Effects of Microengineered Interactions on Liver-Specific Gene Expression

描述(由申请人提供)：肝脏生物学方面的大量研究工作集中在为肝脏定向细胞疗法、生物人工肝辅助设备和其他应用寻找新的肝细胞来源。重点是研究祖细胞、胎肝细胞或人类胚胎干细胞(ESC)；这些细胞可以首先扩增，然后被诱导表达肝脏特异性功能。最近，我们建立了一种新型的基因工程人胎肝细胞系，该细胞系含有重组的人端粒酶逆转录酶(HTERT)亚单位，能够进行300多个细胞倍增。传统的细胞培养方法一次一个地改变生物诱导剂，需要大量的细胞和大量的时间投资来进行彻底的分析。这些方法不适合于确定在新的胎儿或干细胞来源的细胞系中诱导肝脏表型所需的微环境的精确组成。在这里，我们建议使用基于微制造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，并同时暴露于多种生物刺激。这些新的细胞培养工具将通过提供适当的细胞-细胞和细胞-细胞外基质(ECM)相互作用的组合，在永生化的胎肝细胞中诱导肝脏特异性分化。建议的细胞培养平台将补充一种新的方法，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相结合，从局部工程微环境中提取小群肝细胞，并分析肝脏特异基因的表达。白蛋白、转铁蛋白和α1-抗胰蛋白酶等基因的上调将确认给定的生物刺激组合对肝脏分化具有积极影响。在未来，所提出的微制造装置将被用于诱导人胚胎干细胞向肝系分化。

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.