Effects of Microengineered Interactions on Liver-Specific Gene Expression

描述（由申请人提供）：肝脏生物学的大量研究工作集中在获得用于肝定向细胞治疗、生物人工肝辅助装置和其他应用的新肝细胞来源。重点放在研究祖细胞、胎儿肝细胞或人胚胎干细胞（ESC）上;这些细胞可以首先扩增，然后诱导表达肝脏特异性功能。最近，我们开发了一种新的基因工程人胎肝细胞系，含有重组人端粒酶逆转录酶（hTERT）亚基，并能够超过300个细胞倍增。传统的细胞培养方法一次一种地改变生物诱导剂，需要大量的细胞和相当长的时间投入以进行彻底的分析。这些方法不太适合于定义在新的胎儿或干细胞衍生的细胞系中诱导肝表型所需的微环境的精确组成。在这里，我们建议采用基于微加工和微阵列的细胞培养方法，以允许在精确定义的微环境条件下培养小群细胞，并将其平行暴露于多种生物刺激。这些新的细胞培养工具将通过提供细胞-细胞和细胞-细胞外基质（ECM）相互作用的适当组合来诱导永生化胎肝细胞的肝脏特异性分化。拟议的细胞培养平台将补充一种新的方法，用于在当地环境的背景下评估肝功能。激光捕获显微切割将与实时定量RT-PCR串联使用，以从局部工程化微环境中检索小组肝细胞，并分析肝脏特异性基因的表达。如白蛋白、转铁蛋白和α 1-抗胰蛋白酶等基因的上调将确定生物刺激的给定组合对肝分化具有积极作用。在未来，提出的微加工装置将用于诱导人ESC向肝系分化。

DESCRIPTION (provided by applicant): Considerable research efforts in liver biology are focused on deriving new sources of hepatocytes for liver directed cell therapies, bioartificial liver-assist devices and other applications. Emphasis is being placed on investigating progenitor cells, fetal hepatocytes or human embryonic stem cells (ESC); cells that could first be expanded and then induced to express liver-specific function. Recently, we developed a novel genetically-engineered human fetal hepatocyte cell line containing reconstituted human telomerase reverse transcriptase (hTERT) subunits and capable of more than 300 cell doublings. Traditional cell culture approaches vary biological inducers one-at-a-time, requiring large number of cells and considerable time investment for thorough analysis. These methods are not well suited for defining precise composition of the microenvironment required to induce hepatic phenotype in novel, fetal- or stem cell-derived cell lines. Here we propose to employ microfabrication- and microarraying-based cell culture approaches to allow for small groups of cells to be cultured under precisely-defined microenvironment conditions and to be exposed to multiple biological stimuli in parallel. These novel cell culture tools will be applied to induce liver-specific differentiation in the immortalized fetal hepatocytes by providing an appropriate combination of the cell-cell and cell-extracellular matrix (ECM) interactions. Proposed cell culture platforms will be complemented with a new approach for evaluating hepatic function in the context of the local environment. Laser capture microdissection will be used in tandem with real-time quantitative RT-PCR to retrieve small groups of hepatic cells from locally engineered microenvironment and to analyze expression of liver specific genes. Upregulation in such genes as albumin, transferrin and alpha 1-antitrypsin will indentify a given combination of biological stimuli as having a positive effect on hepatic differentiation. In the future, proposed microfabricated devices will be used to induce differentiation of human ESC toward hepatic lineage.