Human Theca Stem Cell Isolation and Culture

In addition to the hTSCs obtained from the ovary of a 19-year-old patient that was evaluated in the previous study, ovaries from two transgender patients aged 20 and 38 years were collected to ensure reproducibility of the test at different ages. Small antral follicles (3–5 mm) were collected from the three ovaries. Three different age ranges were used in this experiment to evaluate the reproducibility of differentiation of hTSCs into hGCLCs at young and late reproductive ages. The follicles were cut in half, and the oocytes and granulosa cells were removed using a spatula, leaving only the theca layer in the follicle wall. The isolated layer was dissected into small pieces and incubated with 0.5% collagenase type I (Sigma, St. Louis, USA) at 37°C. After 45 min, DMEM/F12 ( Gibco, Grand Island, NY, USA), containing 10% fetal bovine serum albumin (FBS, Gibco, USA) was added to neutralize the enzyme activity. The cell suspension was further purified by passing it through 100 and 40 µm filters (BD Falcon, Mexico) [7], and then cultured in DMEM/F12 supplemented with 10% FBS, 2 mM glutamine (Gibco, UK), 100 U/mL penicillin G (Sigma, USA), 100 µg/mL streptomycin (Sigma, USA), 20 ng/mL epidermal growth factor (EGF; Royan Biotech, Tehran, Iran), 10 ng/mL basic fibroblast growth factor (bFGF; Royan Biotech, Tehran, Iran), and 10 ng/mL glial cell line-derived neurotrophic factor (GDNF; Royan Biotech, Tehran, Iran) at 37°C in a 5% CO2 incubator. Cells that reached 70% confluence in the third passage were considered hTSCs (Fig. 8).

Fig. 8

Stages of isolation of human small antral follicles from ovaries. a Ovary isolated from patients. b Cutting and scratching the ovary. c Follicle size 3-5 mm. d Enzymatic digestion of follicles

Preparation of human follicular fluid

Follicular fluid samples were obtained from four ICSI-treated patients (age range: 20–38 years) with male factor infertility and were centrifuged at 2000 rpm for 10 min to remove cells. The cell supernatants were subjected to incubation at 56°C for 30 min to inactivate enzyme function, followed by storage at -20°C.

Differentiation of hTSCs to hGCLCs

To differentiate hTSCs to hGCLCs, 7 × 104 cells were seeded in DMEM/F12 supplemented with 5% FBS, 5% human follicular fluid, 2 mM L-glutamine, and 2 mM penicillin–streptomycin (Sigma) for 40 days [22]. The medium was refreshed every three days, and cells that became spherical were isolated using a Pasteur pipette and considered hGCLCs, as previously established in our study [9].

Preparation of cumulus cells

Cumulus cells were obtained from three patients who underwent ICSI treatment at the Royan Institute. The age range of these patients was 19 to 38 years, and they were diagnosed with male factor infertility. Following oocyte retrieval, the cumulus masses were carefully separated from COCs using hypodermic needles and treated with 300 μg/ml hyaluronidase for 30 s. Dissection medium (Minimum essential medium alpha (α-MEM, Sigma, St Louis, MO, USA)) supplemented with 10% FBS was added for enzyme inactivation, and cells were further washed using centrifugation at 200 × g for 10 min. The pellets were cultured for one day in DMEM/ F12 supplemented with 10% FBS, 2 mM glutamine (Gibco), 100 U/mL penicillin G (Sigma), and 100 µg/mL streptomycin (Sigma), and incubated at 37°C and 5% CO2 [23].

Follicle-like structures

This experiment aimed to determine whether placing hGCLCs next to somatic cells would increase the developmental potential of hGCLCs. To accomplish this, hGCLCs and somatic cells (hTSCs and cumulus cells) were combined. One-tenth of germ cells and all somatic cells, with equal proportions of cumulus cells and hTSCs, were used to make follicle-like structures. Phytohemagglutinin (PHA-P; Sigma) was added to create a final concentration of 35µg/mL to promote cell cohesion. The culture media was Waymouth MB752/1 (Gibco), supplemented with 0.23 mM pyruvic acid (Sigma, Japon), 50 mg/L streptomycin sulfate, 75 mg/L penicillin-G (Sigma), and 10% FBS. Samples were cultured at 37°C in an atmosphere of 5% CO2 and 95% air. Each 10 µl droplet of a 6-cm suspension culture dish contained five follicle-like structures [10].

Evaluation of the viability of hTSCs, hGCLCs and follicle-like structures

Trypan blue staining was used to assess cell viability. Living cells are impermeable to trypan blue, whereas dead cells absorb it. A 0.4% concentration of trypan blue was mixed with an equal volume of the cell suspension and added to a well in a 96-well plate. The mixture was gently pipetted to ensure proper distribution. For hTSCs, 10 μl of the stained cell suspension was placed on a hemocytometer slide to count the cells.

Evaluation of size and morphology of hTSCs, hGCLCs and follicle-like structures

The transition in morphology from a spindle shape to a spherical shape, as well as the size of hGCLCs and follicle-like structures, were assessed at various stages of differentiation. These evaluations were conducted using an inverted microscope, and measurements were recorded.

Evaluation of DAZL and GDF9 protein expression using immunocytochemistry

hGCLCs and follicle-like structures were evaluated for the DAZL and GDF9 proteins, specific markers for germ cells and oocyte. To confirm that the antibodies were specific for DAZL and GDF9 proteins, the secondary antibodies were used alone as negative controls. Images were taken using a fluorescence microscope (Eclipse 50i; Nikon, Japan) and analyses and merge were performed with ImageJ Tool- kit software (version 1.46r, National Institutes of Health) and Java software executing on 32-bit architecture (version 1.6.0–20, Oracle). hTSCs and mouse oocytes (GV and MII) were used as negative and positive controls, respectively.

Samoles were fixed with a 4% paraformaldehyde solution for 30 min and permeabilized with 0.05% Triton X-100 in PBS for 10 min at room temperature. The cells were then incubated overnight at 4°C with DAZL (rabbit polyclonal, Abcam, city, USA, 1:200 (in blocking buffer) and GDF9 (goat polyclonal antibody, Santa Kruz, USA, 1: 100 ratio). The samples were washed with a PBS/Tween solution (0.05%) for 15 min. The cells were incubated for one hour at 37°C with FITC rabbit anti-goat secondary antibody at a 1:200 dilution for DAZL antibody and FITC goat anti-donkey secondary antibody at a 1:200 dilution for GDF9 antibody. Nuclei were stained with 1 µg/mL 4’,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 5 min. For negative controls of staining, the secondary antibody was used alone. The image intensity was quantified using ImageJ software (version 1.42q, Wayne Rasband, NIH, Bethesda, USA).

Statistical analysis

Statistical analysis was performed using the SPSS software version 22.0. Continuous variables were expressed as a mean ± standard deviation, while stratified variables were expressed as numbers (percentage). The Kolmogorov–Smirnov test was used to check the normality of the studied variables. Due to the groups' dependence, a repeated measures test was used to examine the three groups and the corresponding post hoc tests were used for significant variables. Paired t-tests were used to evaluate the two groups. A p-value of less than 0.05 was considered statistically significant.