Neonatal desensitization supports long-term survival and functional integration of human embryonic stem cell-derived mesenchymal stem cells in rat joint cartilage without immunosuppression

Abstract

Immunological response hampers the investigation of human embryonic stem cells (hESCs) or their derivates for tissue regeneration in vivo. Immunosuppression is often used after surgery, but exhibits side effects of significant weight loss and allows only short-term observation. The purpose of this study was to investigate whether neonatal desensitization supports relative long-term survival of hESC-derived mesenchymal stem cells (hESC-MSCs) and promotes cartilage regeneration. hESC-MSCs were injected on the day of birth in rats. Six weeks after neonatal injection, a full-thickness cylindrical cartilage defect was created and transplanted with a hESC-MSC-seeded collagen bilayer scaffold (group d+s+c) or a collagen bilayer scaffold (group d+s). Rats without neonatal injection were transplanted with the hESC-MSC-seeded collagen bilayer scaffold to serve as controls (group s+c). Cartilage regeneration was evaluated by histological analysis, immunohistochemical staining, and biomechanical test. The role of hESC-MSCs in cartilage regeneration was analyzed by CD4 immunostaining, cell death detection, and visualization of human cells in regenerated tissues. hESC-MSCs expressed CD105, CD73, CD90, CD29, and CD44, but not CD45 and CD34, and possessed trilineage differentiation potential. Group d+s+c exhibited greater International Cartilage Repair Society (ICRS) scores than group d+s or group s+c. Abundant collagen type II and improved mechanical properties were detected in group d+s+c. There were less CD4+ inflammatory cell infiltration and cell death at week 1, and hESC-MSCs were found to survive as long as 8 weeks after transplantation in group d+s+c. Our study suggests that neonatal desensitization before transplantation may be an efficient way to develop a powerful tool for preclinical study of human cell-based therapies in animal models.

FIG. 1.

Characterization of mesenchymal stem cells (MSCs) from human embryonic stem cells (hESCs). (A) Morphology of MSCs derived from hESCs. (B) Fluorescence-activated cell sorter analysis of the expression of cell surface markers in hESC-derived cells. (C–E) Chondrogenic, adipogenic, and osteogenic differentiation of hESC-derived cells as indicated by Safranin O staining, oil red staining, and alizarin red staining [scale bars: 500 μm in (A), 50 μm in (C, D), and 200 μm in (E)].

FIG. 2.

Macrophotographs and ICRS scores of the defects in 3 groups 4 and 8 weeks after transplantation. Macrophotographs showed the defects in 3 groups 4 (A–C) and 8 (D–F) weeks after transplantation. (A, D) hESC-MSC-seeded collagen bilayer scaffold-treated group with neonatal desensitization (group d+s+c); (B, E) collagen bilayer scaffold-treated group with neonatal desensitization (group d+s); (C, F) hESC-MSC-seeded collagen bilayer scaffold-treated group without neonatal desensitization (group s+c). (G) International Cartilage Repair Society (ICRS) scores of groups d+s+c, d+s, and s+c 4 and 8 weeks after transplantation (maximum score=14) (*P<0.05 for group d+s+c vs. group vs. s+c at week 4 and 8; *P<0.05 for group d+s vs. group s+c at week 8). Values are represented as mean±standard deviation. Dotted circles indicate defects in three groups respectively.

FIG. 3.

