Stem cell transplantation extends the reproductive life span of naturally aging cynomolgus monkeys

Ethical statement

The collection of human ovaries was approved by the Human Ethics Committees of the First Affiliated Hospital of Zhengzhou University (2020-KY-221), the Peking Union Medical College Hospital (JS-2815), and the Hospital for Reproductive Medicine Affiliated to Shandong University ([2022] LSZ 01) and was conducted in compliance with approved institutional guidelines. The study has been performed following the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

The experiments involving cynomolgus monkeys were conducted according to the Principles for the Ethical Treatment of Non-Human Primates and were approved in advance by the Institutional Animal Care and Use Committee of the Institute of Zoology (IOZ-IACUC-2021-178). All applicable institutional and national guidelines for the care and use of animals were followed in this study.

All experiments were conducted according to the standard operating procedure of the Institute of Zoology.

Source of human ovaries

Physiologically normal human ovaries were obtained from 26 women. These women suffered from uterine diseases and underwent hysterectomy and bilateral salpingo-oophorectomy to prevent poor prognosis caused by estradiol secretion and the risk of metastatic lesions spreading to the ovaries after surgery. The exclusion criteria for donors referred to a previous study⁴⁰. Briefly, donors previously exposed to chemotherapy, or with previous ovarian surgery were excluded from the study. Additionally, ovarian pathology such as endometriomas and cystic masses of the ovary >2 cm was excluded from the study. Specimens with gross or microscopic evidence of ovarian pathology were also excluded from the study. Medical records, surgical pathology reports, and operative reports were reviewed. The ovary of a 23-year-old woman, who died of brain herniation, was donated from Peking Union Medical College Hospital.

Source of cynomolgus monkeys

All cynomolgus monkeys (Macaca fascicularis) were of Southeast Asian origin. The animals were maintained at ~25 °C on 12 h light–12 h dark cycle and raised at the Xieerxin Biology Resource with the accreditation of the laboratory animal care facility of the Institute of Zoology. All animals were fed a commercial diet twice daily while vegetables and fruits once daily, with tap water ad libitum, under careful veterinary oversight. Before the experiment, none of the animals had a clinical or experimental history that would affect physiological aging or increase their susceptibility to diseases. Cynomolgus monkeys were anesthetized with ketamine (10–12 mg/kg) before surgery. A total of 10 cynomolgus monkeys aged 18–23 y were selected. The ovarian sections of young monkeys (3 y) were from the remaining samples of our previous study¹.

Cell lines

M cells were generated by the National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences as described previously³⁹. Firstly, clinical human embryonic stem cells (hESCs, Q-CTS-hESC-2)⁶⁴ were dissociated into small clumps to form human embryoid bodies (hEBs) for 5 days. Subsequently, hEBs were transferred onto plates and cultured for additional 14 days. The hEBs outgrowth cells were dissociated and passaged continuously in M-cell Medium consisting of α-MEM (Gibco, 12561-049) supplemented with 5% KOSR (Gibco, A3020902), 1% Ultroser G (Pall corporation, 15950-017), 1× L-glutamine (Gibco, A12860-01), 1× NEAA (Gibco, 11140050), 5 ng/mL bFGF (R&D systems, 233-FB) and 5 ng/mL TGF-β (Peprotech, 96-100-21-10). After 5 passages, ~4 weeks, M cells were harvested. They displayed a fibroblastic morphology and expressed canonical MSC-specific surface markers.

KGN cells, a human granulosa tumor cell line, were cultured as previously described in DMEM/F12 medium (GIBCO, 11320033) supplemented with 10% fetal bovine serum (FBS) (GIBCO, 10091-148), 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO, 15070063) at 37 °C in 5% CO₂. All the cell cultures tested negative for mycoplasma contamination.

Transwell culture system

KGN cells were plated in the lower chambers of transwell plates (Corning, 3450), and then were treated with 100 µM H₂O₂ for 24 h, followed by 45 µM H₂O₂ for 24 h to induce cellular senescence. Subsequently, M cells were seeded into the upper chambers of the transwell plates. After 5 days, the KGN cells in the lower chamber were harvested for apoptosis, ROS, and RT-qPCR analyses.

