Molecular mechanisms involved in adipose tissue-derived stromal cell hepatic conversion

Background: Adipose tissue-derived stromal cells (ATSCs) hold great promises in regenerative medicine, due to their easy retrieval, high proliferative capacity, and multi-lineage differentiation potential. In the last decade, several studies have reported the plasticity of ATSCs toward a hepatic fate. Nonetheless, the molecular mechanisms underlying the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. Aim: In this study, we compared the full genome expression profiles of ATSCs cultured for 4 weeks under pro-hepatogenic conditions to undifferentiated ATSCs, in order to depict the molecular events involved in ATSC hepatic transdifferentiation. Methods: Molecular analysis was performed using the Affymetrix human focus arrays. Sets of differentially expressed genes were functionally categorized in order to understand which pathways drive the hepatic conversion and interesting target genes were validated by Q-PCR. Results: We showed that ATSC-derived hepatocyte-like cells activate several genes associated with specific liver functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted the downregulation of several transcripts involved in stemness maintenance along with genes associated with a mesenchymal phenotype. Conclusion: Taken together, our data suggest that the in vitro system used in this study drove ATSCs toward a hepatic conversion through a subtle regulation of molecular pathways controlling stem cell properties and lineage commitment that promote mesenchymal-epithelial-transition. Adipose tissue was obtained from 3 patients undergoing partial abdominoplasty. Adipose tissue-derived stromal cells (ATSC) were isolated according to standard procedures, using the in vitro adherence property of these cells. At passage culture 4, ATSC were submitted to an in vitro hepatogenic regimen, consisting of the sequential addition of growth factors. After 1 month of in vitro differentiation, cells were harvested and their transcriptome was compared to control ATSC.

背景：脂肪组织来源基质细胞（Adipose tissue-derived stromal cells，ATSCs）因易于获取、增殖能力优异且具备多向分化潜能，在再生医学领域拥有广阔的应用前景。近十年来，多项研究已报道ATSCs向肝细胞系分化的可塑性。然而，其从间充质表型向上皮表型转化的分子机制仍尚未明确。
目的：本研究将促肝细胞生成培养条件下培养4周的ATSCs与未分化ATSCs的全基因组表达谱进行对比，旨在阐明ATSCs肝细胞转分化过程中的分子事件。
方法：采用Affymetrix人类基因聚焦芯片开展分子分析。对筛选得到的差异表达基因进行功能分类，以明确驱动肝细胞转化的信号通路，并通过定量聚合酶链式反应（Quantitative PCR，Q-PCR）验证潜在靶基因的表达。
结果：本研究证实，ATSCs来源的肝细胞样细胞可激活一系列与肝脏特异性功能相关的基因，包括蛋白质代谢、先天免疫应答调控以及有毒化合物生物降解相关基因。此外，芯片分析结果显示，与干细胞干性维持相关的多项转录本以及间充质表型相关基因均出现下调。
结论：综合来看，本研究采用的体外培养体系通过精细调控控制干细胞干性特征与谱系定向的分子通路，推动ATSCs发生间充质-上皮转化（Mesenchymal-Epithelial Transition），最终实现向肝细胞系的转化。
本研究获取的脂肪组织来自3名接受部分腹壁成形术的患者。ATSCs的分离依照标准流程完成，利用这类细胞的体外贴壁特性进行纯化。于第4代培养时，将ATSCs置于包含生长因子顺序添加的肝细胞定向分化方案中进行体外培养。经过1个月的体外分化后，收集细胞并将其转录组与对照组未分化ATSCs进行对比分析。