Animals and ethics
Thirty male adult Balb/C mice (30±5 g) were provided from Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, and two female eGFP+/+Balb/C mice were gifted from Royan Institute and were housed in the laboratory under the controlled condition of a temperature of 22±2°C, the humidity of 55±5% and lightening cycle of 12 h light/dark. They were fed ad libitum standard diet. All the manipulations were performed in accordance with the regulations of working with laboratory animals and approved by the Ethical Committee of Astana Medical University, Astana, Kazakhstan (Protocol No. 10).
Isolation and culture of BM-MSC and AT-MSC
BM-MSCs were isolated from femurs of two eGFP+/+Balb/C mice by modification of the previously reported method (24). In sterile condition and after soaking the separated thigh of mice in 70% ethanol, the skin has been removed. After cutting the two ends of the thigh bone, the thigh with the muscles was put in a blue-color sampler tip which its upper half parts had been cut. The tips were placed in 1.5 mL micro-tubes. Then all micro-tubes were centrifuged at 1500 ×g for 15 min at 37°C. The collected BM at the end of micro-tubes were mixed with 1 mL of the Dulbecco’s modified eagle medium (DMEM) without fetal bovine serum (FBS, Gibco, U.S.A.) and penicillin-streptomycin antibiotics (Gibco, U.S.A.). Then, the BM suspension was transferred to a 25 cm2 flask containing DMEM medium along with 10% FBS and 1% penicillin-streptomycin. The flask was incubated at 37°C, standard humidity, and 5% CO2 concentration. The culture medium was changed 24 h after the beginning of incubation, then every 72 h under sterile conditions, to remove unattached cells and debris. After increasing the density of the cells adhering to the flask to 80%, the cells were passaged using trypsin enzyme. For this purpose, the trypsin enzyme was added to the flask for 3 min to separate the cells sticking to the flask floor. Then, 2 mL of 10% FBS culture medium was poured in to the flask for neutralizing the trypsin enzyme effect. Collecting cells separated from the medium in the first passage was performed and continued till the third passage.
AT-MSCs were provided using ovarian AT of the same mice used in the previous step. Briefly, AT was minced into tiny parts. The AT parts were explanted in a T75 flask. After 15 min of pasting the explants by drying the attachment surface of the explant and flask, they were gently covered with a drop of FBS. The explanted AT were incubated for 48 h in an incubator containing 5% CO2 at 37°C and saturated humidity. Then DMEM with 10% FBS and 1% penicillin and 1% streptomycin were gently added. By daily monitoring of the border of attachment surface of AT explants and flask, 5 days after explanting and observation of AT-MSCs, the explants were removed by tapping the bottom of flask and media replacement. Then, DMEM containing 10% FBS, 2 mM of L-glutamine (Invitrogen, Netherlands), 1% penicillin and 1% streptomycin were added and kept in a CO2 incubator. The sub-culturing of cells was continued till the third passage.
Flow cytometry characterization of MSCs
To confirm the isolation of BM-MSCs and AT-MSCs and the non-appearance of hematopoietic stem cells, flow cytometry analysis was used. In this regard, the non-appearance of hematopoietic stem cell-specific surface marker expression (CD34 and CD45) and the existence of specific surface area marker expression on BM-MSCs and AT-MSCs (CD44 and CD105) in the isolated cells based on the method previously described were considered (27). Briefly, 5×105 cells of the third passage were incubated with specific individual monoclonal antibodies, conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in 250 mL phosphate-buffered saline (PBS) for half an hour at ambient temperature in the dark. CD34, CD45, CD44, and CD105 primary antibodies were used and cells were diluted using 4 mL PBS. The diluted cells were centrifuged, and re-suspended in 600 mL solution containing PBS–formaldehyde 2%. Then, they were studied using a four-color FACS Calibur flow cytometer (BD bioscience), and obtained data were analyzed by FlowJo software (FlowJo LLC, Ashland, OR, USA). Immunoglobulin G (IgG)1 FITC and IgG1 PE monoclonal antibodies were applied as isotype controls.
Osteogenic, chondrogenic, and adipogenic characterization of MSCs
In order to determine the potential of osteogenic, chondrogenic, and adipogenic differentiation of AT-MSC and BM-MSC, the cells of third passage were used. MSCs were in 6-well plates. After fusion, 70% of the cells were grown in an osteogenic, chondrogenic, or adipogenic environment for 3 weeks. For osteogenic differentiation, the MSCs at 90% confluence were exposed to an osteogenic differentiation kit (Stem Cell Technology, Iran). Changing the medium was performed two times per week for 3 weeks. After 21 days, osteogenic differentiation was confirmed by Alizarin red staining (Stem Cell Technology, Iran). For chondrogenic induction, MSCs at the confluence of 90% were exposed to a chondrogenic differentiation kit (Stem Cell Technology, Iran). Induction continued 3 weeks and chondrogenic induction was confirmed by Alcian blue staining (Stem Cell Technology, Iran). For adipogenic induction, MSCs at the confluence of 90% were exposed to adipogenic differentiation kit (Stem Cell Technology, Iran). Induction continued 3 weeks and induction of adipogenic was confirmed by Oil Red O staining (Stem Cell Technology, Iran). MSCs without differentiation media were stained simultaneously with three protocols as control.