Follicle identification and counting

Ovaries were obtained right after surgical removal. In the case of elective surgical removal, a small part of the ovary was removed and prepared separately for review by a surgical pathologist. The remainder of the ovary was then re-weighed. The difference in weights recorded was used to determine the fraction of the whole ovary that was available for use in this study (the first fraction, f1). Each ovary was first cut into 1-mm slabs perpendicular to the long axis using a slicer designed by our lab¹. Approximately one-third of the slabs were selected out of the total generated slabs (the second fraction, f2) using systematic random sampling rules. Systematic random sampling rules require that an interval for sampling be set, a random position within that interval be determined for the initial sample, and then sequential samples be selected at that fixed interval. The selected slabs were embedded in paraffin wax (Leica, 39601006) following immersion through a graded series of alcohol solutions (70%, 80%, 90%, 100%, and 100%). The slabs were then cut into 5-μm-thick sections using a rotary microtome (Leica, Leica 2135). Subsequently, five serial sections from every 50 sections (the third fraction, f3) were collected from each slab and stained with H&E. The five H&E-stained serial sections were scanned using a PerkinElmer Vectra Polaris (Leica, Leica Aperio VESA8).

Follicles were classified according to the morphological criteria described by Charleston⁴⁰. Primordial follicles were defined as those containing a single layer of flattened granulosa cells while primary follicles were defined as those containing a single layer of cuboidal granulosa cells without any flattened granulosa cells. Primordial and primary follicles were counted when a clearly defined oocyte nucleolus was observed. Follicles that had already been counted were excluded from the subsequent adjacent section.

Raw counts (Q) for five serial sections of follicles were then converted to an estimate of the total number (N) in the entire ovary by the following equation (where Q is the raw count for each serial set of sections):

Secondary follicles were defined as having an enlarged oocyte surrounded by at least a partial or complete second layer of cuboidal GCs but no more than four complete layers of cuboidal GCs. Antral follicles were characterized by the presence of areas of follicular fluid (antrum) or a single large antral space. As the volume of secondary follicles is significantly larger than that of primordial and primary follicles, the secondary follicles appear in more tissue sections and tend to be overcounted when using the same follicle counting method for primordial and primary follicles. Therefore, we compared the relative number of secondary follicles in different female ovaries by selecting 1/3 slabs and counting follicles in one out of every 50 serial sections.

For growing follicle counting of monkeys, one ovary per group was analyzed, and 3 sections were taken from each ovary, with a 50-section interval between each selected section. The entire section was used to count the number of growing follicles.

Ultrasound evaluation of ovaries and uteruses

The ultrasonic examination was performed before and after treatment to evaluate the size of the ovary, and the endometrial thickness. Monkeys were examined in the supine position, using CX50 compact Xtreme ultrasound system (Philips Ultrasound, Inc.). The mean diameter of the ovaries was calculated as follows: D = [L (maximal length) + W (maximal width)] ∕ 2.

M-cell transplantation and saline injection

Cynomolgus monkeys were anesthetized with ketamine (10–12 mg/kg) before surgery. M cells (100 μL per ovary, 5 × 10⁶ cells, treated group) or 0.9% saline (100 μL per ovary, control group) were injected into the ovary of monkeys by laparoscopy. The solution was injected into the ovary using 25-g needles (World Precision Instruments, Inc, 3030217) by two senior medical physicians.

Superovulation, ICSI, and embryo culture

As described in a previous study⁶⁵, female cynomolgus monkeys received twice daily intramuscular injections of rhFSH (GONAL-F, Merck Serono) for 8 days, beginning at days 1–3 of the menstrual cycle, then received injection of rhCG (OVIDREL, Merck Serono) on day 9. Oocytes were aspirated laparoscopically at 32–35 h after rhCG administration. MII oocytes (with the first polar body) were kept in hamster embryo culture medium-10 (HECM-10) until ICSI. The fertilized embryos were then cultured in 50-µL drops of HECM-10 containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO₂, with the medium changed every other day.

H&E staining

The ovarian sections were deparaffinized and rehydrated using a graded alcohol series (100%, 95%, 85%, and 75%). After a brief wash in distilled water, the slides were incubated in a hematoxylin solution for 4 min. The sections were then washed with running tap water for 10 min to remove excess hematoxylin. Next, the sections were incubated in 1% acid alcohol for 5 s and washed with running tap water for 3 min. This step was followed by incubation in eosin counterstain for 8 min, dehydration in a graded alcohol series (95%, 100%, and 100%), and immersion in xylene. Finally, the slides were sealed with coverslips using Cytoseal-60 (Stephens Scientific, USA).

