Student Research Committee, Tabriz University of Medical Sciences, 5166615573, Tabriz, Iran.
Stem Cell Research Center, Tabriz University of Medical Sciences, 516615731, Tabriz, Iran.
Stem Cell Res Ther. 2021 Oct 21;12(1):550. doi: 10.1186/s13287-021-02617-x.
Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs).
The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction.
When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells.
The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.
硬脂酰辅酶 A 去饱和酶 1（SCD1）是从头合成脂肪酸所必需的。通过脂肪酸酰化过程，该酶协调参与细胞发育和分化的蛋白质的翻译后修饰。在这项研究中，我们使用生化方法、免疫染色和共价标记来评估调节不饱和脂肪酸的小分子是否会影响人诱导多能干细胞（iPSC）的早期内胚层分化。
在含有激活素 A 和定义的胎牛血清的内胚层诱导培养基中，将 hiPSC 在不同时间点用 SCD1 抑制剂培养。使用流式细胞术评估细胞周期和三个胚层（内胚层、中胚层和外胚层）的产量。使用 Western blot 和 RT-PCR 评估内胚层和多能性标记物以及 Wnt 信号通路蛋白的表达。使用点击化学反应评估总蛋白酰化。
当第一天抑制 SCD1 时，分化结束时具有内胚层特征的细胞群体减少。此外，早期 SCD1 抑制保留了 hiPSC 的特性，防止其向中胚层或外胚层谱系转移。同样，仅在第一天用 SCD1 抑制剂处理细胞会降低β-连环蛋白基因表达和点击化学测定中的荧光发射强度。用油酸共同处理可有效挽救细胞免受这些影响。在内胚层诱导的后两天中晚期用抑制剂处理对内胚层特异性标记物或荧光强度没有任何显著影响。用人类胚胎干细胞也获得了可重复的结果。
小分子 SCD1 抑制剂减弱了 Wnt/β-连环蛋白信号通路，通过拮抗内胚层分化的启动，维持 hiPSC。SCD1 活性在多能干细胞内胚层分化中的即时需求可能与内胚层来源器官的疾病和代谢失调有关。研究设计和主要结果的示意图表示。激活素 A 通过 Smad2/3/4 诱导内胚层特征，并增加 SCD1 的表达。SCD1 可以产生 MUFA，然后修饰 Wnt 分子。MUFA 酰化/激活的 Wnts 分泌到靶细胞上与相应的受体相互作用。Wnt 与受体相互作用后，β-连环蛋白在细胞质中积累并易位到细胞核中。然后，β-连环蛋白增加内胚层标记物 Sox17 和 CXCR4 的表达。
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