Preparation of BMCM
In order to obtain the BMCM, BM-MSCs at the third passage were cultured at a density of 106 cells in a T75 flask. The BM-MSCs at 80% confluence were rinsed three times with PBS and 10 mL of FBS-free DMEM media were replaced. Collection of the media was performed following 48 h incubation and they were filtrated using a 0.2-µm filter to eliminate cellular debris. The BMCM were stored at -80°C until injection or isolation and confirmation of EVs. EVs were evaluated by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).
Isolation of EVs of BMCM by kit
The CM was collected and centrifuged in 2000 ×g and 4°C for 30 min. The supernatant was harvested and then added to precipitant reagent of Exovista kit (PerciaVista Co., Iran) with 1:1 ratio. The mixture was kept at 4°C for 14 h. To continue, the mixture was centrifuged in 10000 ×g and 4°C for 1 h. The EVs’ pellet was observed at the bottom of the falcon and collected for further analysis.
SEM imaging of EVs of BMCM
SEM imaging was performed to show EVs in the isolated BMCM (28). The isolated EVs were fixed with 2% formaldehyde in PBS. Then, 10 𝜇L drop of the sample was placed on poly-l-lysine coated slides and was allowed to air dry for 30-45 min. The sample was washed with phosphate buffered saline (PBS). Then, the sample post-fixed with 2.5% glutaraldehyde for 30 min. After that, the sample was washed another time for 10 min. Then the sample was stained with 1% osmium tetroxide for 20 min. The sample was washed again, immediately. At the next step, the sample was dehydrated with accelerated percentage of alcohol (25, 50, 70, 90, 100, 100, and 100% alcohol). Then the sample was placed into desiccator for drying. The sample was coated with layer of gold. The grid was examined on a FESEM TESCAN MIRA3 machine (TESCAN Co., Czech Republic) operating at resolution of 1.2 nm at voltage of 30 kV.
TEM imaging of EVs of BMCM
TEM imaging was performed to confirm the presence of EVs in BMCM. The procedure was done based on the previous study (29). In details, 6 𝜇L drop of the fixed sample was placed on a formvar Carbon film coated on 200 mesh copper grid (EMS) for 10 min. Excess liquid was absorbed with filter paper. Then, the grid was washed quickly with 100 𝜇L MilliQ water and then blotted to remove excess liquid. At the final stage, the grid was placed on 30 𝜇L of 1.5% uranyl acetate (w/v) for 12 s. The grid was examined by PHILIPS CM120 transmission electron microscope (Netherlands) operating at an accelerating voltage of 100 kV.
Induction of azoospermia
In order to model the experimental groups, the male adult Balb/C mice were randomly divided into six equal groups (Figure 1A).
An intact control group (n = 6): the mice did not receive busulfan and treatment.
Azoospermia group (n = 6): the left testicles of mice received busulfan injections without treatment and were sampled 60 days following the last busulfan injection.
BMCM therapy group (n = 6): the right testicles of mice received busulfan injections and then treated with BMCM and were sampled 60 days following the last busulfan injection.
BM-MSC therapy group (n = 6): the right testicles of mice received busulfan injections and then treated with BM -MSC and were sampled 60 days following the last busulfan injection.
AT-MSC therapy group (n = 6): the right testicles of mice received busulfan injections and then treated with AT-MSC and were sampled 60 days following the last busulfan injection.
Spontaneous healing group (n = 6): the mice received busulfan injections and were left without any treatment and were sampled 120 days following the last busulfan injection.
Then, azoospermia was induced in mice of all groups except those of the intact control group. To induce azoospermia in mice, busulfan was used according to previous reports (24). Briefly, the azoospermia groups were received two doses of busulfan (10 mg/kg, Busilvex®; Pierre Fabre Medicament Boulogne, France) with 21 days’ interval. Thirty-five days following the second injection, their testes were removed and fixed in 10% formalin buffer solution for histomorphometry studies. The epididymis was collected from all groups and after incubating in PBS, spermatozoa were evaluated by flow cytometry. The testes of control group mice, which were not received busulfan, were assessed through similar procedures.
