DeLaForest Ann, Quryshi Afiya F, Frolkis Talia S, Franklin Olivia D, Battle Michele A
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI, United States.
Front Med (Lausanne). 2020 Feb 19;7:44. doi: 10.3389/fmed.2020.00044. eCollection 2020.
Three-dimensional gastrointestinal organoid culture systems provide innovative and tractable models to investigate fundamental developmental biology questions using human cells. The goal of this study was to explore the role of the zinc-finger containing transcription factor GATA4 in gastric development using an organoid-based model of human stomach development. Given GATA4's vital role in the developing mouse gastrointestinal tract, we hypothesized that GATA4 plays an essential role in human stomach development. We generated a human induced pluripotent stem cell (hiPSC) line stably expressing an shRNA targeted against GATA4 (G4KD-hiPSCs) and used an established protocol for the directed differentiation of hiPSCs into stomach organoids. This model system, informed by studies in multiple non-human model systems, recapitulates the fundamental processes of stomach development, including foregut endoderm patterning, specification, and subsequent tissue morphogenesis and growth, to produce three-dimensional fundic or antral organoids containing functional gastric epithelial cell types. We confirmed that GATA4 depletion did not disrupt hiPSC differentiation to definitive endoderm (DE). However, when G4KD-hiPSC-derived DE cells were directed to differentiate toward budding SOX2+, HNF1B+ posterior foregut spheroids, we observed a striking decrease in the emergence of cell aggregates, with little to no spheroid formation and budding by GATA4-depleted hiPSCs. In contrast, control hiPSC-derived DE cells, expressing GATA4, formed aggregates and budded into spheroids as expected. These data support an essential role for GATA4 during the earliest stages of human stomach development.
三维胃肠类器官培养系统提供了创新且易于处理的模型,可利用人类细胞来研究基本的发育生物学问题。本研究的目的是使用基于类器官的人类胃发育模型,探索含锌指转录因子GATA4在胃发育中的作用。鉴于GATA4在发育中的小鼠胃肠道中起着至关重要的作用,我们假设GATA4在人类胃发育中也起着关键作用。我们构建了一个稳定表达靶向GATA4的短发夹RNA的人类诱导多能干细胞(hiPSC)系(G4KD-hiPSCs),并使用已建立的方案将hiPSCs定向分化为胃类器官。这个模型系统是在多个非人类模型系统研究的基础上建立的,概括了胃发育的基本过程,包括前肠内胚层的模式形成、特化以及随后的组织形态发生和生长,从而产生包含功能性胃上皮细胞类型的三维胃底或胃窦类器官。我们证实,GATA4的缺失不会破坏hiPSC向定形内胚层(DE)的分化。然而,当将G4KD-hiPSC来源的DE细胞定向分化为出芽的SOX2+、HNF1B+前肠后球状体时,我们观察到细胞聚集体的出现显著减少,GATA4缺失的hiPSC几乎没有形成球状体和出芽。相比之下,表达GATA4的对照hiPSC来源的DE细胞按预期形成聚集体并芽生出球状体。这些数据支持了GATA4在人类胃发育的最早阶段起着关键作用。