Homing and restorative effects of bone marrow-derived mesenchymal stem cells on cisplatin injured ovaries in rats

Abstract

Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment. However, current available treatment regimens are not optimal. Emerging evidence suggests that bone marrow-derived mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues, but the homing and restorative effects of BMSCs on chemotherapy injured ovaries are still not clear. In this study, we found that granulosa cell (GC) apoptosis induced by cisplatin was reduced when BMSCs were migrated to granulosa cells (GCs) in vitro. Chemotherapy-induced POF was induced by intraperitoneal injection of cisplatin in rats. BMSCs labeled with enhanced green fluorescent protein (EGFP) were injected into the rats via the tail vein to investigate the homing and distribution of BMSCs in vivo. The number of BMSCs in the ovarian hilum and medulla was greater than in the cortex, but no BMSCs were found in the follicles and corpus lutea. In addition, the BMSCs treatment group's antral follicle count and estradiol levels increased after 30 days, compared with the POF group. Hence, our study demonstrates that intravenously delivered BMSCs can home to the ovaries, and restore its structure and function in POF model rats.

Keywords: bone marrow-derived mesenchymal stem cells; cisplatin; homing; intravenous injection; premature ovarian failure.

Fig. 1

Morphology and phenotypes of BMSCs: (A) Third passage BMSCs showed fibroblastic morphology; (B) Fluorescent microscopic view of BMSCs transfected with Ad-EGFP; (C) Flow cytome try revealed that BMSCs were CD29⁻, CD90⁻ and CD34⁻. Scale bars, 20 μm.

Fig. 2

Morphology and phenotypes of GCs, and the effects of cisplatin on GCs: (A) The morphology of normal first passage GCs; (B) The morphology of GCs injured by cisplatin; (C) FSHR expression in GCs; (D) CCK-8 assays indicated that cisplatin significantly injured GCs, compared to normal, ^**P < 0.01. Scale bars, 20 μm.

Fig. 3

Co-culture with BMSCs, induced apoptosis in GCs: (A) Normal GCs; (B) Apoptotic rate of GCs in the cisplatin group; (C) Co-cultured with BMSCs.

Fig. 4

BMSCs migrated to cisplatin injured GCs: (A) BMSCs migrated to normal GCs in 24 h; (B) BMSCs migrated to cisplatin injured GCs; (C) The number of migrated BMSCs every 200× magnification field (n = 5), ^**P < 0.01. Scale bars, 20 μm.

Fig. 5

Serum levels of estradiol (E₂), the number of antral follicles and corpus lutea in the three groups (control, POF, and BMSCs treated; n = 5), 15 and 30 days after administering BMSCs. Data were expressed as means ± SD in numerous experiments; 15 days (control vs. POF P < 0.01; control vs. BMSCs P < 0.01; POF vs. BMSCs P > 0.05); 30 days (control vs. POF P < 0.01; control vs. BMSCs P < 0.05; POF vs. BMSCs ^**P < 0.01).

Fig. 6

Histologic analysis of the ovaries after transplanting BMSCs. The ovaries of the control group (A), POF group (B), and BMSCs treated group (C); (D–F) the primary follicle, secondary follicle, antral follicle and corpus luteum (CL) of the BMSCs treated group. Scale bars, 100 μm.

Fig. 7

EGFP-positive BMSCs in tissue sections: (A–F) transplanted BMSCs in the ovaries after 15 days; (A, D) BMSCs in the ovarian hilum; (B, E) BMSCs in the ovarian medulla; (C, F) BMSCs in the ovarian cortex, surrounding follicles; (G–I) transplanted BMSCs in the ovaries after 30 days; (G, H) BMSCs surrounding follicles, (I) BMSCs surrounding corpus lutea; (J–L) BMSCs in the kidneys, heart and liver. Scale bars, 50 μm.

Fig. 8

Apoptosis of GCs in the ovaries: (A) no apoptotic GCs were observed in the healthy antral follicles; (B, C) apoptotic GCs were observed in the atretic follicles. Scale bars, 50 μm.