DEDIFFERENTIATION OF NON-HEMATOPOIETIC TISSUE BY GENETIC MANIPULATION AND ITS ACQUISITION OF PLASTICITY AND HEMATOPOIETIC TRANSDIFFERENTIATION

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We explored the possibility that genetic manipulation may enhance the efficiency of transdifferentiation of non-hematopoietic tissue. Transient overexpression of Msx1 in muscles was reported to generate abundant mononuclear cells (MNCs) capable of differentiation into myotubes, chondrocytes, adipocytes and osteocytes. Since virtually all of AAV vector-mediated transgenes exist as a non-integrated form, they gradually disappear as the host cells divide. We took advantage of this feature of AAV vectors ; i.e. muscle-derived MNCs lose Msx1 transgenes through multiple cell divisions after dedifferentiation. We postulated that a proper differentiation cue might redirect muscle-derived undifferentiated MNCs into a hematopoietic lineage. AAV5 vector expressing Msx1 (AAV-msx1) was injected into tibialis anterior muscles of C57BL/6 mice. Flow cytometric analysis revealed that MNCs from AAV-msx1-treated muscles contained a considerable number of cells expressing hematopoietic stem cell markers. To evaluate hematopoietic activity, MNCs were cultured in methylcellulose medium. After AAV-msx1 injection, colony-forming cells in the muscles were gradually increased, reaching a peak at 3 wk. In vivo hematopoietic reconstitution activity was also evaluated by transplanting MNCs from AAV-msx1-treated muscles of Ly5.2 mice to irradiated congenic mice. Efficient engraftment of Ly5.2 cells was observed, and these transplants showed a very high chimerism of Ly 5.2. Furthermore, in the secondary bone marrow transplantation from the former mouse to a Ly5.1/5.2 heterozygous recipient, the donor cell chimerism was even higher. These results suggest that enforced Msx1 expression can reprogram muscle cells into multipotential cells capable of differentiation into a hematopoietic lineage as well. This novel technology would be applied to the treatment of acquired bone marrow failure using genetically-normal hematopoietic stem cells derived from patient muscles.