Amniotic fluid, follicular fluid and oocyte sample collection
All amniotic fluid cell samples were obtained via amniocentesis performed after the 18th week of pregnancy for routine prenatal diagnosis. The indications were advanced maternal age, a familial or personal history of birth defects, or any foreseen risk of the fetus carrying a chromosomal anomaly or inherited condition. The cytogenetic analyses revealed normal karyotypes for all donors. The mean maternal age was 36 years, with a range of 33–38 years of age, and the mean gestational age was 19 weeks, with a range of 16–22 weeks. Samples of follicular fluid (5 ml) were separately collected from three women (range 29–40 years of age) who were undergoing oocyte pick up for in vitro fertilization and embryo transfer (IVF-ET) to overcome male factor infertility. Ten GV oocytes with fertilization failure were collected from four women (range 32–40 years of age) who were undergoing IVF-ET. This study was carried out with the approval of the Ethics Committee of the International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai, China, and informed consents were obtained from all donors.
Preparation of follicular fluid
Three samples of follicular fluid (5 ml each) were mixed, pooled and centrifuged at 4000 g for 5 min. The supernatant was filtered (0.22 μm pore size) to eliminate contaminating cells potentially present in follicular fluid, aliquoted and stored at −20°C until use. All experiments were performed with the same batch.
Human amniotic fluid cell culture and differentiation
Amniotic fluid samples were centrifuged individually at 4000 × g for 5 minutes at room temperature and the supernatant was discarded. Cellular components were grown in DMEM/F12 medium (Gibco, Grand Island, NY, USA) containing 15% ES-FBS (Gibco, Grand Island, NY, USA), 1% glutamine and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA), supplemented with bFGF (4 ng/ml, Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 atmosphere. After expansion to confluence (5–7 d), AFSCs formed clones. Then, some clones were used to prepare a single cell suspension by gentle trypsinization. In addition, some clones were picked up and cultured under standard EB media supplemented with 5% human follicular fluid for 7–14 days to promote EB formation. Then, EBs were maintained in differentiation medium which contain germ cell maturation factor cocktail (FAC)  for 7–14 days by replacing half the medium every 3–4 days. The FAC medium consisted of standard EB media supplemented with a germ cell factor cocktail containing: human SCF 100 ng/ml, SDF1 20 ng/ml, bFGF 20 ng/ml, BMP4 50 ng/ml (all R&D Systems, Minneapolis, Minnesota, USA), N-acetylcysteine 1 mg/ml, forskolin 5 mM, retinoic acid 1 mM (all Sigma, St. Louis, Missouri, USA) and CYP26 inhibitor R115866 1 mM (Johnson & Johnson, Brunswick, New Jersey, USA).
RNA extraction and real-time qPCR analysis
Total RNA extraction from samples was performed using the RNeasy Mini Kit (Qiagen, Chataworth, CA, USA). Five hundred ng of total RNA from each sample was used in reverse transcription (RT) using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Real-time RT-qPCR was carried out on cDNA using IQ SYBR Green (Bio-Rad, Hercules, CA, USA) on the Mastercycler ep realplex (Germany). All reactions were performed in a 25-μl volume according to the kit. Primer sequences are listed in Additional file 1: Table S1. Cycling conditions were as follows: 1) for ZPA, ZPC, GDF9, STELLA, STRA8, and DAZL 94°C for 2 min, then 94°C for 30 sec, 60°C for 30 sec, 72°C for 45 sec, 28 cycles, then 72°C for 10 min; for OCT4, CD133, CD117, HLA-DR, BLIMP1, VAZA, SCP1, SCP3, 18 s RNA, 94°C for 2 min, then 94°C for 30 sec, followed by 28 cycles at 53°C for 30 sec and 72°C for 45 sec, finalizing at 72°C for 10 min; and, 2) for NANOG and cMOS, 94°C for 2 min, then 94°C for 30 sec, followed by 28 cycles at 60°C for 30 sec and 72°C for 45 sec, and finalizing at 72°C for 10 min. The amplification efficiency of different genes was determined relative to 18sRNA (∆C t = Ctgene-Ct18sRNA). The quantity of mRNA in each sample was calculated by the comparative (∆∆Ct = ∆Ctgene-∆Ctcontrol) value method. The fold change in gene expression relative to the control was calculated by 2-∆∆Ct . Data were obtained by averaging the results from three independent experiments.
The expression of OCT4 and CD117 was evaluated on both single cell suspensions obtained from hAFCs as well as on cells from EBs. A total of 1 × 106 Cells were suspended in 2% BSA/PBS and labeled with the corresponding antibodies, as follows: 1) mouse anti-human OCT3/4 conjugated with Alexa Fluor 488 (eBioscience, San Diego, CA, USA) and 2) mouse anti-human PE-labeled CD117 (eBioscience, San Diego, CA, USA). Identification of Oct4+ and CD117+ cells was performed using a FC500 flow cytometer (Beckman Coulter, Miami, FL, USA) and analyzed by Beckman Coulter CXP software.
