Characterization and optimization of in vitro culture conditions of adult stem cells for clinical cell therapy

Adipose and dental tissues are attractive and abundant sources of stem cells with therapeutic applicability in diverse medical fields for the repair and regeneration of acute and chronically damaged tissues. Mesenchymal stem cells (MSCs) are found in a variety of tissues including bone marrow, adipose tissue and dental tissues, and reside within the tissue in a hierarchical arrangement maintaining their proliferative potential and differentiation stage. Importantly, unlike the human bone marrow stromal/stem stem cells (BMSCs) that are present at low frequency in the bone marrow, adipose stem cells (ASCs) can be retrieved in high number from either liposuction aspirates or subcutaneous adipose tissue fragments and can easily be expanded in vitro. Also, the dental stem cell sources (DSCs) can easily be retrieved in high numbers from third molars representing an appealing source of MSCs owing to the fact that third molars are an expendable tissue that can be removed with minimal surgical stress. ASCs and DSCs display properties similar to those observed in BMSCs and, upon induction, undergo at least osteogenic, chondrogenic and adipogenic differentiation in vitro. Furthermore, ASCs have been shown to be immunoprivileged, prevent severe graft-versus-host disease in vitro and in vivo and to be more genetically stable in long-term culture than BMSCs. They have also proven applicability in other functions, such as providing hematopoietic support and gene transfer. Nevertheless, expansion of ASCs and DSCs is necessary prior to performing clinical studies. Currently, standard in vitro cell expansion techniques utilize reagents of animal origin as part of the cultivation procedures. Due to critical safety issues, use of animal-derived reagents in clinical applications is not a suitable option, since severe immune response may develop in the recipient. Other possible risks for the recipient related to the introduction of animal-derived reagents include viral or bacterial infections, prions, and unidentified zoonoses. In this study, the renewal and differentiation capacity of stem cells from different adult tissues were evaluated in vitro. Stem cells from impacted third molars such as dental pulp stem cells, dental follicle cells and periodontal ligament stem cells, and also stem cells from adipose tissue were investigated. Furthermore, for ASCs, the culture conditions were developed and optimized to meet the GMP (Good Manufacturing Practice) standards set by the European Union. In the final part of the study, the clinical applicability of adipose stem cells was evaluated in a bone tissue engineering case study. The results show, that the cells from the dental pulp, follicle, and periodontal ligament exhibit mesenchymal stem cell-like characteristics and show capacities for chondrogenic and osteogenic differentiation thus suggesting usability in hard tissue engineering applications in the craniofacial area. Furthermore, albeit ASCs showed comparable surface marker expression profiles and multipotential differentiation capacity in different culture conditions demonstrating characteristics of mesenchymal stem cells, fundamental differences in genes central for cell cycle progression and cell differentiation were detected. Clinical stem cell therapy studies using ASCs are under way, which call for a strong focus on the safety, reproducibility and quality of transplanted in vitro expanded stem cells. The results from the study suggests that cells from various adult tissue sources may be utilized for clinical cell therapy, but that the choice of culturing supplements has considerable effects on the cells at gene level. Thus, the culturing conditions may have significant implications on the cell product aimed for clinical therapy. The results show that proper characterization of culture conditions with different, yet specific, analyzing methods is necessary for cells intended for clinical stem cell therapy. Nevertheless, additional pre-clinical safety and efficacy studies will be needed before the promise of the adult stem cells can be fully realized.