DYR0100 human iPSCs (SCSP-1301) were obtained from Professor Xiaoyan Ding (Stem Cell Bank, Chinese Academy of Sciences, Shanghai, China). All experiments using DYR0100 iPSCs were approved by the Shanghai University of Traditional Chinese Medicine Research Ethics Board (Shanghai, China). The DYR0100 iPSCs were cultured in Dulbecco's modified Eagle's medium:F12 (1:1) supplemented with 15% KnockOut™ Serum Replacement, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM beta-mercaptoethanol, penicillin (25 U/ml)-streptomycin (925 mg/ml), Reconstituting Human Basic Fibroblast Growth Factor (15 ng/ml), Reconstituting Human Epidermal Growth Factor (15 ng/ml). The aforementioned cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were incubated in a humidified incubator containing 5% CO₂ at 37°C. DYR0100 iPSCs were induced to differentiate into OGLCs for 12 days, according to a multistage protocol using appropriate cell growth factors and hormones (Table I). Briefly, the iPS cells were stimulated with all-trans-retinoic acid (atRA) for 48 h, then atRA was removed. The iPS cells were cultured in fresh medium supplemented with E2, FSH and human growth hormone (hGH) for 48 h. The iPS cells were then cultured in fresh medium supplemented with anti-Müllerian hormone (AMH), estradiol, FSH and hGH for 48 h. Finally, the iPS cells were cultured in fresh medium supplemented with AMH, estradiol, inhibin α, inhibin β and transforming growth factor-β for 96 h.

As described in our previous studies (2–4), hebetic female C57BL/6 mice (n=30; age, 5 weeks) were obtained from the Shanghai University of Traditional Chinese Medicine with approval from the Institutional Animal Care and Use Committee. To generate a murine model of POF, the mice were administered a single intraperitoneal injection of 70 mg/kg cyclophosphamide (Sigma-Aldrich, St. Louis, MO, USA). The mice were divided into two groups: A negative control group (n=15) treated with phosphate-buffered saline (PBS), and an experimental group (n=15) transplanted with iPSC-derived OGLCs. A total of 1 week after the POF models were established, each mouse received an injection of 50 µl cells (~1×10⁷ cells/µl), which were stained with DiI red fluorescent dye (Beyotime Institute of Biotechnology, Hangzhou, China), or 50 µl PBS. The final experiments in both groups were conducted 21 days post-transplantation. All mice were sacrificed by cervical dislocation following this treatment and additional experiments were conducted.

An AKP assay kit (Beyotime Institute of Biotechnology) was conducted, according to the manufacturer's protocol. Briefly, the cells were washed twice with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min. Dye solution was added to the cells, which were incubated at 37°C for 60 min until the AKP-positive cells were stained deep blue. The staining reaction was terminated following the addition of PBS. Digital images were captured using a fluorescence microscope (DMI3000; Leica Microsystems, Inc., Buffalo Grove, IL, USA).

As previously described (3,4), the cultured cells or ovarian tissue sections were washed three times with PBS and were fixed with 4% paraformaldehyde for 30 min. After blocking with blocking solution (Beyotime Institute of Biotechnology), the cells were incubated with primary antibodies (Table II) overnight at 4°C, followed by a 30 min incubation with cyanine 3- or fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibodies (1:200; cat. no. C0992; Sigma-Aldrich) and 5 µg/ml 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) at room temperature. Subsequently, the cells and tissues were thoroughly washed with Tris-buffered saline containing 0.5% Tween-20 (Sigma-Aldrich) and were visualized by fluorescence microscopy (DMI3000; Leica Microsystems, Inc.).

As previously described (3,4), fresh ovarian tissues were washed three times with PBS and were fixed with 4% paraformaldehyde for 30 min. The tissue specimens were then dehydrated in a graded ethanol series, vitrified in xylene, and paraffin embedded. Serial 6-µm sections were prepared and stained with H&E. Images were captured using a DMI3000 microscope (Leica Microsystems, Inc.).

