Designing a Microenvironment Niche for Liver-Specific Differentiation of hESCs

描述（由申请人提供）：获得新的肝细胞来源对于进一步推进肝脏定向细胞疗法、生物人工肝辅助装置和毒理学研究至关重要。对肝细胞新来源的需求源于人肝细胞的总体短缺、可用于实验的人细胞的次优条件以及与采用异种（鼠或猪）肝细胞相关的潜在危害。理想的肝细胞来源应该能够进行群体扩增;因此，重点放在研究祖细胞或人胚胎干细胞（hESC）上，这些细胞可以首先扩增，然后诱导表达肝脏特异性功能。然而，迄今为止，将hESC向肝谱系驱动的成功是有限的，尝试在体外诱导肝特异性分化导致仅小百分比的干细胞表达肝特异性标志物如白蛋白和11-抗胰蛋白酶。干细胞分化研究中采用的传统细胞培养方法每次改变一种生物诱导剂，需要大量细胞和相当长的时间投资进行彻底分析。此外，这些培养技术不太适合于工程化诱导干细胞中的肝表型所需的微环境的精确组成（例如细胞间接触和细胞表面相互作用）。本提案的目标是设计一个微环境生态位，有利于干细胞向肝脏表型的体外分化。为了收敛于肝特异性干细胞龛的组成，我们建议开发微加工细胞培养表面，其中hESC与细胞外基质（ECM）蛋白，生长因子和其他细胞（例如，成人肝细胞或非实质肝细胞）的相互作用将被精确定义和测试，在理性和“组合”的方式。此外，我们将开发含生长因子的支架，作为体外人胚胎干细胞分化研究和干细胞移植实验的桥梁。将携带干细胞的肝素水凝胶微结构植入免疫缺陷小鼠中，并表征这些构建体中细胞的存活/功能。该提案的成功完成将导致更好地理解hESC的肝脏特异性分化所需的外源性信号，提高驱动干细胞向肝脏表型的效率，并开发用于干细胞移植的新型仿生支架。总的来说，拟议的项目将使hESC在肝脏相关疗法开发中的未来应用成为可能。

DESCRIPTION (provided by applicant): Deriving new sources of hepatocytes is critical for further advancement of liver-directed cell therapies, bioartificial liver-assist devices and toxicology studies. The need for new sources of hepatocytes stems from the overall shortage of human hepatocytes, suboptimal condition of human cells available for experiments and potential hazards associated with employing xenogenic (murine or porcine) liver cells. An ideal source of hepatocytes should be capable of population expansion; therefore, emphasis is being placed on investigating progenitor cells or human embryonic stem cells (hESC), cells that could first be expanded and then induced to express liver-specific function. However, success in driving hESC toward hepatic lineage has been limited so far, with attempts to induce liver-specific differentiation in vitro leading to only a small percentage of stem cells expressing such liver-specific markers as albumin and 11-antitrypsin. Traditional cell culture approaches employed in stem cell differentiation studies vary biological inducers one-at-a-time, requiring large numbers of cells and considerable time investment for thorough analysis. In addition, these culture techniques are not well-suited for engineering the precise composition of the microenvironment (e.g. intercellular contacts and cell-surface interactions) required to induce the hepatic phenotype in stem cells. The goal of the present proposal is to design a microenvironment niche conducive to in vitro differentiation of stem cells toward the liver phenotype. In order to converge on the composition of liver-specific stem cell niche, we propose to develop microfabricated cell culture surfaces where the interactions of hESC with extracellular matrix (ECM) proteins, growth factors and other cells (e.g. adult hepatocytes or nonparenchymal liver cells) will be precisely defined and tested in both rational and "combinatorial" fashion. In addition, we will develop growth factor- containing scaffolds as a way of bridging in vitro hESC differentiation studies and stem cell transplantation experiments. Stem cell-carrying heparin hydrogel microstructures will be implanted in immunodeficient mice and survival/function of cells within these constructs will be characterized. Successful completion of this proposal will lead to better understanding of the exogenous cues required for the liver-specific differentiation of hESC, improved efficiency in driving stem cells towards the hepatic phenotype and development of novel biomimetic scaffolds for stem cell transplantation. Overall, the proposed project will enable future applications of hESCs in the development of liver-related therapies.