doi: 10.1002/ctm2.70052.
Mesenchymal stem cells with an enhanced antioxidant capacity integrate as smooth muscle cells in a model of diabetic detrusor underactivity
Affiliations
- PMID: 39390754
- PMCID: PMC11467036
- DOI: 10.1002/ctm2.70052
Item in Clipboard
Mesenchymal stem cells with an enhanced antioxidant capacity integrate as smooth muscle cells in a model of diabetic detrusor underactivity
Clin Transl Med.
2024 Oct.
No abstract available
Conflict of interest statement
D‐.M.S. cofounded Cell2in, a company focused on developing FreSHtracer‐based GRC assays. The other authors declare that no conflicts of interest exist.
Figures
Transcriptome profiling of diabetic DUA following the MSC therapy. (A) Transcriptome analysis of bladder tissues from a preclinical study that demonstrated the effectiveness of M‐MSCs for treating diabetic DUA. (B) Principal component analysis (PCA) of the transcriptomes of the indicated groups. (C,D) The ten most highly enriched pathway maps identified by MetaCore analysis (C) and GSEA with a representative enrichment plot for glutathione (GSH) metabolism (D) in the comparison of transcriptomes between the STZ‐DUA and sham groups. (D) The bubble plot was presented by order of normalised enrichment score (NES) through size values by −Log2(FDR). As results of GSEA, the STZ‐DUA group was enriched with gene sets related to GSH metabolism (normalised enrichment score [NES] = 2.291) and drug metabolism by cytochrome p‐450 (CYP450) (NES = 2.327), as well as immunological disorders such as graft‐versus‐host disease (GVHD) (NES = 2.062) and systemic lupus erythematosus (NES = 2.051). (E) Heatmap of the differentially expressed genes (DEGs) generated using the pheatmap R package. (F) Mfuzz clustering analysis that identified four clusters of DEGs based on their patterns of expression. Mean expression values of each cluster are highlighted in black. The degree of membership of each gene for the cluster is presented as gradual membership values. (G) Gene ontology (GO) analysis (biological process, cellular component, and molecular function) of cluster‐3, which was upregulated in diabetic DUA, but their expression was normalised by M‐MSC therapy. In the bubble plot, the abscissa GeneRatio represents the proportion of enriched genes relative to the total number of genes. (H) qPCR analysis for the expression of genes involved in GSH synthesis, utilisation, NADPH oxidase, nitric oxide synthetase, or the inflammatory response. Expression is presented as the percentage relative to rat Gapdh expression (n = 5). (I) Quantitative analysis of immunofluorescence staining of GSH (upper panel) or oxidative stress (lower panel) related proteins in three locations (urothelium, muscle and serosa) of bladders from the indicated groups. Representative staining results for these proteins are available in Figure S4A–E. Statistical analyses were performed using a two‐way ANOVA with the Bonferroni post hoc test. *
p < 0.05, **
p < 0.01, ***
p < 0.001 relative to the STZ‐DUA group. The exact p‐values and number of replicates are specified in Data S1. DUA, Detrusor underactivity; MSC, mesenchymal stem cell; STZ, streptozotocin; GSEA, Gene‐set enrichment analysis.
Improved in vivo therapeutic efficacy of PFO‐MSCs. (A) Schematic diagram of preclinical studies conducted in this study, which included (i) N‐acetylcysteine (NAC) alone, (ii) NAC combined with M‐MSCs, and (iii) PFO‐MSC therapies. The therapeutic outcomes of these treatments were evaluated using the awake cytometry, which allows long‐term evaluation of the bladder function in free‐moving animals. (B,C) Representative awake cystometry results (B) and quantitative voiding data (C) of sham‐operated and STZ‐DUA rats injected with the vehicle control (phosphate‐buffered saline, PBS) or naïve hUC‐MSCs or PFO‐MSCs (5.0 × 105). Transplantation of naïve cultured hUC‐MSCs or PFO‐MSCs (5.0 × 105) ameliorated the defective bladder functions in diabetic DUA, resulting in increases of micturition pressure (MP), maximum pressure, and bladder voiding efficiency (BVE) as well as decreases of micturition interval (MI), micturition volume (MV), bladder capacity (BC), and residual volume (RV). Importantly, compared with naïve cultured hUC‐MSCs, animals injected with PFO‐MSCs demonstrated significant enhancements of bladder function parameters, validating the improved therapeutic efficacy of these cells. (D) Hematoxylin and eosin staining (upper panel; magnification, 100×; scale bar, 200 µm), Masson's trichrome staining (middle panel; magnification, 100 ×; scale bar, 200 µm), and Toluidine blue staining (lower panel; magnification, 400×; scale bar, 100 µm) of bladder sections from the indicated groups at 1 week after injection of PFO‐MSCs. (E) Quantification of histological staining from five animals per group. (F) Quantitative analysis of immunofluorescence staining of Gpx2 and Gclc proteins in bladder sections of the indicated groups. (G,H) Representative images (G) and quantification (H) of bioluminescence activities of Nano‐lantern‐expressing UC‐MSCs (1.0 × 105) subjected to normal (naïve) culture or the PFO procedure at the indicated day after transplantation (DAT) in the STZ‐induced diabetic mice model. (G) Representative images were obtained at 15 min after intraperitoneal injection of 150 µg/mL coelenterazine (200 µL), a substrate of Renilla luciferase. (H) Relative bioluminescence signals at the indicated DAT are presented as the relative values to 1 DAT value at the PFO‐MSC group. Nano‐lantern: a chimera of enhanced Renilla luciferase and Venus fluorescent protein with highly efficient bioluminescence resonance energy transfer. Notably, the animals injected with PFO‐MSCs showed significantly brighter bioluminescence throughout the entire experimental period than those injected with naïve hUC‐MSCs, indicating the in vivo engraftment capacity of PFO‐MSCs was superior. (I) Quantification results of immunostaining for hB2M throughout rat diabetic bladder sections from the indicated groups at 1 week after injection of PBS vehicle, naïve hUC‐MSCs, or PFO‐MSCs. All quantitative data are presented as the mean ± standard error of the mean (SEM) (n = 5). Data were analysed by the one‐way (C, E, F) or two‐way (H,I) ANOVA with the Bonferroni post hoc comparison (**
p < 0.01, ***
p < 0.001 relative to the PBS group; #
p < 0.05, ##
p < 0.01, ###
p < 0.001). DUA, Detrusor underactivity; MSC, mesenchymal stem cell; STZ, streptozotocin; PFO,
P
rimed/
F
resh/
O
CT4; hUC, human umbilical‐cord.