BM-MSCs and AT-MSCs transplantation
Preparing an injecting set for a fine cell injection has been previously described (30). The mice in cell and CM therapies’ groups (n=18, AT-MSC therapy, BM-MSC therapy, and BMCM therapy groups) were surgically allotransplanted (31). Cell suspensions or CM were mixed with vital stain, sterile trypan blue. The mice were anesthetized with xylazine and ketamine (Alfasan, Netherland) 35 days following the second injection of busulfan. After preparation of abdominal area in dorsal recumbence position, a 1 cm midline abdominal incision was made to reach the peritoneum and testicle. The fat pad and then right testicle were carefully pulled out using iris forceps under a stereomicroscope (Model SZN, Optika, Italy). Simultaneously, a thin sterile plastic black card with a 30° V-neck as a holder was placed under the testicle. A polyethylene tube attached to a one-milliliter syringe, was filled with the cell suspension (106 cells in 100 µL). The tip of the pipette was carefully inserted into the efferent duct, and slowly threaded a few millimeters toward the testes (Figure 1B). The blue suspension was injected as slowly as possible to avoid moving the pipette and the entrance of the blue suspension into the seminiferous tubules was observed (Figure 1C). Finally, the testes were returned back into the abdomen and the abdominal wall was closed. The untreated testicles on the left side were considered as azoospermia groups.
Histological and histomorphometric assessments
Sixty days after treatment, the animals’ testes were removed after euthanizing with ether. Next, they were put in paraffin for histological and histomorphometric assessments after fixing in 10% formalin buffer solution and dehydrating using alcohol. For each testis, five horizontal cross-sections with a thickness of 5 µm were made from appropriate regions and stained with hematoxylin-eosin for histological assessment. Then, they were studied carefully by means of a light microscope (Model CX21, Olympus, Tokyo, Japan) to evaluate the presence of spermatogonia, spermatocytes, and spermatids in all tubules. Five circular cross-sections were provided from different areas of the tubules and the inner, outer, and total diameters of all the tubules were determined using the Dinocapture software (version 2.0, Dino-Eye, San-Chung, Taiwan). Using the mean of two diameters (D1, D2) at right angles, the average diameter of the seminiferous tubules (D) was specified. Using diameter data, cellular (germinal epithelium), luminal, and cross-sectional areas were determined. In the seminiferous tubules, the cross-sectional area (A) was obtained by the equation A = πD2/4, where D is the average diameter of the tubules, and π is 3.142. The number of seminiferous tubule profiles per unit area was also considered. The area of cells was computed through subtraction of the luminal area from the cross-sectional area. In the seminiferous tubules, determination of the numerical density (Nv) was performed using the following equation:
NV = NA/D + T
where NA indicates the number of profiles per unit area, D displays the average diameter of the seminiferous tubules, T shows the average section thickness, and indicates the number of tubules per unit volume.
The spermatogenesis index including the presence of spermatogenic cells throughout the testicular tissue, affecting the number of cell layers, cell types, and the existence of spermatids in the tubules was studied and the spermatogenic potential of testes was rated based on the modified scale of 0 to 7 (31) as following: 0, no spermatogonia; 1, the existence of only spermatogonia; 2, the presence of spermatocytes; 3, the presence of spermatids up to 25 in each tubule; 4, the presence of 25-50 spermatids per tubule; 5, the appearance of 50-75 spermatids per tubule; 6, the appearance of 75-100 spermatids per tubule; and 7, more than100 spermatids per tubule.
Flow cytometry of enhanced green fluorescent protein expression (eGFP)
Flow cytometry was conducted to prove the presence of eGFP spermatozoa. For this goal, epididymis was collected from all groups and was incubated in 1 mL PBS at 37°C for 15 min. Then, they were re-suspended in a total volume of 1.8 mL, after adding 800 µL of 10% formalin buffer solution, and then stored on ice until analysis with a flow cytometry under illumination in the range of the 360–400 nm that is related to eGFP fluorescence (32). A four-color FACS Calibur flow cytometer (BD bioscience) was used to collect the data and data were analyzed using the CellQuest Pro software package (BD bioscience).
Imaging of eGFP spermatozoa
The imaging of eGFP spermatozoa from the epididymis of treated mice was conducted using an epi-fluorescent microscope (XDS 3FL4, Optika, Italy) to prove the green fluorescent nature of spermatozoa produced in cell therapy groups. For this purpose, a red filter (594 nm) and a green filter (498 nm) were used.
Kolmogorov-Smirnov test was conducted to study the normal distribution of data of histomorphometry indices of seminiferous tubules. All data were presented as means and standard error (mean ± SE) and analyzed using one-way ANOVA and Tukey post-hoc test (SPSS for Windows, version 20, SPSS Inc, Chicago, Illinois, USA). The Mann-Whitney U test was used to assess the spermatogenesis index. A P-value of less than 0.05 was considered statistically significant.