Analysis for mechanisms of self-renewal and differentiation signal transduction in human embryonic stem cells.

人胚胎干细胞自我更新和分化信号转导机制分析。

• 批准号: 13480204
• 负责人: YOKOTA Takashi
• 金额: $ 9.6万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480204/
• 关键词: Cytokine; gp130; LIF; Embryonic stem cell; chimeric cytokine receptor; self-renewal mechanism; transgenic mice; STAT3; 分化; gp130; トランスジェニックマウス; エストロジェン受容体; タモキシフェン; 生殖系列伝達; キメラマウス

The pluripotent phenotype of ES cells is maintained in the presence of LIF. LIF binds to a cell surface receptor complex composed of LIF receptor β and gp130, through which several signaling molecules including MAP kinase and STAT3 are activated. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp!30 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we have constructed a fusion protein composed of the entire region of STAT3 and the ligand binding domain of estrogen receptor. This fusion protein (STAT3ER) was dimerized and activated in the presence of a synthetic ligand 4-hydroxytamoxifen (4HT). When ES cells stably expressing STAT3ER were cultured in the presence of 4HT without LIF and feeder cells, they maintained a morphologically undifferentiated state and expressed undifferentiated state-specific markers (SSEA-1 and alkaline phosphatase). Moreover, ES cells maintained by STAT3ER and 4HT contributed to chimeric mice production when they were injected into blastocysts. These results indicate that STAT3 activation is sufficient to maintain the pluripotency of ES cells. Oct-3/4 transcription factor is expressed in ES cells and ES cells specifically. It is known to be essential for the formation of inner cell mass and for the maintenance of undifferentiated state of ES cells. It is likely that LIF or STAT3 signals inhibit differentiation of ES cells through yet unidentified factor by cooperating with Oct-3/4.

在 LIF 存在的情况下，ES 细胞的多能表型得以维持。LIF 与由 LIF 受体 β 和 gp130 组成的细胞表面受体复合物结合，通过该复合物激活包括 MAP 激酶和 STAT3 在内的多种信号分子。 gp130 在 ES 细胞的自我更新中发挥重要作用 在本研究中，我们检查了 gp!30 促进 ES 细胞自我更新的信号通路。负责STAT3激活的gp130胞质结构域的突变分析对于ES细胞的自我更新是必要的，而SHP2和MAP激酶激活所需的突变分析则是可有可无的。 接下来，我们构建了由STAT3和MAP激酶的整个区域组成的融合蛋白。当 ES 细胞存在合成配体 4-羟基他莫昔芬 (4HT) 时，该融合蛋白 (STAT3ER) 会二聚化并被激活。稳定表达STAT3ER的细胞在没有LIF和饲养细胞的4HT存在下培养，它们维持形态学未分化状态并表达未分化状态特异性标记物（SSEA-1和碱性磷酸酶）此外，STAT3ER和4HT维持的ES细胞有助于嵌合。这些结果表明 STAT3 激活足以维持 ES 的多能性。 Oct-3/4转录因子在ES细胞和ES细胞中特异性表达，已知其对于内细胞团的形成和ES细胞的未分化状态的维持是必需的。信号通过与 Oct-3/4 合作，通过尚未识别的因子抑制 ES 细胞的分化。

项目成果

Sato, A., Nishinakamura, R., Yokota, T., et al.: "Zinc-finger protein Sall2 is not essential for embryonic and kidney development"Mol.Cell.Biol.. 23. 62-69 (2003)

Sato, A.、Nishinakamura, R.、Yokota, T. 等：“锌指蛋白 Sall2 对于胚胎和肾脏发育不是必需的”Mol.Cell.Biol.. 23. 62-69 (2003)

Ishibashi, H., et. al.: "Tool-use learning induces BDNF expression in a selective portion of monkey anterio parietal cortex."Molecular Brain Res.. 102. 110-112 (2002)

石桥，H.，等。

Tanaka, T., Yokota, T., et al.: "Gene expression profiling of embryonic stem cells reveals candidate genes associated with pluripotency and lineage specificity"Genome Research. 12. 1921-1928 (2002)

Tanaka, T.、Yokota, T. 等人：“胚胎干细胞的基因表达谱揭示了与多能性和谱系特异性相关的候选基因”基因组研究。

Nakayama, N., et. al.: "A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages."Dev. Biol.. 232. 372-387 (2001)

中山，N.，等。

Senga, T., Yokota, T., et al.: "STAT3-dependent induction of BATF in M1 cells"Oncogene. 21・53. 8186-8191 (2002)

Senga, T.、Yokota, T. 等：“M1 细胞中 BATF 的 STAT3 依赖性诱导”Oncogene 21·53（2002）。