Glycogen is found in a wide range of organisms, ranging from bacteria to mammals. As a major form of stored glucose, glycogen plays an essential role in maintaining energy homeostasis and cell survival. In this study, we engineered the glycogen metabolism of MSCs to promote glycogen accumulation. We tested several engineering strategies in human HEK 293T cells and primary MSCs derived from mice. Overexpression of phosphorylation-resistant GYS (GYSmut), as well as the co-expression of GYS and UGP, induced intense glycogen. Glycogen engineering enhanced the starvation resistance of MSCs and improved their viability post-implantation. Compared with the control group, the engineered MSCs exhibited a significantly enhanced therapeutic effect on PF, indicating the importance of glucose metabolism regulation for MSC-based therapy.
Glycogen-engineered MSCs may serve as chassis cells for further applications by enhancing cell viability post-implantation, and our glycogen engineering strategies also have the potential to be applied to other therapeutic cells. Transient expression of GYSmut by mRNA transfection may be a safer alternative than lentiviral transduction, avoiding the risks of chromosomal modification. LNP-mediated circular mRNA transfection would further improve engineering efficiency (Huang et al., 2024).
We investigated the effect of glycogen engineering on the transcriptome of MSCs by RNA-seq, which revealed large-scale gene transcription changes. In future studies, complex regulatory networks may be elucidated at the levels of proteomics and metabolomics. Previous studies have revealed the role of glucose metabolism regulation in MSCs’ immunoregulatory properties (Contreras-Lopez et al., 2020; Luo et al., 2023). The broader impact of glycogen engineering on MSCs remains to be elucidated. We tested the therapeutic efficacy of glycogen-engineered MSCs in the bleomycin-induced PF mice model. Their efficacy in other disease models is still to be investigated.
Within the context of MSC-based cellular therapies, administered cells were distributed to various microenvironments characterized by variable oxygen concentration, nutrient availability, and immune responses. While in vivo animal models offer a more physiologically relevant platform for validation, they are less convenient for detailed investigation due to multifactorial influences, making it challenging to fully understand the precise kinetic properties and adaptation dynamics of the implanted cells, which is a limitation of this study.
In previous studies, complex gene circuits were introduced to enhance the efficacy of various cell-based therapies, including MSCs and CAR-T cells (Allen et al., 2022; Cheng et al., 2019), and some research also revealed the advantages of metabolic engineering (ME) (Ye et al., 2022). However, in contrast to extensive ME research in prokaryotic organisms, ME of mammalian therapeutic cells has yet to be developed. Here, we demonstrated the great potential of glycogen engineering, offering a basis for future studies. By dynamically controlling essential enzymes, cellular metabolism can be remodeled to achieve optimal survival and therapeutic efficacy post-implantation. Further research in this area will provide additional opportunities for cell therapy, gene therapy, and tissue engineering.