成果報告書詳細
管理番号100013743
タイトル*平成20年度中間年報 「研究用モデル細胞の創製技術開発/分子構成を最適化した人工基底膜によるES細胞の分化誘導制御技術の開発」(H19-H21)
公開日2009/4/24
報告書年度2008 - 2008
委託先名国立大学法人大阪大学
プロジェクト番号P05010
部署名バイオテクノロジー・医療技術開発部
和文要約以下本編抜粋:(1)細胞ごとに最適化された基底膜分子構成の解明とそのデータベース化心臓、肝臓等を主要な標的臓器とし、マウス胚発生の各段階および生体臓器における基底膜の分子構成をより詳細に、細胞種や分化段階と対応させて解析した。
英文要約The goal of our project is to develop a new technology that enables us to induce differentiation of ES cells toward defined cell lineages through manipulation of the basement membrane-like substrata with cumstomized molecular composition. The major achievements made during the fiscal year of 2008 toward this goal are as follows. 1.Molecular composition of the basement membranes of mouse embryos was analyzed by immunohistochemistry in the developing heart and liver. In early stages of heart development, laminins containing alpha4 chain are predominantly expressed at the basement membrane between cardiomyocytes and endothelial cells, followed by expression of laminins containing alpha2 chain in later stages. In the developing liver, laminins containing alpha1 and alpha3 chains are first expressed at liver sinusoidal endothelial cells. Given the predominant expression of alpha4 chain-containing laminins at the capillary endothelial cells, sinusoidal endothelial cells are considered to have unique basement membrane composition. 2. Recombinant proteins of human nidogen-1, nidogen-2, and agrin were successfully expressed and purified. Perlecan was also purified from human choriocarcinoma cells. Attachment of heparan sulfate chains to agrin and perlecan was confirmed. Besides these basement membrane components, the yields of recombinant laminin isoforms have been significantly improved by optimizing transfection conditions and protein purification protocols. We also developed novel collagen scaffolds for reconstitution of basement membranes, combining type I and type IV collagens. The resulting composite collagen gels retain the physical strength of type I collagen gel while possessing the bioactivities of type IV collagen including high-affinity binding to laminins, nidogens, and agrin. 3. The biological activities of types I and IV composite collagen gel (type I/IV composite gel) were compared with those of type I collagen gel using undifferentiated mouse ES cells. Cells plated on the type I/IV composite gel were adherent to the gel and produced many small colonies while cells plated on type I collagen gel were barely adherent to the gel and produced large cell colonies. When LIF was depleted from the culture medium, ES cells on type I/IV composite gel differentiate into epiblastic cells with Fgf5, an epiblast marker, at a relatively high expression level and Flk1, a mesoderm marker, at a relatively low expression level, compared with cells plated on type I collagen gel.
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