Introduction: In recent years, stem cells differentiation into germ cells has focused on infertility topics and reproductive problems. A good alternative for gamete production in clinical trials would be pluripotent cells isolated from adult tissues. Herein, we identified the suitable conditions which promoted human endometrial stem cells (hEnSCs) differentiation into germ cell-like cells by adjusting the best concentration of retinoic acid (RA) in a 2D medium. Afterward, the differentiated cells were cultured in a 3D fibrin scaffold and their viability and properties were evaluated. The optimum concentration of retinoic acid was 10-5 M in which the cells were differentiated after 7 days. Characteristic cell markers such as DAZL, DDX4, and Dppa3 were determined by immunofluorescence and real-time PCR. The mechanical properties of the fibrin scaffold were examined by rheology analysis. After encapsulating the hEnSCs-derived germ cell-like cells in fibrin hydrogel, cell viability and the expression of the specific markers were assessed subsequently. We analyzed the viability of cells using the MTT assay. Also, SEM analysis proved good cell integrity in the scaffold. It was found that hEnSCs were differentiated in medium containing retinoic acid and the markers of the germ cell-like cells were expressed properly in both 2D and 3D medium cultures. In addition, fibrin gel could provide a suitable 3D scaffold for hEnSCs differentiated cells regarding tissue engineering of gonads.
Methods: Laparoscopy procedure was done to obtain the endometrial biopsy samples from donors who had infertility problem and then the hEnSCs were isolated. Consent forms were signed by all patients to be informed of the experiment. The best solution for maintaining sample tissues was Hanks Balanced Salt Solution (HBSS, Invitrogen, and Carlsbad, CA). It was advisable to add 10% fetal bovine serum (FBS, Sigma-Aldrich, USA), and 1% penicillin/streptomycin (pen/strep, Invitrogen, USA) for better result. Collagenase type I (Sigma-Aldrich USA) as an enzyme was used to dissolve the samples at 37ºC and the process took 60 minutes long. Then we neutralized the resulting suspension in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, USA) with 10% FBS added and passed the solution through 70mm filters and 40 mm respectively. Afterward, the suspension was centrifuged and separated the supernatant from the pellet. Finally, DMEM plus 10% FBS with 1% pen/strep added was used in the pellet of suspended human endometrial stem cells. Incubating the pellets of hEnSCs -in-growth under 80% confluency at 37ºC temperature and 5% CO₂ after 24 h, and removing non-adherent cells by medium exchange, the cells were detached by 0.25% trypsin with 1 microMolar EDTA inside the incubator within 5 minutes. Media exchange was done every 3 days. We made use of hEnSCs at the third passage for our experiment.
2.2. Chemical induction for germ cell differentiation
To induce differentiation in the human endometrial stem cells, they were incubated in 37 ºC temperature and in 95% humidity of air and 5% CO2 for 7 days. The medium culture was DMEM (Invitrogen, USA) plus 10% FBS, 1% pen/strep and retinoic acid (Invitrogen, USA) in 4 different concentrations: 5, 10, 15 and 20 µM. Subsequently, the control group was prepared in which the hEnSCs were cultured in DMEM with no RA as a differentiation-inducing factor for the same time. We changed the DMEM medium culture every other day. At the end of the first week, to realize the differentiated cells, identifying characteristics of germ cell-like cells were assessed by immunofluorescence, and real-time PCR techniques.
2.3. Immunocytochemical analyze
We applied immunocytochemical analyze to assess hEnSCs differentiation into germ cell-like cells. We washed the hEnSCs-derived germ cell-like cells with phosphate-buffered saline (PBS, Invitrogen, USA) in low temperature and fixed them for 20 minutes in 4% formaldehyde subsequently. Then, in 15 minutes, we used triton 100-X (Invitrogen, USA) for cells permeability. For blocking nonspecific proteins, we applied 1% bovine serum albumin in PBS for 45 minutes at room temperature.
Primary antibodies include DAZL (Santa Cruz, USA, 1:100) and DDX4 (Abcam, USA, 1:100). We added diluted antibodies to the cells and preserved them at 4ºC overnight. Then we washed the cells three times by PBS (10 min for each time). Then we added secondary antibody Anti-DAZL antibody Rabbit polyclonal IgG (Abcam, USA, 1:100) and secondary antibody Anti-DDX4 antibody Rabbit polyclonal IgG (Abcam, USA, 1:100) for 1 hour at room temperature. Then we washed out excess antibodies twice with PBS. For nucleus staining, we added two drops of DAPI to each box in dark and drew it out after 15 seconds. Again the cells were washed with PBS to be prepared for microscopic observation.
