P84	Generation and characterisation of a novel multipotential murine hematopoietic progenitor cell line using β-catenin gene transfer.
1Chr.Templin, 1D.Kotlarz, 4C.Rathinam, 2C.Rudolph, 3S.Schaetzlein, 3K.L.Rudolph, 2B.Schlegelberger, 4Chr.Klein, 5Chr.Baum, 1B.Schieffer, 1H.Drexler
1Hannover Medical School/Department of Cardiology and Angiology, Hannover, DE; 2Hannover Medical School/Institut of Cell and Molecular Pathology, Hannover, DE; 3Hannover Medical School/Department of Gastroenterology, Hannover, DE; 4Hannover Medical School/Department of Pediatric Haematology and Oncology, Hannover, DE; 5Hannover Medical School/Department of Haematology and Oncology, Hannover, DE.

Background: The generation of hematopoietic progenitor cell lines using gene transfer of defined genetic elements would greatly faciliate molecular and developmental studies of stem cell differentiation. Beta-catenin mediated Wnt-signaling have been suggested to be critically involved in hematopoietic stem cell maintenance. We generated a novel c-kit/Sca-1 cell line (DK-mix) using gene transfer of ß-catenin. Methods and results: C57/BL6 derived lin- hematopoietic stem cells (HSC) were transduced using a VSV-G pseudotyped bicistronic retrovirus expressing β-catenin and eGFP. Beta-catenin transduced HSC survived, proliferated and were maintained as pluripotent immature cells. The cell line was kept in culture and expanded > 9 months with stable expression of c-kit and Sca-1 (> 92 %), without loss of viability or reduction of proliferative potential, whereas eGFP transduced HSCs (control) died after 3 weeks. Upon exposure to selective hematopoietic cytokines, differentiation was found in myeloid and lymphoid cells as assessed by FACS and CFU-assays. To confirm their differentiation potential in vivo, we transplanted DK-mix cells into CD45.1 congenic recipient mice and analyzed the hematopoietic system five weeks later by FACS. We found DK-mix derived cells (CD45.2) in bone marrow, spleen, thymus, lymph node and peripheral blood, differentiating into T-cells, B-cells, NK-cells, dendritic cells, macrophages, granulocytes and erythrocytes. For further characterization of the cell line, we analyzed telomere length (Q-FISH), telomerase activity (TRAP-assay) and spectral karyotyping (SKY-analysis). Preliminary data suggest beneficial functional effects when cells were administered in a ischemia/reperfusion model of mice. Conclusion: Retroviral transduction of murine HSCs using β-catenin-expressing retroviruses provides a genetically stable multipotential progenitor cell line with great potential for therapeutic application post myocardial infarction.

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