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

我们探讨了基因操作可能提高非造血组织转分化效率的可能性。据报道，肌肉中Msx 1的瞬时过表达产生大量能够分化成肌管、软骨细胞、脂肪细胞和骨细胞的单核细胞（MNCs）。由于几乎所有的AAV载体介导的转基因都以非整合形式存在，它们随着宿主细胞分裂而逐渐消失。我们利用了AAV载体的这一特征，即肌肉来源的MNC在去分化后通过多次细胞分裂丢失Msx 1转基因。我们推测，一个适当的分化线索可能会重新定向肌肉来源的未分化的单核细胞到造血谱系。将表达Msx 1的AAV 5载体（AAV-msx 1）注射到C57 BL/6小鼠的胫骨前肌中。流式细胞术分析显示，从AAV-msx 1处理的肌肉的MNC含有相当数量的细胞表达造血干细胞标志物。为了评价造血活性，将MNC在甲基纤维素培养基中培养。注射AAV-msx 1后，肌肉中的集落形成细胞逐渐增多，在3 wk时达到高峰。还通过将来自AAV-msx 1处理的Ly5.2小鼠肌肉的MNC移植到辐射的同类小鼠来评价体内造血重建活性。观察到Ly5.2细胞的有效植入，并且这些移植物显示出非常高的Ly5.2嵌合性。此外，在从前小鼠到Ly5.1/5.2杂合受体的二次骨髓移植中，供体细胞嵌合性甚至更高。这些结果表明，加强Msx 1表达可以重编程肌肉细胞成多能细胞能够分化成造血谱系以及。这项新技术将应用于使用来自患者肌肉的遗传正常的造血干细胞治疗获得性骨髓衰竭。

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.