Forty six-week-old C57BL/6 females were prepared as sterilized recipients by intraperitoneal injection of busulfan (30 mg/kg; resuspended in DMSO) and cyclophosphamide (120 mg/kg resuspended in DMSO) once and were observed for 1 week. Five age-matched females injected with DMSO only were used as non-sterilized controls. All animal procedures were approved by the Institutional Animal Care and Use Committee of Shanghai, and were performed in accordance with the National Research Council Guide for Care and Use of Laboratory Animals.
Transfection and transplantation of human amniotic fluid cells
Once hAFCs had grown to a density of 80–90%, lentivirus vector with enhanced green fluorescent proteinlenti-EGFP (a gift from Tianjin Liu ) was added to cultured cells and incubated at 37°C in a 5% CO2 atmosphere for 24 h. Titration of concentrated supernatants was performed by serial dilutions of vector stocks on 1 × 105 Hela cells followed by fluorescence-activated flow cytometry analysis according to the formula: 1 × 105 Hela cell × % EGFP positive cells × 1000/μl virus. Titers of lentiviral vectors were 1 × 108 - 1 × 109 TU/ml. After growing for another 2 days, hAFCs were examined by fluorescence microscopy. The overall cell transfection rate was determined to be greater than 95%. After lentiviral infection for 2 days, hAFCs were washed three times and trypsinized (0.25% trypsin), neutralized in 10% FBS, washed in PBS and resuspended in the culture medium. Recipients were anesthetized by an intraperitoneal injection of pentobarbital sodium (45 mg/kg body weight). Approximately 6 μl of a single-cell suspension, containing 2–5 × 103 cells (n = 30), or 6 μl of culture medium for control (n = 10), was injected into both ovaries of the sterilized recipient. Microinjection of each ovary was performed as previously described according to Zou et al. .
Two months following ovary injection, recipient and control mice were euthanized by cervical dislocation. Ovaries were dissected and fixed in 4% paraformaldehyde (4°C, overnight), dehydrated through a graded series of ethanol, vitrified in xylene, and embedded in paraffin. Six-μm thick sections were fixed for 5 minutes in 10% buffered formalin, after which endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide in methanol for 30 minutes. Different sections were then incubated in the presence of the following antibodies: mouse anti-human mitochondria (1:300; monoclonal antibody, EMD Millipore, Billerica, Massachusetts, USA); mouse anti-human nuclei (1:100; human nuclei, EMD Millipore, Billerica, Massachusetts, USA); mouse anti-human follicular stimulating hormone receptor (FSHR; 1:100; Abcam, Cambridge, UK); mouse anti-human anti-mullerian hormone (AMH; 1:30 AbD Serotec, Kidlington, Oxford, UK). A peroxidase kit (Vector Laboratories, Burlingame, CA, USA) was used following the manufacturer’s manual. Peroxidase substrate was developed using the DAB (3,39-diaminobenzidine) substrate kit (Vector Laboratories). Slides were counterstained with hematoxylin QS (Vector Laboratories) and were either mounted with low viscosity aqueous mounting medium (Scytek Laboratories, Logan, UT, USA) or dehydrated and mounted with VectaMount Permanent Mounting Medium (Vector Laboratories).
Both individual hAFCs and EBs in suspension were fixed in 4% paraformaldehyde for 15 to 20 min at room temperature, and then washed twice (10 min each) with 1 × PBS. Cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and then washed twice with 1 × PBS. After blocking in 10% skim milk solution for 30 min, cells were incubated with the anti-OCT-4A (rabbit anti-human; 1:200; Santa Cruz, CA, USA), anti-Blimp1 (goat anti-human; 1:200; Santa Cruz, CA, USA), anti-DAZL (goat anti-human; 1:500; Santa Cruz, CA, USA), anti-STELLA (goat anti-human 1:200, Santa Cruz), anti-ZPC (rabbit anti- human; 1:200; Santa Cruz, CA, USA), or anti-SCP3 (rabbit anti-human; 1:800; Abcam, Cambridge, UK) antibodies, for 1 h, at room temperature.
Ovaries from recipient and control mice were fixed with the Tissue-Tek® OCT7™ Compound (Sakura Finetek Middle East, Dubai, United Arab Emirates) and sliced in 5-μm thick sections. Slides were washed twice with PBS and kept in blocking solution for 30 min at room temperature. Slides were then incubated with rabbit polyclonal anti-GFP (1:200; Chemicon, Billerica, Massachusetts, USA) at 4°C, overnight.
Cells or sections were washed three times with 1 × PBS, and probed with FITC-labeled IgG (1:200, Santa Cruz, CA, USA). Fluorescence images were taken using a Leica DMI3000 microscope (Wetzlar, Germany).
Means for relative gene expression were compared by ANOVA using Microsoft Excel software. Statistical significance was set at P < 0.05.