seen in human patients affected by BPD. The results show that the model for chronic lung damage has been properly implemented; specific staining for lacZ performed three weeks post-transplant confirmed for hAFS cells a bronchiolar homing. After four weeks transplantation LacZ positive cells have been detected inside alveolis. Finally, an important phenomenon of damage repair was observed in the treated animals as compared to untreated controls.
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