Figures and data
Schematic illustration of the strategy used to enhance the therapeutic efficacy of MSCs through glycogen engineering.
(A) Engineering glycogen metabolism of MSCs by overexpressing essential enzymes of glycogen synthesis. (B) Engineered MSCs use glycogen as an energy supply after implantation, improving cell viability and therapeutic efficacy.
Construction of glycogen engineering strategies.
(A) Essential enzymes of glycogen synthesis. (B) Tests of essential enzyme combination strategies in HEK293T cells by transient transfection. The glycogen content of each group was measured. (C) Periodic acid-Schiff (PAS) staining of cells expressing GYSmut and GYS-UGP revealed significant accumulation of glycogen granules.
Construction of glycogen-engineered MSCs.
(A) Glycogen content of GYSmut MSCs. (B) PAS staining of GYSmut MSCs, showing glycogen granules (red). (C) Survival of engineered MSCs under DPBS (starvation) treatment in vitro (N = 8). (D) Residual glycogen content of GYSmut MSCs after DPBS (starvation) treatment for 48 hours. (E) Viability of GYSmut MSCs according to the CCK8 assay. (F, G) Adipogenic differentiation potential of GYSmut MSCs, assessed by oil red O staining and qPCR detection of Lpl expression (n=3, unpaired t-test P-value < 0.0001). (H) GO enrichment analysis of differentially expressed genes (DEGs) between GYSmut MSCs and the GFP control. (I) KEGG analysis of DEGs. (J) Gene set enrichment analysis (GSEA) of the DEGs.
Survival of implanted glycogen-engineered MSCs.
(A) Schematic illustration of the strategy used to assess the survival of MSCs by detecting Gaussia luciferase activity in the homogenate of lungs. (B) Changes of Gaussia luciferase activity in the two groups on day 7 post-implantation (3 mice each group, unpaired t-test P-value = 0.044). (C) Live imaging of Akaluc luciferase activity in the two groups implanted with GYSmut-Akaluc MSCs and control cells (5 mice each group). One mouse from the control group died on day 7, and one mouse from the GYSmut-Akaluc group died on day 11.
Therapeutic efficacy of glycogen-engineered MSCs.
(A) Survival and body weight changes of PF mice treated with GYSmut MSCs and control cells (10 mice each group, mean ± SEM, 2-way ANOVA P-value=0.0001), (B) Representative lung tissue sections stained with H&E and Masson’s trichrome. NC group is healthy mice. (C) Collagen deposition and preserved alveolar size (quantified by mean linear intercept, MLI) of lung tissue sections (6 mice each group).
(A) GYSdelc promoted glycogen accumulation. (B) Co-expressing GYSmut and UGP further promoted glycogen accumulation.
Impacts of glycogen engineering on MSCs.
(A) Cell volume change of engineered MSCs, assessed through flow cytometry. (B) PAS and hematoxylin staining of GYSmut MSCs. Glycogen (red) is distributed in cell nucleus (blue) and cytoplasm. (C) Glycogen distribution of GYSmut and GYS-GYG MSCs. Scale bar, 50 μm. (D) Starvation resistance test under hypoxia. (E) Impacts of glycogen engineering on transcriptome of MSCs.
KEGG and GSEA analysis of implanted MSCs in our previous research.
GFP MSCs were intratracheally administered to PF mice and collected through flow cytometry. Single-cell sequencing showed downregulation of glucose metabolism.
Quantification of akaluc activity of in vivo imaging post-implantation.