Single‐cell transcriptome profiling of engrafted PFO‐MSCs. (A) Representative immunofluorescence (magnification, 400×; scale bar, 200 µm) micrographs of co‐staining of hB2M (red) and α‐smooth muscle actin (α‐SMA, blue), a muscle marker in the indicated bladder sections of STZ‐DUA rats at 1 week after transplantation. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). (B,C) PCA (B) and volcano plot (C) comparing five cultured (Cultured_#1–5) and ten engrafted (Engrafted_#1–10) single‐cell transcriptomes. (D) The number of upregulated and downregulated genes with the indicated cut‐off values of |log2(FC)| > 2 or 4 and p < 0.05. Accordingly, ∼2000 DEGs, including 834 upregulated and 1203 downregulated DEGs, were identified in engrafted cells compared with cultured cells. (E) Heatmap of DEGs in single‐cell transcriptomes generated using the pheatmap R package with nine gene clusters. Cluster‐1 was downregulated and cluster‐5 was upregulated in engrafted cells compared with cultured cells. (F) Mfuzz clustering analysis that identified nine clusters of DEGs based on their patterns of expression. Mean expression values of each cluster are highlighted in black. (G) GO analysis (biological process, cellular component, and molecular function) of cluster‐5, which was upregulated in engrafted cells compared with cultured cells. In the bubble plot, the abscissa GeneRatio represents the proportion of enriched genes to the total number of genes. Notably, GO analysis of cluster‐5 genes indicated that the single‐cell transcriptomes of engrafted cells were characteristically represented by pathways related to skeletal muscle tissue development and cellular stress such as the DNA damage checkpoint and mitogen‐activated protein kinase (MAPK) cascade. (H) GSEA with a representative enrichment plot for the immune response and growth factors in the comparison of single‐cell transcriptomes between the cultured and engrafted groups. Accordingly, engrafted cells were enriched with gene sets related to the immune response including macrophage subtypes (NES = 1.726) and B lymphocyte maturation (NES = 1.841), as well as growth factors including epidermal growth factor (EGF) (NES = 1.877) and transforming growth factor beta (TGFB) signalling (NES = 1.749), compared with cultured cells. DUA, Detrusor underactivity; MSC, mesenchymal stem cell; STZ, streptozotocin; PFO,
P
rimed/
F
resh/
O
CT4; hUC, human umbilical‐cord; PCA, principal component analysis; DEG, differentially expressed gene; GSEA, Gene‐set enrichment analysis; NES, normalised enrichment score.
Upregulation of MET and PD‐L1 in engrafted M‐MSCs. (A) Bubble plot of qPCR results for putative biomarkers characterising engrafted PFO‐MSCs in diabetic DUA, which are related to myogenesis, immunomodulation, and engraftment processes. (B) qPCR analysis validating expression of biomarker genes representing each process (Cultured n = 10, Engrafted n = 30). Expression is presented as the percentage relative to human GAPDH expression. All quantitative data are shown as the mean ± SEM. Statistical significance was examined by the non‐parametric Mann–Whitney test (*
p < 0.05, **
p < 0.01, ***
p < 0.001). (C,D) Immunostaining of MET (C) or RHOA (D) (green) as well as hB2M (red) in bladder sections of an STZ‐DUA rat at 1 week after injection of PFO‐MSCs. Two independent images are shown (magnification, 400×; scale bar, 200 µm). (E,F) Representative fluorescence (magnification, 400×; scale bar, 200 µm, E) and confocal (magnification, 1000×; scale bar, 10 µm, F) micrographs of co‐staining of PD‐L1, an immunomodulation marker (red), and hB2M (green) in bladder sections of the indicated groups. Nuclei were stained with DAPI (blue). DUA, Detrusor underactivity; MSC, mesenchymal stem cell; STZ, streptozotocin; PFO,
P
rimed/
F
resh/
O
CT4.
References
-
- van Koeveringe GA, Vahabi B, Andersson KE, Kirschner‐Herrmans R, Oelke M. Detrusor underactivity: a plea for new approaches to a common bladder dysfunction. Neurourol Urodyn. 2011;30:723‐728. - PubMed
-
- Yu HY, Lee S, Ju H, et al. Intravital imaging and single cell transcriptomic analysis for engraftment of mesenchymal stem cells in an animal model of interstitial cystitis/bladder pain syndrome. Biomaterials. 2022;280:121277. - PubMed