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. 2020 Jun 26;11(1):255.
doi: 10.1186/s13287-020-01769-6.

Recovery of ovarian function by human embryonic stem cell-derived mesenchymal stem cells in cisplatin-induced premature ovarian failure in mice

Sook Young Yoon  1 , Jung Ah Yoon  1 , Mira Park  2 , Eun-Young Shin  2 , Sookyung Jung  3 , Jeoung Eun Lee  3 , Jin Hee Eum  1 , Haengseok Song  2 , Dong Ryul Lee  3   2 , Woo Sik Lee  1 , Sang Woo Lyu  4
Affiliations

Recovery of ovarian function by human embryonic stem cell-derived mesenchymal stem cells in cisplatin-induced premature ovarian failure in mice

Sook Young Yoon et al. Stem Cell Res Ther. .

Abstract

Background: Clinical use of mesenchymal stem cells (MSCs) requires a uniform cell population, and their harvesting is invasive and produces a limited number of cells. Human embryonic stem cell-derived MSCs (hESC-MSCs) can differentiate into three germ layers and possess immunosuppressive effects in vitro. Anticancer treatment is a well-known risk factor for premature ovarian failure (POF). In this study, we investigated the effect of hESC-MSC on recovery of ovarian function in cisplatin-induced POF in mice.

Methods: Female mice received intraperitoneal cisplatin for 10 days. On day 12, CHA15-derived hESC-MSCs were transplanted into the mice by tail vein injection. An injection of PBS served as the negative control. Ovaries were removed 28 days after transplantation for assessment of ovarian histology, immunostaining, and fertility testing by superovulation and in vitro fertilization. hESC-MSC transplantation into mice with cisplatin-induced damage restored body weight and ovary size.

Results: Mean primary and primordial follicle counts in the hESC-MSC group were significantly improved compared to the PBS group (P < 0.05), and counts of zona pellucida remnants, an apoptotic sign in ovarian follicles, were significantly reduced (P < 0.05). TUNEL assays and cleaved PARP immunostaining indicated apoptosis, which led to loss of ovarian stromal cells in negative control mice, while Ki-67 was higher in the hESC-MSC group and in non-cisplatin-treated controls than in the PBS group. Ovulation was reduced in the PBS group but recovered significantly in the hESC-MSC group. Rates of blastocyst formation from ovulated eggs and live births per mouse also recovered significantly in the hESC-MSC group.

Conclusions: hESC-MSC restored structure and function in the cisplatin-damaged ovary. Our study provides new insights into the great clinical potential of human hESC-MSC in treating POF.

Keywords: Chemotherapy-induced premature ovarian failure; Human embryonic stem cell-derived MSC; Ovarian stromal cell apoptosis; Recovery of ovarian function.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Schematic description of the experimental design. Cisplatin (2 mg/kg) was administered by intraperitoneal injection for 10 days. On day 12, hESC-MSCs (5 × 106/mouse) were transplanted by tail vein injection. Experimental analyses were performed after 2 and 4 weeks
Fig. 2
Fig. 2
Generation and characterization of mesenchymal stem cells (MSC) from human embryonic stem cells (hESCs). a The typical morphology of hESC-MSC. Scale bar = 100 μm. b Karyotype of MSCs. c Surface antigen profiling in hESC-MSC by FACS. TRA-1-60 and SSEA4 are markers of pluripotency, CD34 and CD45 are hematopoietic markers, and CD29, CD44, CD90, and CD105 are MSC markers. d hESC-MSCs have the capacity to differentiate into adipocytes, osteocytes, and chondrocytes. Experimental details are described in Supplementary Methods. Magnified images of each differentiation are shown at the bottom. Scale bar = 100 μm
Fig. 3
Fig. 3
Improved survival rate and changes in body weight of mice treated with cisplatin by hESC-MSC transplantation and tracking of hESC-MSC distribution in vivo. Total numbers of mice were 72, 97, and 75 in the control (CON), PBS, and hESC-MSC treatment groups, respectively. b, c Body weights were measured each day during cisplatin administration and again 2 and 4 weeks after hESC-MSCs transplantation. *P < 0.05, **P < 0.001. d Tracking of hESC-MSC distribution in vivo by Molday ION B with Prussian blue staining. e Nested PCR detection of human Sry gene. The control gene was mouse GAPDH (mgapdh). MM, molecular marker; L, liver; M, muscle; O, ovary; U, uterus; S, spleen
Fig. 4
Fig. 4
Recovery of ovarian structure by hESC-MSC transplantation in cisplatin-induced ovarian failure. a Ovarian histology was analyzed 2 and 4 weeks after transplantation using H&E staining. Scale bar = 200 or 50 μm. PrFs in insets were captured from other section. b Ovaries were removed from mice in the control, cisplatin + PBS, and cisplatin + hESC-MSC transplantation groups. Scale bar = 2 mm. c Total number of follicles per ovary. d Percentages of each follicle type per ovary. *P < 0.05, **P < 0.001; CL, corpus luteum; AF, antral follicle; SF, secondary follicle; PF, primary follicle; PrF, primordial follicle; and ZPR, zona pellucida remnant
Fig. 5
Fig. 5
Effect of hESC-MSCs on ovarian stromal and granulosa cells injured by cisplatin. a Immunofluorescence of Ki-67 (red) represents granulosa cell proliferation in the ovary. b TUNEL detection (green) demonstrates apoptotic signals in ovarian stromal cells. a, b Blue represents DAPI-stained nuclei. Scale bar = 100 μm. c, d Western blot results with relative band intensities calculated from three different blots
Fig. 6
Fig. 6
Rescue of ovarian function by hESC-MSC transplantation after cisplatin injury. a Ovulated eggs from control, PBS, and hESC-MSC-transplanted mice at 4 weeks. b The average number of eggs ovulated per mouse. c Ovulation rate, calculated as the fraction of the number of mice in the group that ovulated. d Quality of ovulated eggs. e Embryonic development to blastocyst from ovulated eggs of treated mice following in vitro fertilization. f Percentage of fertilization with pronuclear zygotes, two cells, and blastocyst formation and hatching after in vitro fertilization. g Hatching blastocysts were immunostained for Oct3/4 (green) of the ICM, actin-phalloidin (red) in TE, and nuclear staining with DAPI (blue). h Total cell numbers in ICM and the TE and the ratio of TE cells to ICM cells. Scale bar = 100 μm (e) and 10 μm (g)
Fig. 7
Fig. 7
Fertility restoration by hESC-MSC transplantation in mice with cisplatin-induced ovarian failure. a Reproductive outcomes (offspring obtained) in three mating experiments with fertile males. Mating occurred 4 weeks after hESC-MSC transplantation. Mean litter size per pregnant mouse for generation F1 (b) or F2 (c). Data represent mean ± SEM
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