ESTABLISHMENT OF FEEDER- AND SERUM-FREE CULTURE SYSTEM OF ES CELLS AND ITS APPLICATION TO PROPAGATE ES CELLS FROM INBRED MICE EMBRYOS

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 11558099
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 展開研究
• Research Field: Laboratory animal science
• Research Institution: RIKEN (THE INSTITUTE OF PHYSICAL AND CHEMICAL RESERCH) (2001); Osaka University (1999-2000)
• Principal Investigator: NIWA Hitoshi RIKEN CENTER FOR DEVELPMENTAL BIOLOGY, LABORATORY FOR PLURIPOTENT CELL STUDIES, TEAM LEADERNETWORK DEVELOPMENT, TEAM LEADER (RESEARCHER), 多能性幹細胞研究チーム, チームリーダー(研究職) (80253730)
• Project Period (FY): 1999 – 2001
• Keywords: ES cells / serum / feeder cells / proliferation

Research Abstract

We found the autocrine activity derived from ES cells to support ES self-renewal in the absence of serum and feeder cells, and named it KSRS. Since we could not identify KSRS by expression cloning and testing known candidates, we tried to purify it from the serum that contains the same activity. Now purification has almost finished and we will analyze the purified fraction by mass-spectrometry.
We have already confirrned that this activity can support self-renewal of 129-derived ES cells by culturing them for 1 week with semi-purified fraction followed by blastocyst injection, resulting formation of chimeric mice. Moreover, this serum-free culture condition allowed propagation of ES cells from C57BL6 blastocysts very efficiently in the presence of feeder cells, so now we are trying to remove feeder cells from this condition by choosing appropriate matrix that allows efficient attachment of blastocysts.
We investigated about the role of TGF-b superfamily on ES cell growth by modulation of Smad signaling pathway. Since Smad7 overexpression repress proliferation of ES cells and this negative effect can be relieved, the Smad signal would has physiological function to stimulate ES cell proliferation.

Research Products

• [Publications] Tashiro, F. et al.: "Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system"Human Gene Therapy. 10. 1845-1852 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Burdon, T. et al.: "Signalling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells"Cells Tissues Organs. 165. 131-143 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hotz Vitaterna, M. et al.: "Differential regulation of mammalian Period genes and circadian rhythmicity by cryptochromes 1 and 2"Proc. Natl. Acad. Sci. USA. 96. 12114-12119 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Miyawaki, K. et al.: "Glucose intolerance caused by a defect in the entero-insular axis : A study in qastric inhibitory polypeptide receptor knockout mice"Proc. Natl. Acad. Sci. USA. 96. 14843-14847 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Niwa, H. et al.: "Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells"Nat. Genet.. 24. 372-376 (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kawamoto, S. et al.: "A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination"FEBS Letters. 470. 263-268 (2000)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Sato, H. et al.: "Impaired fertility in female mice lacking urinary trypsin inhibitor"Biochem. Biophys. Res. Commun.. 281. 1154-1160 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Higaki, K. et al.: "Isolation of NPC1-deficient chinese hamster ovary cell mutants by gene trap mutagenesis"J. Biochem.. 129. 875-880 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Niwa, H.: "Molecular mechanism to maintain stem cell renewal of ES cells"Cell Struct. Funct.. 26. 137-148 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yamane, T. et al.: "Wnt signaling regulateshemopoiesis through stromal cells"J. Immunol.. 167. 765-772 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hashimoto, H. et al.: "Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide(PACAP)"Proc Natl Acad Sci U S A.. 98. 13355-13360 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Niwa, H. et al.: "Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells"Mol. Cell. Biol.. 22. 1526-1536 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Tashiro, F, Niwa, H. and Miyazaki, J.-l.: "Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system"Human Gene Therapy. 10. 1845-1852 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Burdon, T., Chambers, I., Niwa, H., Stracey, C. and Smith, A. G.: "Signalling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells"Cells Tissues Organs. 165. 131-143 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hotz Vitaterna, M., Selby, C. P., Todo, T. Niwa. H. Thompson, C., Fruechte, E. M., Hitomi, K., Thresher, R. J., Ishikawa, T, Miyazaki, J.-l., Takahashi, J. S. and Sancar, A.: "Differential regulation of mammalian Period genes and circadian rhythmicity by cryptochromes 1 and 2"Proc.Natl.Acad.Sci.USA. 96. 12114-12119 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Miyawaki, K., Yamada, Y., Yano, H., Niwa. H., Ban, N., lhara, Y., Kubota, A., Fujimoto, S., Kajikawa, M., Kuroe, A., Tsuda, K., Hashimoto, H., Yamashita, T., Jomori, T., Tashiro, F, Miyazaki, J.-l. and Seino, Y.: "Glucose intolerance caused by a defect in the entero-insular axis : A study in gastric inhibitory polypeptide receptor knockout mice"Proc. Nati. Acad. Sci. USA. 96. 14843-14847 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Niwa, H., Miyazaki, J-I. And Smith, A.G.: "Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells news&views, Stewart, C.L. : Oct-4, Scene 1 : the dream of mouse deveropment."Nat. Genet.. 24. 328-330 (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kawamoto, S., Niwa, H. Tashiro, R, Sano, S., Kondoh, G., Takeda, J., Tabayashi, K. and Miyazaki, J.-I.: "A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Ore-mediated recombination"FEBS Letters. 470. 263-268 (2000)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Sato, H., Kajikawa, S., Kuroda, S., Horisawa, Y., Nakamura, N., Kaga, N., Kakinuma, C., Kato, K., Morishita, H., Niwa. H. and J.-l. Miyazaki: "Impaired fertility in female mice lacking urinary trypsin inhibitor"Biochem. Biophys. Res. Commun.. 281. 1154-1160 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Higaki, K., Ninomiya, H.Sugimoto, Y., Suzuki, T., Taniguchi, M. Niwa. H. Pentchev, P. G., Vanier, M. T. and Ohno, K.: "Isolation of NPC1-deficient Chinese hamster ovary cell mutants by gene trap mutagenesis"J. Biochem.. 129. 875-880 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Niwa. H.: "Molecular mechanism to maintain stem cell renewal of ES cells"Cell Struct. Funct.. 26. 137-148 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yamane, T., Kunisada, T., Tsukamoto, H., Yamazaki, H., Niwa. H. Takada, S., Hayashi, S.-l.: "Wnt signaling regulateshemopoiesis through stromal cells"J. Immunol.. 167. 765-772 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hashimoto, H., Shintani, N., Tanaka, K., Mori, W., Hirose, M., Matsuda, T., Sakaue, M., Miyazaki, J. Niwa. H. Tashiro, R, Yamamoto, K., Koga, K., Tomimoto, S., Kunugi, A., Suetake, S. and Baba, A.: "Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP)."Proc Nati Acad Sci U S A. 98. 13355-13360 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Niwa. H. Masui, S., Chambers, I., Smith, A. G. and Miyazaki, J.-l.: "Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct3/4 in embryonic stem cells."Mol. Cell. Biol. 22. 1526-1536 (2002)
  • Description: 「研究成果報告書概要(欧文)」より