Development of efficient hematopoietic stem cell proliferation systems using our own established ES cells of small monkey, common marmoset

使用我们自己建立的小猴、狨猴的ES细胞开发高效的造血干细胞增殖系统

• 批准号: 17390279
• 负责人: TANI Kenzaburo
• 金额: $ 9.86万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390279/
• 关键词: Common marmoset; ES cell; tal1; scl; glycophorin antibody; human fetal liver; pseudotype lentiviral vector; cDNA expression library; leukemia cell line; tall; SSEA4抗体; tet-on; アデノウイルスベクター; 造血前駆細胞; NOGマウス

Toward the goal of establishing efficient hematopoietic stem cells and hematopoietic differentiation system from our own established common marmoset(CM) embryonic stem(ES) cells, we first established tall/scl gene transduced CMES (tal1/scl) cell lines. Tall/scl gene transduction was found to differentiate CMES cells efficiently to hematopoietic cells in vitro in our previous studies. We first of all found in vitro differentiating capacity of the tal1/scl cells to lymphocytes and megakaryocytes, although the efficiency was low. On the other hand, our in vivo studies using immune deficient NOG mice demonstrated very poor hematopoietic reconstitution with intramarrow injected tall/scl cells because of early lung tumor production and death. Then we tried to find out second gene which may assist the hematopoietic differentiation of tall/scl cells in vivo. For this purpose, we constructed human fetal liver cDNA expression VSV-G pseudotyped lentiviral vector library. This library consisted of more than 8x10^7 individual clones with average cDNA length of 2.1kb. DNA sequencing analysis of several fractions showed 60% of them contained full length cDNAs. When we produced virus using the library with 293T cells, 100% of cells demonstrated gene transduction. Average length of the gene transduced cDNA was about 1 kb. Flow cytometric analysis with antiglycophorin antibody showed the gene transduced cells expressed glycophorin, which is known to be one of the highly abundantly expressed genes in human fetal liver, properly. Using this novel human fetal liver cDNA library, we have already cloned several candidate genes which promotes hematopoiesis of tall/scl cells or induces cytokine independency of cytokine dependent leukemia cell lines. By the identification of genes which differentiate ES cells efficiently to hematopoietic cells in vitro and in vivo, safer and more effective cell supplementation therapy would be realized in future.

为了从我们建立的普通绒猴（common marmoset，CM）胚胎干细胞（embryonic stem，ES细胞）中建立高效的造血干细胞和造血分化系统，我们首先建立了转tall/scl基因的CMES（tal 1/scl）细胞系。我们前期的研究发现Tall/scl基因转导能有效地诱导CMES细胞体外分化为造血细胞。我们首先发现tal 1/scl细胞在体外具有向淋巴细胞和巨核细胞分化的能力，尽管分化效率较低。另一方面，我们使用免疫缺陷NOG小鼠的体内研究表明，由于早期肺肿瘤产生和死亡，骨髓内注射的tall/scl细胞的造血重建非常差。在此基础上，我们试图寻找可能在体内辅助tall/scl细胞造血分化的第二个基因。为此，我们构建了人胎肝cDNA表达VSV-G假型慢病毒载体文库。该文库包含超过8 × 10^7个单个克隆，平均cDNA长度为2.1kb。几个组分的DNA测序分析表明，其中60%含有全长cDNA。当我们使用293 T细胞的文库生产病毒时，100%的细胞表现出基因转导。基因转导的cDNA平均长度约为1 kb。抗血型糖蛋白抗体的流式细胞仪分析显示，基因转导的细胞表达血型糖蛋白，这是已知的高度丰富表达的基因之一，在人胎肝，正确。利用这个新的人胎肝cDNA文库，我们已经克隆了几个促进tall/scl细胞造血或诱导细胞因子依赖性白血病细胞系细胞因子非依赖性的候选基因。通过在体外和体内鉴定出能有效诱导ES细胞分化为造血细胞的基因，将为将来实现更安全、更有效的细胞补充疗法奠定基础。

项目成果

最新医学・第61巻・第6号 がん領域におけるドラッグデリバリーシステム(DDS)DDSと細胞療法

最新医学第61卷第6期药物输送系统（DDS）DDS和癌症领域的细胞治疗

• 发表时间: 2006
• 作者: 中村 貴文;谷 憲三朗
• 通讯作者: 谷 憲三朗

Serial analysis of gene expression in progressing and regressing mouse tumors implicates the involvement of RANTES and TARC in antitumor immune responses.

对进展和消退小鼠肿瘤中基因表达的系列分析表明 RANTES 和 TARC 参与抗肿瘤免疫反应。

• 发表时间: 2006
• 期刊: Mol Ther 14
• 作者: Nakazaki;Y.;Tani;K.;et al.
• 通讯作者: et al.

Antiangiogenic activity of BAI1 in vivo:: implications for gene therapy of human glioblastomas

• DOI: 10.1038/sj.cgt.7700898
• 发表时间: 2006-04-01
• 期刊: CANCER GENE THERAPY
• 影响因子: 6.4
• 作者: Kang, X;Xiao, X;Tani, K
• 通讯作者: Tani, K

Sustained molecular remission by non-myeloablative stem cell transplantation after autologous hematopoietic stem cell transplantation in a patient with multiple myeloma.

多发性骨髓瘤患者在自体造血干细胞移植后通过非清髓性干细胞移植实现持续分子缓解。

• 发表时间: 2005
• 期刊: Leuk Lynphoma 46
• 作者: Nakashima;Y.;Tani;K.;et al.
• 通讯作者: et al.

Tall/scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset(Callithrix jacchus)ES cells

使用慢病毒载体转导 Tall/scl 刺激普通狨猴 (Callithrix jacchus) ES 细胞高效分化造血细胞

• 发表时间: 2006
• 期刊: Stem Cells 24
• 作者: Kurita,R.;Sasaki,E.;Yokoo,T.;Hiroyama,T.;Takasugi,K.;Izawa,K.;Dong,Y.;Hashiguchi,T.;Soda,Y.;Maeda,T.;Suehiro,Y.;Tanioka,Y.;Nakazaki,Y.;Tani,K.
• 通讯作者: Tani,K.