ONDERZOEK VAN HET POTENTIEEL VAN hAFSC VOOR PERINATALE PATHOLOGIEËN

ABSTRACT Stem cells potential in regenerative medicine has gained attention in recent years. Stem cells obtained from different tissues retain the ability to self-renew and differentiate into several cell types. Amniotic fluid (AF) is a promising source of stem cells being accessible at routine amniocentesis whilst raising few ethical concerns . Not only for they have high proliferative capacity and they differentiate into cells from all the three germ layers not forming teratomas when injected in vivo. In the first part of this PhD project, a reproducible isolation protocol was established to derive monoclonal population of AFSC from amniocentesis samples collected between the 15th till the 22nd gestational week. Of the 120 colonies analysed all expressed the canonical mesenchymal stem cell markers and could differentiate into three mesenchymal lineages (adipogenic, osteogenic and chondrogenic). No link between morphology, marker expression and differentiation potential was observed, showing the heterogeneity of AFSC during development. In view of the clinical use of AFSC, effective cryopreservation of AFSC or fresh AF was investigated. Seven cryopreservation protocols were analysed in order to freeze AFSC and fresh AF. The best three protocols were also tested in a collaborating facility with no stem cell expertise proving their reproducibility. One of these applied the good manufacture practice (GMP)-legislation and so can be used in a clinical setting. Thawed stem cells retained their markers expression and differentiation potential. The second part of this PhD focused on the regenerative potential of AFSC, in particular in skeletal muscle and kidney regeneration. Our research group is interested in novel therapies for congenital diaphragmatic hernia (CDH). To tackle the issue of the diaphragmatic defect we investigated the myogenic potential of AFSC in vitro and in vivo. Since no myogenic precursor are found into the AF, AFSC were screened for pericyte markers in particular mesangioblast (Mab). Mab is a pericyte subpopulation founded in the skeletal muscle that can differentiated into myoblasts in vitro and in vivo. AFSC positively expressed pericyte markers and they were able to participate into the formation of new myotubes when cultivated with murine myogenic cells. In addition, AFSC showed a paracrine action on injured myotubes in a transwell assay. Specific growth factors and remodelling proteinase were modulated by AFSC. Same expression pattern was observed when AFSC were injected into an injured muscle with also a reduction in fibrosis process. Since the majority of AF during the second trimester is composed of foetal urine, characterisation of AFSC for renal markers was evaluated. Several AFSC populations showed renal-like characteristics with the expression of renal progenitor markers by qPCR, IF and flow cytometry. Renal-like AFSC were tested in an acute kidney injury (AKI) model and showed their renoprotective effect. When AFSC were genetically manipulated to over-express the therapeutic protein vascular endothelial growth factor (VEGF), this renoprotective effect was maximized. Moreover, we noticed an optimal therapeutic window as an exacerbation of the injury was observed when a high dose of VEGF-AFSC was administered. In conclusion this study has shown a reproducible isolation protocols to derive AFSC and suitable freezing procedures to store AFSC for future research and clinical applications. Moreover, our data suggest that a AFSc subtype with pericytic characteristics has the ability to modulate muscle regeneration in vitro and in vivo. Further to this, a monoclonal population of AFSC with renal-like characteristics were identified and showed renoprotective effect in AKI. This effect was increased when VEGF-AFSC were used, showing the possibility to use AFSC as a delivery of therapeutic protein.nrpages: 196status: publishe