Prevention of chemotherapy-induced premature ovarian insufficiency in mice by scaffold-based local delivery of human embryonic stem cell-derived mesenchymal progenitor cells

Abstract

Background: Premature ovarian insufficiency (POI) is one of the most serious side effects of chemotherapy in young cancer survivors. It may not only reduce fecundity but also affect lifelong health. There is no standard therapy for preserving ovarian health after chemotherapy. Recently, administration of embryonic stem cell-derived mesenchymal progenitor cells (ESC-MPCs) has been considered a new therapeutic option for preventing POI. However, the previous method of directly injecting cells into the veins of patients exhibits low efficacy and safety. This study aimed to develop safe and effective local delivery methods for the prevention of POI using two types of bioinspired scaffolds.

Methods: Female mice received intraperitoneal cisplatin for 10 days. On day 11, human ESC-MPCs were delivered through systemic administration using intravenous injection or local administration using intradermal injection and intradermal transplantation with a PLGA/MH sponge or hyaluronic acid (HA) gel (GEL) type of scaffold. PBS was injected intravenously as a negative control. Ovarian function and fertility were evaluated 4 weeks after transplantation. Follicle development was observed using hematoxylin and eosin staining. The plasma levels of sex hormones were measured using ELISA. Expression levels of anti-Müllerian hormone (AMH) and ki-67 were detected using immunostaining, and the quality of oocytes and embryos was evaluated after in vitro fertilization. The estrous cycles were observed at 2 months after transplantation.

Results: The local administration of human ESC-MPCs using the bioinspired scaffold to the backs of mice effectively prolonged the cell survival rate in vivo. The HA GEL group exhibited the best recovered ovarian functions, including a significantly increased number of ovarian reserves, estrogen levels, and AMH levels and decreased apoptotic levels. Furthermore, the HA GEL group showed improved quality of oocytes and embryos and estrous cycle regularity.

Conclusions: HA GEL scaffolds can be used as new delivery platforms for ESC-MPC therapy, and this method may provide a novel option for the clinical treatment of chemotherapy-induced POI.

Keywords: Bioinspired scaffold; Cell therapy; Chemotherapy-induced premature ovarian insufficiency; Embryonic stem cell-derived mesenchymal progenitor cells; Hyaluronic acid; Local delivery; Oncofertility.

Fig. 1

Scaffold characterization; PLGA sponge, and HA gel. A Optical images of the two types of scaffold. B Scanning electron microscopic images of each scaffold (× 200 magnification, scale bar = 200 μm). C Percentage of remaining mass of the scaffolds and D pH changes in scaffolds during in vitro degradation at 37 °C for 28 days (n = 3). E Live and dead staining images before implantation (scale bar = 100 μm)

Fig. 2

Schematic illustrations of the experimental design and changes in body weight of mice. A Experimental scheme for in vivo experiments. B Body weights were measured daily during cisplatin administration and then measured every other day after implantation of human MPCs. C Body weights of the different groups were the same on the day of transplantation. D Body weights at 1, 2, 3, and 4 weeks after cell therapy. Different superscript letters indicate a significant difference (p < 0.05); Normal, normal control group; POI, premature ovarian insufficiency; I.V., intravenous; I.D., intradermal; Sponge, PLGA sponge type of scaffold; GEL, HA gel type of scaffold

Fig. 3

Recovery of the ovarian structure by transplantation of human ESC-MPCs in the CIP model. A Gross morphology of ovaries. Scale bar = 5000 μm. B Weight of ovaries was measured at 4 weeks after cell therapy. C Ovarian histology was analyzed by H&E staining. Scale bar = 200 or 25 μm. The arrow heads indicate primordial follicles. D–G The number of follicles at different stages (D, primordial; E, primary; F, secondary; G, antral) was counted and compared in the six groups at 4 weeks after human ESC-MPC therapy. H The number of zona pellucida remnants per ovary. I Total number of follicles per ovary. J Percentage of follicles at different stages per ovary. The different superscript letters indicate significant differences (p < 0.05); Primordial, primordial follicle; Primary, primary follicle; Secondary, Secondary follicle; Antral, antral follicle; ZPR, zona pellucida remnant

Fig. 4

Human ESC-MPC therapy reduces ovarian damage in CIP mice. A Immunofluorescence for AMH (red) and Ki-67 (green). DAPI (blue) represents follicle growth and granulosa cell proliferation in the ovary. Scale bar = 200 μm. B Relative mRNA expression by qRT-PCR analysis for Amh normalized to β-actin. Fold changes were measured by the 2^−ΔΔCT method, n = 3. Serum levels of E2 (C) and FSH (D). E Apoptosis levels in ovaries were evaluated by the expression levels of cleaved PARP using western blot analysis. F Western blot results with relative protein levels of cleaved PARP controlled to α-TUBULIN calculated from three different blots. The different superscript letters indicate significant differences (p < 0.05); Amh, anti-Müllerian hormone; E2, estrogen; FSH, follicular stimulation hormone

Fig. 5

Transplantation of human ESC-MPCs with HA gel improves the quality of oocytes and the development of embryos. A Representative images of spindle morphology in normal and abnormal MII oocytes. DAPI (blue), Spindle (red). Scale bars = 20 μm. B Percentages of spindle defects in the Normal, POI, and GEL groups. C Representative images of embryos after 120 hours of culture in vitro. Scale bars = 50 μm. D The average developmental efficiency of embryos in the Normal, POI, and GEL groups. The percentage was calculated using the number of embryos that reached the blastocyst stage. Developmental rates were statistically analyzed by Student’s t test. E Representative image of blastocysts stained with anti-OCT4 (ICM) and DAPI (total cells) in the normal, POI, and GEL groups. Scale bars = 20 μm. F (a) ICM and total number of cells in the embryo. (b) The percentage of ICM to total cells in blastocysts. The different superscript letters indicate significant differences (p < 0.05); BL, blastocyst; MII, MII stage oocyte; 2C, 2-cell; ICM, inner cell mass

Fig. 6

Transplantation of hESC-MPCs with HA gel improved the regularity of the estrous cycle in CIA mice. A Representative images of each stage of the estrous cycle. The estrous cycle stage was determined by vaginal cytology. B, C Representative examples of (B regular or C irregular) estrous cycling patterns from four individual mice. D The percentage of mice with regular (red) or irregular (black) cyclicity was detected at 8 weeks after cell therapy