Expansion and tagging of hematopoietic stem cells

A controlled and timely restricted expansion of hematopoietic stem cells (HSCs) without compromising their stem cell potential would be the ultimate solution to provide sufficient number of cells for cell replacement therapy in various diseases. In the last years we developed a reliable technology for expansion of fibroblast and endothelial cells from mouse and human. In this system an autoregulated Dox dependent regulatory module is used for tightly controlled expression of the proliferation gene(s). The focus of this project is laid on expanding hematopoietic stem/progenitor cells (HSCs) by means of conditional, i.e. reversible expression of proliferation genes. We explored if and in which way a timely restricted expression of these genes supports the maintenance of the in vivo potential of HSCs for differentiation and long-term repopulation. Upon infection of mouse lineage negative CD34-Sca1+c-Kit+(LSK) cells with various Dox-controlled proliferator cassettes, LSK cell lines have been established that could be cultivated in vitro for up to ten months without loosing the stem cell markers. Moreover, they were able to differentiate and form colonies in Methocult media, indicating that the protocol does support differentiation. Transplantation is currently being followed to evaluate further the grafting potential of these cells. In order to genetically modify these cells in a predictable manner, we evaluate the exploitation of recombinase based methods for defined genetic modification of the immortalized hematopoietic (stem) cells. For this purpose, immortalized HSCs were generated that display a defined chromosomal tag supporting recombinase mediated cassette exchange (RMCE). Results from these studies will be presented.