Immunostaining of ovarian tissues and KGN cells

The ovarian sections were deparaffinized and rehydrated using 100%, 95%, 85%, and 75% alcohol. After washing in distilled water, antigen retrieval was conducted by boiling the slides for 15 min in Tris-EDTA buffer and cooling the slides for 2 h. After washing three times with phosphate-buffered saline (PBS), the sections were blocked with 3% bovine serum albumin (BSA, Sigma–Aldrich, A1933) for 1 h at 25 °C. Then, the sections were incubated with primary antibodies at 4 °C overnight. After washing thrice with PBS, the sections were incubated with the corresponding secondary antibodies and DAPI (Thermo Fisher Scientific, USA, D3571) for 1 h at 25 °C. The slides were sealed with coverslips using Cytoseal-60 (Stephens Scientific, USA). A laser scanning confocal microscope (Carl Zeiss, Germany, LSM 880) was used to capture images. Three sections were selected per ovary, with each section spaced 50 slices apart and 5 regions were randomly selected for each section. The assessors were blinded to the treatment group when performing the image capture.

The KGN cells were fixed in 4% PFA for 30 min and permeabilized in 0.5% Triton X-100 (Sigma) for 30 min. After being washed in 0.1% BSA three times, the KGN cells were blocked with 5% BSA in PBS solution for 1 h and incubated with primary antibodies overnight at 4 °C. The KGN were then incubated with corresponding secondary antibodies for 1 h and counterstained with DAPI for 15 min. The images were captured using Leica Stellaris.

The primary antibodies included: Rabbit anti-γ H2A.X (Abcam, ab81299, 1:200), Rabbit anti-Ki67 (Abcam, ab15580; 1:200), Rabbit anti-PPARG (Cell Signaling Technology, 2345t; 1:200), Rabbit anti-PRDX4 (Abcam, ab184167; 1:200), Rabbit anti-RDX (Abcam, ab52495; 1:200), Rabbit anti-P21 (Cell Signaling Technology, 2947 P, 1:200), Rabbit anti-P53 (Santa Cruz, sc-126, 1:200). The secondary antibodies included: Alexa Fluor 568 donkey anti-rabbit antibody (Thermo Fisher Scientific, A10042; 1:200), Alexa Fluor 568 donkey anti-mouse antibody (Thermo Fisher Scientific, A10037; 1:200).

Masson’s trichrome staining of ovarian tissues

Fibrosis was detected by Masson’s trichrome staining kit (Solarbio, G1340). The ovarian sections were deparaffinized and rehydrated using 100%, 95%, 85%, and 75% alcohol. After washing in distilled water, the sections were stained with an iron hematoxylin working solution for 5 min. Next, the sections were washed with distilled water and incubated in an acid-alcohol solution for 5 s. The sections were then washed with tap water for 5 min. Next, the sections were stained in a ponceau-acid fuchsin solution for 8 min, rinsed in distilled water, and incubated in phosphotungstic acid for 1 min. The sections were then directly transferred (without rinsing) to an aniline blue solution and stained for 20 s. This step was followed by a brief rinse in distilled water and fixation in 1% acetic acid solution for 1 min. After washing in distilled water, the sections were dehydrated quickly through 95% alcohol and 100% alcohol and cleared in xylene. Finally, the sections were mounted on a resinous mounting medium. Fibrotic areas are indicated in the blue-stained regions. Red areas denote muscle fibers, cytoplasm, cellulose, keratin, and red blood cells. Quantification of the fibrotic area was performed using ImageJ software (Softonic). Each picture was separated into different colors. Subsequently, an appropriate channel was chosen, and the appropriate threshold value was applied to all pictures. One ovary per group was analyzed, and 3 sections were taken from each ovary for Masson’s staining, with a 50-section interval between each selected section. The entire section was used to calculate the fibrosis area. Therefore, the fibrosis calculation formula is the fibrotic area /total area in the whole section × 100%.

Sex hormone assays

Blood samples were collected from 10 cynomolgus monkeys aged 18–23 y. At the time of collection, serum specimens were aliquoted and stored at –80 °C until the sex hormone assays were performed. The serum levels of E2 (Roche, 06656021190) and P4 (Roche, 07092539190) were measured by Cobas6000 (Roche). Before and after 3-, 6-, and 8-month treatment, we collected blood samples every 4 days for 6 weeks. Then we analyzed the E2 and P4 levels at day 1 (corresponding to the early follicular phase) of the menstrual cycle because the sex hormone levels were relatively stable during this period⁴⁵.

CCK8 assay

The experiments were performed following the instructions of Cell Counting Kit-8 (LABLEAD, China). Briefly, a total of 2 × 10³ viable cells were plated in each well of the 96-well plates (Corning). After incubation for the first 24 h, the viable cell number was then calculated every 24 h for three consecutive days. To determine the number of viable cells, the optical density value at 450 nm was detected with BioTex Power Wave XS (BioTex, USA).

