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

通过基因操作实现非造血组织的去分化及其可塑性和造血转分化的获得

• 批准号: 16390281
• 负责人: OZAWA Keiya
• 金额: $ 7.68万
• 依托单位: JICHI MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390281/
• 关键词: Dedifferentiation; Transdifferentiation; Plasticity; Msx1; AAV vector; Skeletal muscle; Hematopoietic system; Stem cell; AVVベクター; Dedifferentiation; Transdifferentiation; Plasticity; Msx1; AAV vector; Skeletal muscle; Hematopoietic system; Stem cell; AVVベクター

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.

我们探讨了遗传操作可以提高非造成杂物组织转分化的效率的可能性。据报道，肌肉中MSX1的瞬时过表达产生了能够分化为肌管，软骨细胞，脂肪细胞和骨细胞的丰富的单核细胞（MNC）。由于几乎所有AAV矢量介导的转基因都以非集成形式存在，因此随着宿主细胞的分裂，它们逐渐消失。我们利用了AAV矢量的这一功能；即肌肉衍生的跨国公司在去分化后通过多个细胞分裂失去MSX1转基因。我们假定适当的分化提示可能会将肌肉衍生的未分化的MNC重新定向为造血谱系。将表达MSX1的AAV5载体注入C57BL/6小鼠的胫骨前肌。流式细胞仪分析表明，来自AAV-MSX1处理的肌肉的MNC包含大量表达造血干细胞标记的细胞。为了评估造血活性，将MNC培养在甲基纤维素培养基中。注射AAV-MSX1后，肌肉中的菌落形成细胞逐渐增加，达到3周的峰值。还通过从AAV-MSX1治疗的LY5.2小鼠的AAV-MSX1处理的肌肉中移植MNC来评估体内造血重构活性。观察到有效的Ly5.2细胞的植入，这些移植物显示出非常高的嵌合为5.2。此外，在从前小鼠到LY5.1/5.2杂合子的次级骨髓移植中，供体细胞嵌合感甚至更高。这些结果表明，强制执行的MSX1表达可以将肌肉细胞重新编程为能够分化为造血谱系的多能细胞。这项新技术将应用于使用源自患者肌肉的遗传正常造血干细胞来治疗获得的骨髓衰竭。