Histological evaluation of 3 groups 4 weeks after transplantation with hematoxylin and eosin staining (H&E) (A–F), Safranin O staining (G–L), and their histological scores (M, N). (A, D, G, J) hESC-MSC-seeded collagen bilayer scaffold-treated group with neonatal desensitization (group d+s+c); (B, E, H, K) collagen bilayer scaffold-treated group with neonatal desensitization (group d+s); (C, F, I, L) hESC-MSC-seeded collagen bilayer scaffold-treated group without neonatal desensitization (group s+c); (M, N) Both cartilage region and total histological scores of group d+s+c (cartilage: 5.67±2.93, total: 18.5±3) were higher than those of group d+s (cartilage: 4.33±1.53, total: 13.67±2.75) and group s+c (cartilage: 2.33±1.53, total: 11.83±3.82) (*P<0.05 for group d+s+c vs. both group d+s and group s+c regarding the total histological score) [scale bars: 500 μm in (A–C) and (G–I), 200 μm in (D–F) and (J–L)]. Rectangle box indicates the area shown in the under column with higher magnification respectively. The edge of the defect is indicated by the black arrows.

FIG. 4.

Histological evaluation of 3 groups 8 weeks after transplantation with H&E staining (A–F), Safranin O staining (G–L), and their histological scores (M, N). (A, D, G, J) Group d+s+c; (B, E, H, K) group d+s; (C, F, I, L) group s+c; (M, N). In group d+s+c, the total histological score (26.63±3.86) was significantly higher than both group d+s (20.63±2.75) and group s+c (14.50±2.00) (*P<0.05 for group d+s+c vs. both group d+s and group s+c). Besides that, its cartilage score was also significantly higher than group s+c (10.88±3.59 vs. 2.17±0.58, P<0.05) [scale bars: 500 μm in (A–C) and (G–I), 200 μm in (D–F) and (J–L)]. Rectangle box indicates the area shown in the under column with higher magnification respectively. The edge of the defect is indicated by the black arrows.

FIG. 5.

Immunohistochemical staining and biomechanical evaluation of the repaired tissues 8 weeks after transplantation. Immunohistochemical staining of collagen II in the repaired tissues in group d+s+c (A, D), group d+s (B, E), and group s+c (C, F). Abundant collagen II was exhibited in group d+s+c while hard to detect in group d+s and group s+c. (G) Biomechanical evaluation of the repaired tissues by mechnical test. Values were represented as mean±standard deviation (n≥5 in all groups). The repaired tissue in group d+s+c exhibited a significantly higher compressive modulus than that in group d+s (0.056±0.008 MPa vs. 0.041±0.004 MPa, *P<0.05), though still lower than the normal cartilage tissue (0.056±0.008 MPa vs. 0.089±0.014 MPa, *P<0.05) [scale bars: 500 μm in (A–C), 100 μm in (D–F)].

FIG. 6.

Survival of hESC-MSCs within cartilage after transplantation. (A) hESC-MSCs were stained with DiI before transplantation; (B) hESC-MSCs stained with DiI were visualized by the tracking system 10 days after surgery. (C–F) Cooled charge-coupled device analysis demonstrated with neonatal desensitization (group d+s+c), and fluorescence signal was detected at week 4 (C) and 8 (E) after surgery, while the fluorescence signal was undetectable in group s+c (D, F). (G, H) Immunofluorescence staining for the human nucleus in the regenerated tissues was positive in group d+s+c 4 weeks after transplantation.

FIG. 7.

Reduced inflammatory cell infiltration 1 week after transplantation in group d+s+c. Macroscopic images of the joints collected from group d+s+c (A) and group s+c (B). (C–H) Inflammatory cell infiltration by CD4 staining in group d+s+c and group s+c, respectively, and its quantitative results (I) (*P<0.05). Images were taken from the overall view (C, D), within the defect (E, F) and interface areas between the defect and normal tissues (G, H), abundant CD4+ cells were found within the interface areas between normal and defect tissues in group s+c [scale bars: 200 μm in (C, D), 50 μm in (E–H)]. Insets indicate the area and its location within the defect.

FIG. 8.

Less cell death 1 week after transplantation in group d+s+c. Cell death detection by the TUNEL kit in group d+s+c (A, C, E) and group s+c (B, D, F). Abundant cell death was found on the defect surface (B), between the defect and normal tissue (D), and within the defect (F) in group s+c (scale bars: 50 μm). Insets indicate the area and its location within the defect.