eMSC used in our experiments were obtained from the MSC collection of the Department of Intracellular Signaling and Transport of the Institute of Cytology RAS, Russia. These cells were established from menstrual blood with endometrium fragments obtained from healthy woman at the age of 27 years [22]. The cells were plated in 6-cm Petri dishes (Corning, Corning, NY, USA) in DMEM/F12 medium with 10% fetal calf serum (FCS) (HyClone, Logan, UT, USA), 1% antibiotic–antimycotic mixture, and 1% glutamaxandand cultivated for 3–7 days. The cells were subcultured with EDTA/trypsin solution.

Human embryonic cell line (ESC) C910 [23] was obtained from the Department of Intracellular Signaling and Transport of the Institute of Cytology RAS, Russia. These cells was maintained in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) on Petri dishes covered with mitomycin-treated eMSC as feeder layer. The cells were subcultured mechanically.

Spheroid formation

Spheroids were formed from eMSC at the 4–6th passages using the hanging drop technique [18]. 5000–7000 cells per 35 μL were placed in drops on the cover of 96-well plates and inverted then over single wells. Cells spontaneously aggregated in hanging drops for 48 h were then transferred for 24 h in dishes coated with 2-hydroxyethyl methacrylate (HEMA; Sigma-Aldrich, St. Louis, MO, USA). Single cell suspension was obtained by spheroid treatment with 0.05% trypsin/EDTA and used to monitor spheroid cell properties.


Immunophenotyping (CD marker expression) of monolayer eMSC and eMSC spheroids was performed with an Epics XL flow cytometer (Beckman Coulter, Brea, CA, USA). The single cell suspension was obtained using 0.05% trypsin/EDTA. 1 × 106 cells were suspended in 1 mL of PBS with 5% FCS. FITC-conjugated antibodies to CD34, CD 44, CD45, CD90, CD 146, HLA-1, and phycoerythrin (PE)-conjugated antibodies to CD73, CD105, CD140b, and HLA DR were applied.

Adipogenic differentiation

2 × 10




were seeded in Petri dishes coated with 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA). When the cells reached about 80% confluence, 1 mM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM isobutyl-methyl-xanthine (IBMX; Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL human recombinant insulin (Sigma-Aldrich, St. Louis, MO, USA) and 100 mM indomethacin were added. In this medium, the cells were differentiated for 3–5 weeks with a half volume of the medium changed every 2–3 days. Lipid drops were visualized with Oil Red staining (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Adipogenic differentiation was also tested with RT-PCR. Primers for adipogenic differentiation are shown in the Table 



Table 1

Primer sequences for control and target genes and q-PCR conditions



93 °C, 20 s, 61 °C, 20 s 72 °C 30 s



Liu et al. 2016 [44]




93 °C, 20 s, 62 °C, 20 s 72 °C 30 s



Kunisch et al. 2009 [45]




93 °C, 20 s, 62 °C, 20 s 72 °C 30 s



Jankowski et al. 2003 [46]




93 °C, 20 s, 62 °C, 20 s 72 °C 30 s



Shafiee et al. 2011 [47]




93 °C, 20 s, 62 °C, 20 s 72 °C 30 s



Bahrambeigi et al. 2012 [48]




93 °C, 20 s, 59 °C, 20 s 72 °C 30 s



Ponnaiyan et al. 2014 [49]




93 °C, 20 s, 67 °C, 20 s 72 °C 30 s



Liang et al. 2015 [50]




93 °C, 30 s, 60 °C, 30 s 72 °C 30 s



Kozhukharova et al., 2009 [23]




93 °C, 30 s, 63 °C, 30 s 72 °C 30 s



Liedtke et al., 2007 [51]




93 °C, 30 s, 60 °C, 30 s 72 °C 30 s



Koshkin et al. 2016 [52]




93 °C, 30 s, 70 °C, 30 s 72 °C 30 s



Alekseenko et al., 2012 [53]


Osteogenic differentiation

2 × 104 cells/cm2 were seeded in Petri dishes coated with 0.1% gelatin. After the cells reached 100% confluence 100 nM dexamethasone, 10 mM β glycerol phosphate, and 0.2 mM ascorbate- 2- phosphate were added. In this medium, the cells were differentiated for 3–5 weeks with a half volume of the medium changed every 2–3 days. Then, the cells were fixed with 70% cold ethanol for 1 h and stained with Alizarin Red, pH 4.1 (Sigma-Aldrich, St. Louis, MO, USA). Osteogenic differentiation was also tested with RT-PCR. Primers for osteogenic differentiation are shown in the Table 1.

