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

基本信息

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

项目摘要

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.

我们探索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 Msx1 的瞬时过度表达会产生丰富的单核细胞 (MNC)，能够分化为肌管、软骨细胞、脂肪细胞和骨细胞。由于几乎所有 AAV 载体介导的转基因都以非整合形式存在，因此它们会随着宿主细胞分裂而逐渐消失。我们利用了AAV载体的这一特性；即肌肉来源的 MNC 在去分化后通过多次细胞分裂失去 Msx1 转基因。我们假设，适当的分化线索可能会将肌肉来源的未分化 MNC 重定向到造血谱系。将表达 Msx1 (AAV-msx1) 的 AAV5 载体注射到 C57BL/6 小鼠的胫骨前肌中。流式细胞分析显示，来自 AAV-msx1 处理肌肉的 MNC 含有大量表达造血干细胞标记物的细胞。为了评估造血活性，在甲基纤维素培养基中培养 MNC。注射AAV-msx1后，肌肉中的集落形成细胞逐渐增多，在3周时达到峰值。还通过将经 AAV-msx1 处理的 Ly5.2 小鼠肌肉中的 MNC 移植到受辐射的同系小鼠中来评估体内造血重建活性。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 Ly 5.2 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，强制表达 Msx1 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。这项新技术将利用来自患者肌肉的基因正常的造血干细胞来治疗获得性骨髓衰竭。

项目成果

期刊论文数量（83）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fragl, a homolog of altemative replication factor C subunits, links replication stress surveillance with apoptosis.

Fragl 是替代复制因子 C 亚基的同源物，将复制应激监视与细胞凋亡联系起来。

DOI：
发表时间：
2005
期刊：
Proc. Natl. Acad. Sci. U.S.A. 102・27
影响因子：
0
作者：
Ishii;H
通讯作者：
H

Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transpene expression.

通过阴离子交换层析从 1 型腺相关病毒载体原液中去除空衣壳可增强 transpene 表达。

DOI：
发表时间：
2006
期刊：
Mol. Ther. 13・4
影响因子：
0
作者：
Urabe;M.
通讯作者：
M.

Large-scale production of recombinant viruses by use of a large culture vessel with active gassing.

使用具有主动通气功能的大型培养容器大规模生产重组病毒。

DOI：
发表时间：
2005
期刊：
Hum. Gene Ther. 16
影响因子：
0
作者：
Okada;T.;Nomoto;T.;Yoshioka;T.;Nonaka-Sarukawa;M.;Ito;T.;Ogura;T.;Iwata-Okada;M.;Uchibori;R.;Shimazaki;K.;Mizukami;H.;Kume;A.;Ozawa;K.
通讯作者：
K.

Hematopoietic microchimerism in sheep after in utero transplantation of cultured cynomolgus embryonic stem cells.

培养的食蟹猴胚胎干细胞宫内移植后绵羊的造血微嵌合。