Looking for the holy well of tissue regeneration: isolation, characterization and differentiation into hard tissues of multipotent cells from human placenta

The improvement of life quality and lifespan lengthening in Western countries leads as a consequence a more and more urgent need for organs and tissues replacement. As organs and tissues derived from heterologous donors are not easily available and, moreover, oblige to a life-spanning use of immunosuppressive drugs, effective alternatives are needed. The aim of this work was the investigation of the regenerative potential nested in placenta, as an ethically acceptable and promising source of multipotent cells for tissue repair. A protocol is reported that allows the isolation from placenta of a homogeneous population of human chorionic multipotent stromal cells (hCMSCs) by the combination of plastic adherence and subsequent cell sorting of CD34neg cells. This population displays the typical characteristics of mesenchymal stem cells and exhibits clonal properties, as testified by the analysis of surface markers and doubling time. As for the differentiation potential, evidence was provided on the ability of hCMSCs to differentiate into osteoblastic cells as well as into adipoblasts and chondroblasts. In particular, concerning chondrogenic differentiation, culture protocols were useful to get the induction of the expression in hCMSCs of typical chondrogenic genes, but some work still has to be done in order to obtain a clear hyaline cartilage to satisfy the target of articular cartilage regeneration. Moreover hCMSCs possess an osteogenic potential if cultivated in appropriate conditions. When a canonical osteoinductive medium was used, differentiation towards the osteogenic lineage was scanty and only small calcified nodules were visualized. In order to improve these results, cells were grown in the presence of collagen or Bio-oss® as substrates. Collagen didn't influence gene expression of hCSMCs, while the presence of Bio-oss®, especially if combined with an osteogenic medium, allowed better differentiation and maturation of cells into osteoblasts or osteocytes. In experiments performed on a rat model of critical size defect, hCMSCs confirmed their ability to settle and proliferate on Bio-oss® scaffolds. Moreover those cells were able to fill the created defect by forming a soft compact tissue. On the whole, data collected in this PhD thesis suggest that placenta could be an effective and abundant source of cells for tissue regeneration.