2.4. Real-time PCR
We applied real-time PCR to assess the expression of genes DAZL, Dppa3, DDX4, which were produced by retinoic acid induction. We used RNX Plus (Signage, Tehran, Iran) to derive RNAs from cells. In order to omit other DNAs, we applied DNAs Ι (Fermenta, Canada) in the residual culture flask. Afterward, we separated complementary DNA (cDNA) from extracted RNAs. This was done based on the protocol of BioRT cDNA First Strand Synthesis Kit. Then we applied the following procedure for real-time – PCR:
First, the Amplicon was transferred to a tube as a prepared master mix. The genes primer sequences which we applied are demonstrated in Table 1. The software we used was Eco real-time PCR software and performed as below:
at 95ºC, enzyme activation for 15 minutes, at 95ºC, denaturation for 10 seconds, at 56ºC annealing for 20 seconds, at 72ºC, synthesis for 25 seconds. Finally, we plated the specimens after 35 cyclings, in 2% agarose gel electrophoresis and SYBER staining was performed subsequently. Furthermore, we used HPRT as a housekeeping gene.
2.5. Fibrin gel preparation
We prepared fibrin gel by solving 1.5 mg/mL bovine fibrinogen (Sigma-Aldrich, USA) in M199 Media (Sigma-Aldrich, USA) and 15 mL thrombin (120 U/mL in 1M sodium buffer, Sigma-Aldrich, USA). Then we add the solution to a 24-well plate and left it for 15 minutes at room temperature. Subsequently, we put it at 37ºC in an incubator for 1 hour to form the construction of a 3D mesh network (Asmani et al. 2013). After this waiting time, we added 2×105 cells/mL of differentiated germ cell-like cells to 500mL of prepared fibrinogen solution (3 mg/mL) and mixed carefully. Following, we put the cell-fibrinogen solution into the 24-well plate and added 15 mL thrombin to the cell-fibrinogen solution and placed it in an incubator for 1–2 hours till gel formation.
2.6. Rheology features of fibrin hydrogel
Fibrin is a kind of viscoelastic biomaterial, a substance with viscous and elastic components. Its viscoelastic quality differs upon on clot biochemical and structural properties (Weisel 2004). Through the polymerization process, while the network is forming, finite clot stiffness is detected at first. We conducted Rheology tests by a remoter, Paar Physica MCR300 (Anton-, Ashland, VA, USA). We accomplished the test under a custom-made hood (n=3) in order to reduce dehydration of the mixtures during the test. The diameter of parallel plates was 25mm and the distance between was 0.05mm. The frequency sweeps which were conducted on the hydrogel samples in gel form was between 0.1 and 10 Hz. Before measurement, we adjusted the temperature of the plate to 37º C where the hydrogel was placed directly.
2.7. Cell viability test by MTT analyze
Viability of germ cell-like cells derived hEnSCs were analyzed in conventional 2D medium and 3D as well. Then they are compared by 3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) in 1, 3, and 5 days after cell seeding. This was done to analyze the effects of fibrin 3D scaffold on the differentiated cells. Each day, 4 hours was taken to incubate the cells in 600 ml solution of 5 mg/mL MTT. Finally, to dissolve formazan crystals sufficiently, 400 mL DMSO was added. Afterward, we did test the aliquots by an enzyme-labeled (BioTek, USA) prior to pipetting into a 96-well plate. Spectrometry with UV was measured the absorbance of each well at 570 nm.
2.8. Evaluation of cultured cells Morphology by scanning electron microscopy, SEM
We examined the cell morphology by SEM on the third day of culture. We washed the scaffolds with PBS and fixed them in a 2.5% glutaraldehyde solution at 48º C for 1 hour. Then we rinsed the fixed samples with PBS two times and made them dry in ethanol with gradient concentrations subsequently. Ethanol gradient was increased step by step; 30, 50, 70, 80, 90, 95, and 100% for the best result. Then we freeze-dried the sample in vacuum for 2 hours, coated the cells with gold spray and observed them by SEM (Hitachi S4160, Japan) at a voltage of 15 kV.
Results: The project was evaluated by using flow cytometry for detection CD146, CD90 as stem cell markers as in the isolated endometrial stem cells.
In the next step to investigate the ability of human endometrial adult stem cells to differentiate into the germ cell-like cells, the endometrial stem cells were induced by retinoic acid by the optimum concentration of 10-5 M after 7 days.
Then, characteristic cell markers such as Dazl, DDX4, and Dppa3 were determined by immunofluorescence and real-time PCR assays. After encapsulating the hEnSCs-derived germ cells in a fibrin gel, cell differentiation and viability were assessed by culturing for 7 days subsequently.
Structural and mechanical properties of the fibrin scaffold were examined by rheological analysis and the porosity was examined by SEM. Also, the viability of cells was analyzed using the MTT assay.
Conclusion: Our study showed the potency of hEnSCs to differentiate into germ cell-like cells in the adjusted concentration of RA, 10µM after 7 days of culture. Also, the study demonstrated the biocompatibility of the differentiated cells in fibrin hydrogel. Furthermore, differentiation of hEnSCs into germ cell-like cells was proved by exploring germ cell markers using immunofluorescence staining for DAZL and DDX4 and real-time PCR for DAZL, DDX4, and Dppa3. Also seeding the differentiated cells in fibrin scaffold, defined a constructive influence of fibrin as a 3D scaffold on differentiated cells.