EdU assay

According to the manufacturer’s instructions (Ribobio, C10310-1), KGN cells were incubated with 5-ethynyl-20-deoxyuridine (EdU) for 2 h. After being fixed with 4% PFA for 30 min and permeabilized with 0.5% Triton X-100 for 10 min, the cells were incubated with 1× Apollo®567 reaction cocktail for 30 min. The cell nuclei were finally stained with DAPI for 10 min. The images were captured using Zeiss LSM 880.

Beta-galactosidase staining

SA-β-gal staining was done using the SA-β-gal staining kit (Beyotime, C0602) according to the manufacturer’s instructions. Briefly, 1. Remove growth media from the cells. 2. Rinse the plate one time with 1× PBS. 3. Add 1 ml of Fixative Solution. Allow cells to fix for 10–15 min at room temperature. 4. Rinse the plate two times with 1× PBS. 5. Add 1 ml of the β-Galactosidase Staining Solution to each well. 6. Incubate the plate at 37 °C at least overnight in a dry incubator (without CO₂). 7. While the β-galactosidase is still on the plate, check the cells under a microscope (200× total magnification) for the development of blue color and take images. 8. For long-term storage of the plates, remove the β-Galactosidase staining solution and overlay the cells with 70% glycerol.

siRNA-mediated knockdown of PPARG, PRDX4, and RDX

si-PPARG, si-PRDX4, si-RDX, and non-targeting si-NC were transfected into KGN cells using jetPRIME Transfection Reagent (Polyplus, 114-01). At 48–96 h after transfection, cells were collected for downstream analyses. All siRNAs were synthesized by TsingkeBiotechnology Co., Ltd.

Flow cytometry analysis

For the measurement of cellular ROS, cells were collected and stained with 10 μM DCFH-DA (Solarbio, D6470) for 30 min at room temperature, and then the signals were quantified in a BD LSRFortesa flow cytometer. For analysis of apoptosis, cells were collected freshly and stained with Annexin V-EGFP and PI using an Annexin V-EGFP Apoptosis Detection Kit (Beyotime, C1062M). Then, the apoptotic cells were quantified in a BD LSRFortesa flow cytometer.

For the characterization of M cells, cells were harvested and blocked with 2% BSA (Sigma–Aldrich, B2064) for 20 min at room temperature. Then, the cells were stained with fluorescein-conjugated antibodies for 40 min at room temperature in 1% BSA. After incubation, cells were washed 3 times with PBS and analyzed with MoFlo (Beckman, Brea, CA, USA) and associated software. The antibodies used for flow cytometry were as follows: PE-conjugated mouse anti-human CD29 (Biolegend, San Diego, CA, USA; 303004), PE-conjugated mouse anti-human CD73 (BD Biosciences, San Jose, CA, USA; 550257), PE-conjugated mouse anti-human CD90 (eBioscience, San Diego, CA, USA; 12-0909-42), PE-conjugated mouse anti-human CD105 (Biolegend, 323206), PE-conjugated mouse anti-human CD34 (BD Biosciences, 555822), PE-conjugated mouse anti-human CD45 (BD Biosciences, 560957), PE-conjugated mouse anti-human HLA − DR (BD Biosciences, 555561), and the PE-conjugated mouse IgG1 (BD Biosciences, 551436) as an isotype control.

RT-qPCR

Total RNA was extracted from cells and tissues using TRIzol reagent (Invitrogen, 15596018). The RNA was reverse-transcribed into cDNA by reverse transcriptase (Vazyme, R333-01) and then subjected to an RT-qPCR assay using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TAKARA, RR820A). All quantified values were normalized to the endogenous GAPDH expression levels. The sequences of the primers used for RT-qPCR are listed in Supplementary Table S6.

