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在诱导多能干细胞衍生的肝样细胞中鉴定法尼醇X受体调节的肝肠混合状态。

Identification of an FXR-modulated liver-intestine hybrid state in iPSC-derived hepatocyte-like cells.

作者信息

Nell Patrick, Kattler Kathrin, Feuerborn David, Hellwig Birte, Rieck Adrian, Salhab Abdulrahman, Lepikhov Konstantin, Gasparoni Gilles, Thomitzek Antonia, Belgasmi Katharina, Blüthgen Nils, Morkel Markus, Küppers-Munther Barbara, Godoy Patricio, Hay David C, Cadenas Cristina, Marchan Rosemarie, Vartak Nachiket, Edlund Karolina, Rahnenführer Jörg, Walter Jörn, Hengstler Jan G

机构信息

Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, 44139 Dortmund, Germany.

Department of Genetics, University of Saarland, 66123 Saarbrücken, Germany.

出版信息

J Hepatol. 2022 Nov;77(5):1386-1398. doi: 10.1016/j.jhep.2022.07.009. Epub 2022 Jul 19.

Abstract

BACKGROUND & AIMS: Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLC) have enormous potential as a replacement for primary hepatocytes in drug screening, toxicology and cell replacement therapy, but their genome-wide expression patterns differ strongly from primary human hepatocytes (PHH).

METHODS

We differentiated human induced pluripotent stem cells (hiPSC) via definitive endoderm to HLC and characterized the cells by single-cell and bulk RNA-seq, with complementary epigenetic analyses. We then compared HLC to PHH and publicly available data on human fetal hepatocytes (FH) ex vivo; we performed bioinformatics-guided interventions to improve HLC differentiation via lentiviral transduction of the nuclear receptor FXR and agonist exposure.

RESULTS

Single-cell RNA-seq revealed that transcriptomes of individual HLC display a hybrid state, where hepatocyte-associated genes are expressed in concert with genes that are not expressed in PHH - mostly intestinal genes - within the same cell. Bulk-level overrepresentation analysis, as well as regulon analysis at the single-cell level, identified sets of regulatory factors discriminating HLC, FH, and PHH, hinting at a central role for the nuclear receptor FXR in the functional maturation of HLC. Combined FXR expression plus agonist exposure enhanced the expression of hepatocyte-associated genes and increased the ability of bile canalicular secretion as well as lipid droplet formation, thereby increasing HLCs' similarity to PHH. The undesired non-liver gene expression was reproducibly decreased, although only by a moderate degree.

CONCLUSION

In contrast to physiological hepatocyte precursor cells and mature hepatocytes, HLC co-express liver and hybrid genes in the same cell. Targeted modification of the FXR gene regulatory network improves their differentiation by suppressing intestinal traits whilst inducing hepatocyte features.

LAY SUMMARY

Generation of human hepatocytes from stem cells represents an active research field but its success is hampered by the fact that the stem cell-derived 'hepatocytes' still show major differences to hepatocytes obtained from a liver. Here, we identified an important reason for the difference, specifically that the stem cell-derived 'hepatocyte' represents a hybrid cell with features of hepatocytes and intestinal cells. We show that a specific protein (FXR) suppresses intestinal and induces liver features, thus bringing the stem cell-derived cells closer to hepatocytes derived from human livers.

摘要

背景与目的

多能干细胞(PSC)来源的肝样细胞(HLC)在药物筛选、毒理学和细胞替代治疗中作为原代肝细胞的替代物具有巨大潜力,但其全基因组表达模式与原代人肝细胞(PHH)有很大差异。

方法

我们通过确定内胚层将人诱导多能干细胞(hiPSC)分化为HLC,并通过单细胞和批量RNA测序以及互补的表观遗传学分析对细胞进行表征。然后我们将HLC与PHH以及公开可用的人胎儿肝细胞(FH)体外数据进行比较;我们通过慢病毒转导核受体FXR和激动剂暴露进行生物信息学指导的干预,以改善HLC分化。

结果

单细胞RNA测序显示,单个HLC的转录组呈现一种混合状态,即肝细胞相关基因与在PHH中不表达的基因(主要是肠道基因)在同一细胞中协同表达。批量水平的过度表达分析以及单细胞水平的调控子分析确定了区分HLC、FH和PHH的调控因子集,暗示核受体FXR在HLC功能成熟中起核心作用。FXR表达与激动剂暴露相结合增强了肝细胞相关基因的表达,并增加了胆小管分泌和脂滴形成的能力,从而增加了HLC与PHH的相似性。不期望的非肝脏基因表达虽有一定程度降低,但仍有再现性。

结论

与生理性肝细胞前体细胞和成熟肝细胞不同,HLC在同一细胞中共表达肝脏和混合基因。对FXR基因调控网络的靶向修饰通过抑制肠道特征同时诱导肝细胞特征来改善其分化。

简要概述

从干细胞生成人肝细胞是一个活跃的研究领域,但其成功受到干细胞来源的“肝细胞”与从肝脏获得的肝细胞仍存在重大差异这一事实的阻碍。在此,我们确定了差异的一个重要原因,具体而言,干细胞来源的“肝细胞”代表一种具有肝细胞和肠道细胞特征的混合细胞。我们表明一种特定蛋白质(FXR)抑制肠道特征并诱导肝脏特征,从而使干细胞来源的细胞更接近人肝脏来源的肝细胞。

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