High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model

Abstract

Background: Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo.

Methods: Flow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites.

Results: FreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs' function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O'Driscoll scores showing that more hyaline cartilage was formed on the defects.

Conclusion: FreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.

Keywords: Cartilage regeneration; Chondral defect; FreSHtracer; Glutathione; Mesenchymal stem cells.

Fig. 1

MSCs with high GSH levels isolated using FreSHtracer had enhanced stem cell function. A Structure of the FreSHtracer backbone and its fluorescence spectral changes upon reaction with GSH. B F₅₁₀/F₅₈₀ ratio from flow-cytometric analysis of MSCs according to cellular GSH levels (n = 8 each). C mRNA expression of stem cell markers in the hES-MSCs sorted in accordance with the levels of GSH, measured by real-time PCR (n = 5 per group). D (above) Transwell migration assay in hES-MSCs sorted based on GSH levels and in unsorted naïve cells (n = 5 per group). (below) Colony-forming assay in hES-MSCs sorted based on GSH levels and in unsorted naïve cells (n = 5 per group). E Evaluation of oxidant resistance of FreSHtracer-isolated hES-MSCs treated with diamide showing statistical significance when comparing GSH-high hES-MSCs to either naïve or GSH-low hES-MSCs using ANOVA. **p < 0.01, ****p < 0.0001

Fig. 2

MSCs with high levels of GSH displayed enhanced chondrogenic potential. A Morphology of hES-MSC pellets with different GSH levels. B (left) Weight and (right) size of the pellets after 21 days of chondrogenic differentiation (n = 5 respectively). C Safranin-O staining for proteoglycan and immunohistochemistry staining for collagen type II after chondrogenic differentiation for 21 days using GSH-high and GSH-low hES-MSCs. D mRNA expression of chondrogenic markers in the hES-MSCs measured by real-time PCR (n = 5 per group). ****p < 0.0001

Fig. 3

SDSCs, UC-MSCs, and BM-MSCs with high GSH levels show enhanced chondrogenesis. A Morphology of SDSC, UC-MSC and ADSC pellets with different GSH levels (B) (above) Weight and (below) size of the pellets after 21 days of chondrogenic differentiation (n = 5 per group, respectively). C mRNA levels for chondrogenic (COL2A1, SOX9 and ACAN) and hypertrophic chondrocyte (COLXA1) markers (n = 5 per group). D Safranin-O staining of cell pellets after chondrogenic differentiation induced in SDSCs, UC-MSCs, and ADSCs. E Quantification of the results in (D) according to the positive area of Safranin-O staining (n = 5 each). ****p < 0.0001

Fig. 4

Regenerative potential of injected hES-MSCs varies based on GSH levels. A Macroscopic appearance of the defect lesions of the only-defect group and the treatment group which injected either naïve or GSH-low or -high hES-MSCs at 4, 8, and 12 weeks. B and C Safranin-O staining of the normal cartilage, only-defect and the treatment groups which injected either naïve or GSH-low or -high hES-MSCs at 4, 8, and 12 weeks (40 × , low magnification and 200 × , high magnification, respectively). D O’Driscoll scoring after injection (n = 8 per group). E (left) High-magnification image (200 × objective) to confirm collagen II expression using immunohistochemical staining and (right) quantification of collagen type II expression using scoring system (n = 8 per group). F Immunohistochemical staining of human-specific β2 microglobulin. ****p < 0.0001

Fig. 5

Different cell culture densities alter cellular GSH dynamics and oxidant resistance. A F₅₁₀/F₅₈₀ ratio according to cell density (n = 5 per group). B GSH level changes according to hES-MSCs culture density after diamide treatment (n = 5 per group). C mRNA expression of stem cell markers in hES-MSCs cultured at different cell densities measured by real-time PCR (n = 5 per group). D (above) Transwell migration assay in hES-MSCs cultured at different cell densities (n = 5 per group). (below) Colony-forming assay in hES-MSCs cultured at different cell densities (n = 5 per group). **p < 0.01, ****p < 0.0001

Fig. 6

Histological changes in the chondral defect model after injection of MSCs cultured at low and high density. A Macroscopic appearance of the defect lesions of the only-defect group and the treatment groups which injected either naïve hES-MSCs or hES-MSCs cultured under low- or high-density conditions at 4, 8, and 12 weeks. B and C Safranin-O staining of the only-defect group and the treatment groups which injected either naïve hES-MSCs or hES-MSCs cultured under low- or high-density conditions at 4, 8, and 12 weeks (40 × , low magnification and 200 × , high magnification, respectively). D Scoring of cartilage regeneration parameters using the O’Driscoll scoring system after injection (n = 8 per group) ****p < 0.0001