Preparation of single-cell suspensions for scRNA-seq

Single-cell isolation was performed based on a protocol described previously with modification⁶⁶. In brief, the ‘clean’ ovaries were cut first into 1-mm thick pieces and then minced using a tissue section (Mcllwain Tissue Chopper, The Mickle Laboratory, Guildford, UK). The minces were transferred to a 50-mL conical tube and digested enzymatically in a medium containing α-MEM (Gibco, 12561-056) + 0.04 mg/mL Liberase DH (Roche, 5401054001) + 0.4 mg/mL DNase I (Sigma, DN25) + 1% penicillin/streptomycin (Gibco, 15140-122) for 60 min in a shaker (160 rpm) at 37 °C interspersed with 20–30 pipetting every 15 min. Digestion was terminated by α-MEM media with 10% HSA (Merck Millipore, 823022). After digestion, the cell suspension was filtered by a 40-µm strainer and centrifuged at 300× g for 5 min, followed by gentle removal of the supernatant. For removal of red blood cells, the cell pellet was treated with 3 mL of ACK (ammonium chloride-potassium) lysis buffer for 10 min at room temperature, and then, the treatment was terminated with 27 mL of PBS. The remaining cells were washed twice with PBS and centrifuged at 300× g for 5 min. For the single-cell suspension, the cell pellet was resuspended in PBS and adjusted to a concentration of 700–1200 cells/μL to ensure subsequent cell capture efficiency. Cell concentration and viability were measured with a Countess II automated cell counter (Thermo Fisher, AMQAX1000). The cells were then used for single-cell sequencing.

scRNA-seq library construction and sequencing

According to the manufacturer’s guidelines, cells were loaded onto a 10x Genomics Chromium chip targeting an 8000–10,000 cell ranger. Subsequently, reverse transcription and library preparation were carried out using the 10x Genomics Single Cell v2 kit, following the established 10x Genomics protocol. The constructed libraries were then subjected to sequencing on an Illumina NovaSeq 6000 platform, employing two lanes to generate 150 paired-end reads.

Preprocessing scRNA-seq data

The initial step involved the processing of raw sequencing reads using Cell Ranger (v3.1, 10x Genomics) with default parameters. To facilitate read alignment and quantification, a reference genome was constructed, following the recommended guidelines from 10x Genomics, utilizing the Macaca fascicularis_5.0 genome and annotations obtained from Ensemble (version 102). In summary, the raw reads were aligned against the reference genome and subjected to quality checks. Only uniquely mapped reads were considered for the unique molecular identifier (UMI) counting. Subsequently, gene expression levels were quantified for each observed barcode. Cells exhibiting a significantly lower RNA content were excluded from the subsequent analysis. The filtered feature barcode matrices, generated through Cell Ranger (v3.1, 10x Genomics), were employed for downstream analyses.

Identification of cell populations in scRNA-seq data

In this study, gene expression data were processed using the R package Seurat (v4.0.4)⁶⁶. The raw counts were normalized by scaling to 10,000, and expression levels were defined as log₂ (normalized counts + 1). As part of quality control, cells failing to meet the following criteria were filtered out: (1) the number of detected UMIs was at least 1000; (2) the number of detected genes was at least 500; and (3) the log-transformed ratio of detected genes to detected UMIs was higher than 0.8. Consensus variable features among samples were first identified as previously reported. For each sample, 3000 features were selected, and a consensus list of 3000 features was generated based on overlaps across samples. To remove potential sample/batch effects, Harmony (v0.1.1) was employed for dimension reduction with these consensus features. Subsequently, potential doublets in individual samples were identified using the Python package Scrublet (v0.2.3). Doublets along with cells in clusters exhibiting a high fraction of doublets were filtered out. After stringent quality control, a total of 26,862 cells remained, and the second round of clustering was performed using the new consensus features generated by Seurat.

Identification of DEGs and GO enrichment analysis

The DEGs were identified using the FindAllMarkers or FindMarkers function in Seurat. Genes with an adjusted P-value less than 0.05 were considered DEGs. GO analysis was performed by using the R package clusterProfiler (v4.0.5) with the annotation R package org.Hs.eg.db (v3.13.0).

Transcriptional regulatory network analysis

The pySCENIC Python package (v0.11.2) was adopted to infer the transcriptional regulatory network for GCs and SCs. Briefly, raw counts and a list of known TFs for humans were used as input for network inference (GRNBoost2), followed by the generation of candidate regulons (cisTarget). The activity score of candidate regulons in the individual cell was quantified using AUCell. Activity scores were averaged, scaled, and visualized using a heatmap. The network of the regulon and its targets was visualized using Cytoscape (v3.8.0).

Inference of cell–cell interactions based on scRNA-seq data

For inference of cell–cell interactions between GCs and SCs, the CellPhoneDB Python package (v2.14.0) was adopted.

Statistical analyses

The experimental data were statistically analyzed using one-way ANOVA, two-way ANOVA, or t-test to compare differences between different groups using PRISM software (GraphPad 9 Software). A P-value < 0.05 was considered statistically significant. In all figures, one, two, three, and four asterisks indicate P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

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• Source: https://www.nature.com/articles/s41421-024-00726-4