Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level. HLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays. HLCs derived using the SM protocol (HLC_SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC_SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC_GF), and commercially available HLCs. Similar to HLC_GF, HLC_SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC_SM compared to HLC_GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs. Overall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.

中文翻译：

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使用小分子衍生自人类诱导的多能干细胞的类肝细胞：转录组研究的意义。

源自人类诱导的多能干细胞（iPSC）的肝样细胞（HLC）在毒理学应用以及再生医学中具有广阔的前景。先前对肝细胞分化的努力主要依靠使用生长因子（GFs）在体外条件下概括发育信号。近年来，由于小分子（SMs）在质量和成本方面均具有优势，因此已成为引起细胞命运转变的诱人工具。但是，使用SM衍生的HLC尚未得到很好的表征，尤其是在转录组水平上。使用仅涉及SM的方案将HLC与人iPSC进行了区分，并使用全基因组微阵列通过转录组分析对其进行了表征。使用SM协议（HLC_SM）派生的HLC显示特定的肝标志物表达并显示关键的肝功能。SM驱动的分化的转录组学分析定义了肝细胞分化的轨迹，并表征了肝细胞分化主要阶段中一些关键标记基因的表达。此外，HLC_SM还通过CellNet（一种生物信息学工具，对工程化细胞群与目标细胞类型的相似程度进行量化）进行了评分，并将其与原代人肝细胞（PHH），成年肝组织，胎儿肝组织，使用GF分化的HLC（HLC_GF），以及市售的HLC。与HLC_GF相似，HLC_SM显示出胎儿和成年肝细胞的混合表型，并且与PHH相比，其代谢酶，转运蛋白和核受体的表达相对较低。最后，与HLC_GF和PHHs相比，分析了HLC_SM中差异表达的基因，以鉴定途径和上游转录调节因子，可以潜在地对其进行操作以改善HLC的分化。总体而言，本研究证明了基于SM的肝细胞分化方法的实用性，为了解肝发生和相关基因调控的分子基础提供了新见识，并提出了进一步改善肝细胞分化的方法，以获得可能更成熟的HLC。用于毒理学研究。