Mouse E2 and FSH ELISAs (Shanghai XiTang Biotechnology Co., Ltd., Shanghai, China) were conducted according to the manufacturer's protocols and our previous studies (3,4). Mouse blood plasma was obtained by retro-orbital blood collection (4). Briefly, a microhematocrit tube (BD Biosciences, Franklin Lakes, NJ, USA) was inserted into the mouse lateral canthus at 30 degree angle to the side of the head. Approximately 100 µl blood was collected from each mouse into heparin-coated blood collection tubes (BD Biosciences). Mouse blood was centrifuged at 453 × g at 4°C for 10 min and the supernatant was collected. Briefly, 100 µl mouse E2 or FSH standards at the following concentrations: E2, 8,000, 4,000, 2,000, 1,000, 500, 250 and 125 pg/ml; FSH, 10,000, 5,000, 2,500, 1,250, 625, 312 and 156 pg/ml, or diluted mouse plasma were added to anti-E2 or anti-FSH pre-coated microtest wells and were incubated for 60 min. The plates were then washed three times with PBS, and horseradish peroxidase-conjugated antibodies were added, followed by an enzymatic substrate. Optical absorbance was determined at a wavelength of 450 nm using a BioTek Synergy Mx plate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Each experiment was performed as least three times, and results are presented as the mean ± standard error of the mean. Data was analyzed using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). Where applicable, differences were evaluated by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Pluripotent stem cell biomarkers were detected prior to induction. Cellular immunostaining detected high expression levels of AKP, Oct4 and SSEA-4 (Fig. 1A and B). Subsequently, various cell growth factors (transforming growth factor-β and human growth hormone) and hormones [E2, anti-Müllerian hormone (AMH), inhibin α and inhibin β] were used to induce iPSCs to differentiate into OGLCs at various time points (Fig. 1C). Following 12 days of induction, morphological observations indicated that the original clone-like iPSCs had successfully differentiated into fibroblast-like cells (Fig. 1D). Cell immunofluorescence indicated that prior to induction, iPSCs expressed high levels of pluripotent stem cell markers, including Oct4, Sox2, Nanog and SSEA-4, but did not express OGC markers, including AMH, FSH receptor (FSHR), inhibin α and inhibin β. Conversely, following induction, the expression levels of OGC markers were elevated in granulosa-like cells derived from iPSCs (Fig. 2). These results suggest that iPSCs may be induced to differentiate into granulosa-like cells in vitro, following treatment with a combination of hormones and growth factors.

A total of 21 days post-transplantation the mice were sacrificed. In the control group (PBS-POF), ovarian pathological analysis indicated that normal follicles were almost absent. Ovarian stromal tissue and OGCs exhibited hallmarks of apoptosis and atrophy. Furthermore, ovarian tissue presented varying degrees of bleeding and serious follicular atresia (Fig. 3A). However, compared with the PBS-POF group, in the experimental group (OGLCs-iPSCs-POF), ovarian pathological analysis revealed that ovarian tissue contained some normal mature follicles, with a significant reduction in the numbers of atretic follicles. In addition, the growth state of the OGCs was markedly improved (Fig. 3A). Ovarian tissue weight was increased in the OGLCs-iPSCs-POF group compared with in the PBS-POF group (P<0.05; Fig. 3B). The number of atretic follicles in the OGLCs-iPSCs-POF mice was reduced, as compared with in the PBS-POF mice (P<0.05; Fig. 3C). Furthermore, detection of hormone levels in murine peripheral blood samples indicated that the levels of E2 were elevated in the OGLCs-iPSCs-POF group, whereas the levels of FSH were decreased (Fig. 3D and E; Table III). Furthermore, in the OGLCs-iPSCs-POF group, immunofluorescence indicated that iPSC-derived OGLCs transplanted into POF mice (visualized by red fluorescence) expressed OGC biomarkers (AMH, inhibin α, inhibin β, FSHR; green fluorescence) in ovarian tissue (Fig. 3F). These results suggest that iPSCs-derived OGLCs were capable of repairing ovarian injury and enhancing ovarian function.