Innovative Strategien zur Expansion hematopoietischer Stamm- und Vorläuferzellen im klinisch relevanten Maßstab

Hematopoiesis is a highly regulated process. A large variety of signaling factors, cell surface receptors and extracellular matrix molecules influence and direct cell expansion, maturation, differentiation, and apoptosis. Up to date, this complex network could not be clarified in detail, making it impossible to establish natural hematopoiesis for potential medical use in vitro. Despite this imprecise understanding, what is known, is the important role that bone marrow stromal cells play in aforementioned processes. In this work, the regulatory role of stromal cells during the expansion of hematopoietic stem and progenitor cells, isolated from cord blood, was analysed and finally exploited in order to improve biotechnological processes as for example the large scale expansion of cells. At first, optimal conditions for the production of stroma-conditioned media were established. Thereafter, differences between media, gained from three murine stromal cell lines, were analysed on single protein level. One protein, which could be isolated and identified, turned out to mimick the medium's effect on hematopoietic cells. Second, a novel bioreactor system, based on membrane-separated co-cultivation of murine stromal cells and human hematopoietic stem and progenitor cells, was developed. This special arrangement allows for the exchange of signaling molecules and nutrients through small pores in the membrane, whereas the harvest of stem cells can be performed without contaminating murine stromal cells. After 7 days of cultivation, mononuclear cells, progenitor cells and early progenitor cells could be expanded on average 61-fold, 22-fold and 19-fold, respectively. A small scale test system served for the analysis and further development of a clinical scale bioreactor