Manipal Institute of Regenerative Medicine, Bangalore, Manipal Academy of Higher Education, Manipal, India.
Division of Reproductive Biology, Department of Reproductive Science, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal 576104, India.
Life Sci. 2024 Dec 1;358:123160. doi: 10.1016/j.lfs.2024.123160. Epub 2024 Oct 20.
The knowledge of the molecular players that regulate the generation of endoderm cells is imperative to obtain homogenous population of pancreatic β-cells from stem cells. The Ubiquitin proteasome system (UPS) has been envisaged as a crucial intracellular protein degradation system, but its role in the generation of β-cells remains elusive. Hence, it would be appropriate to unravel the potential role of UPS in endoderm specification and utilize the understanding to generate β-cells from pluripotent stem cells.
The pluripotent stem cells (mESCs, miPSCs and hIPSCs) were subjected to differentiation towards pancreatic β-cells and assessed the proteasomal activity during endodermal differentiation. Pharmacologic agents MG132 and IU-1 were employed to inhibit and activate proteasomal activity respectively at the definitive endoderm stage to investigate its impact on the generation of β-cells. The expression of stage-specific genes were analyzed at transcript and protein levels. We also explored the role of unfolded protein response and UPS-regulated signalling pathways in endodermal differentiation.
We observed decreased proteasomal activity specifically during endoderm, but not during the generation of other lineages. Extraneous proteasomal inhibition enhanced the expression of endodermal genes while increasing the proteasomal activity hindered definitive endodermal differentiation. Proteasomal inhibition at the definitive endodermal stage culminated in an enriched generation of insulin-positive cells. Elevated endodermal gene expression was consistent in mESCs and hIPSCs upon proteasomal inhibition. Mechanistic insight revealed the proteasome-inhibited enhanced endodermal differentiation to be via modulating the YAP pathway.
Our study unravels the specific involvement of UPS in endoderm cell generation from pluripotent stem cells and paves the way for obtaining potential definitive endodermal cells for plausible cellular therapy in the future.
了解调控内胚层细胞生成的分子机制对于从干细胞获得同质的胰岛β细胞群体至关重要。泛素蛋白酶体系统（UPS）被认为是一种重要的细胞内蛋白降解系统，但它在β细胞生成中的作用仍不清楚。因此，揭示 UPS 在内胚层特化中的潜在作用，并利用这一认识从多能干细胞中生成β细胞将是合适的。
将多能干细胞（mESCs、miPSCs 和 hIPSCs）诱导分化为胰岛β细胞，并在向内胚层分化过程中评估蛋白酶体活性。在确定的内胚层阶段，使用药理学试剂 MG132 和 IU-1 分别抑制和激活蛋白酶体活性，以研究其对β细胞生成的影响。分析了转录和蛋白水平的阶段特异性基因表达。我们还探讨了未折叠蛋白反应和 UPS 调节的信号通路在向内胚层分化中的作用。
我们观察到蛋白酶体活性在向内胚层分化过程中特异性降低，而在其他谱系生成过程中则不降低。额外的蛋白酶体抑制增强了内胚层基因的表达，而增加蛋白酶体活性则阻碍了确定的内胚层分化。在确定的内胚层阶段进行蛋白酶体抑制导致胰岛素阳性细胞的生成增加。蛋白酶体抑制后，mESCs 和 hIPSCs 的内胚层基因表达均升高。机制研究揭示，蛋白酶体抑制增强的内胚层分化是通过调节 YAP 通路实现的。
我们的研究揭示了 UPS 在内胚层细胞从多能干细胞生成中的特定作用，并为未来获得潜在的确定内胚层细胞进行可行的细胞治疗铺平了道路。