Decidual differentiation

eMSC were seeded in 24-well plates in the growth medium. After the cells reached 80% density, the medium was exchanged for serum-free medium for 24 h. The medium was then exchanged for the medium with 2% FCS and 1 mM 8-Br-cAMP (Sigma-Aldrich, St. Louis, MO, USA) and then was changed every 3 days. Control cells were cultivated in the same medium but without 8-Br-cAMP. After 7 days, the medium from control and differentiated cells was collected for prolactin and insulin-like growth factor binding protein-1 (IGFBP-1) testing. The assay was done with ELISA kits for prolactin and IGFBP-1 quantitative measurements (Sigma-Aldrich, St. Louis, MO, USA). The amount of prolactin and IGFBP-1 was normalized to the total cell protein determined with the Bradford method.

SA β-galactosidase staining

Cell senescence, was assayed with Senescence β-Galactosidase Staining Kit (Cell Signaling, Danvers, MA, USA). Cells were plated in 3.5-cm Petri dishes. 2–3 days after seeding, the medium was discarded and cells were washed with PBS, and fixed with formaldehyde/glutaraldehyde mixture at room temperature for 10–15 min. Fixed cells were thoroughly washed with PBS and incubated with staining solution at 37 °C without CO2 for 8 h. The pH value of staining solution should be kept at a level of 6.0. The staining pattern was visualized under the light microscope. Senescent cells were identified by the blue color in cytoplasm (SAβGal-positive).

RT-PCR and qRT-PCR assays

To analyze gene expression, total RNA was isolated with RNesy Micro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA was quantified in the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). cDNA was obtained by reverse transcription of RNA using the Revert Aid H Minus First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. It was subsequently amplified with specific primers, using DreamTaq™ PCR Master Mix (2x) (Thermo Fisher Scientific, Waltham, MA, USA) with CycloTemp amplificator. The electrophoresis of amplified products was performed in 2% agarose gel with TAE buffer and ethidium bromide. 100 kb DNA ladder (Fermentas, Vilnius, Lithuania) was used as molecular weight markers. Amplified products were visualized in UV light (302 nm) with Transilluminator and registered with a digital Canon camera (Canon, Tokyo, Japan). For qRT-PCR cDNA was amplified with specific primers, using EvaGreen® dye (Biotium, Fremont, CA, USA) and DreamTaq™ PCR Master Mix (2X) (Thermo Fisher Scientific, Waltham, MA, USA) in the Bio-Rad CFX-96 real time system (Bio-Rad, Hercules, CA, USA), according to the kit’s enclosed protocol. Expression of target genes was normalized to gapdh gene. Primers and reaction conditions are presented in the Table 1. All amplifications were performed in triplicates. Experiments were repeated at least three times.


All experiments were performed with Wistar rats, weight 200–250 g. The animals were maintained in the designated animal care facility with free access to tap water and food. All experimental procedures with animals were performed according to the Institutional Guidelines for the Care and Use of Laboratory Animals. All studies on animals were performed after approval by the Institutional Animal Care and Use Committee of Institute of Cytology RAS (Assurance Identification number F18–00380).

Harvesting of rat bone marrow

Rat bone marrow (BM) was flushed from the femurs and tibias of adult Wistar females with sterile PBS. The cell suspension was filtered through sterile 70-mM Nitex mesh (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and used as transplantation material.

Animal modeling of the Asherman’s syndrome

Adult albino Wistar rat females weighing 200–250 g were used in experiments. Vaginal cytology was performed to evaluate the stage of estrous cycle. A sterile swab was moistened with saline and rotated against the vaginal wall to obtain vaginal cells. Vaginal smears were visualized with the light microscope. Only animals in diestrus were used. Animals were anesthetized by intramuscular injection of Zoletil 100 (Virbac, Carros, France) in a dose 5 mg/kg weight; surgical manipulations were done under aseptic conditions. The animals were fixed in supine position, and the inferior abdomen was sterilized and shaved. An incision of approximately 2.5 cm was made into the inferior abdomen through the skin and underlying layers and uterus horns were pulled out.

0.3 ml of 95% ethanol were injected into both uterine horns and kept for 3 min. Uterine horns cavities were washed with 0.5 ml of PBS solution. Then, the uterus was put back into the abdominal cavity and the abdominal muscles and skin were sutured. About 100 female rats underwent the induction of modeled Asherman’s syndrome (AS). They were randomized into different groups, differing in transplantation material (rat BM, eMSC monolayer, eMSC spheroids) and delivery mode (vein or intrauterine injection). eMSC spheroids were transplanted only into the uterus. Intravenous administration entails the cell trapping in lungs with a high risk of embolism. Animals were subjected to cell therapy 72 h after the uterine injury. Each rat received 0.2 mL PBS (control) or 0.2 mL cell suspension in PBS. Cell suspension contained 107 cells for the vein injection and 106 cells for intrauterine injection. Vein injection was done via the tail vein. For intrauterine transplantation the animals were fixed in the dorsal position. Double sections of skin and muscles were done 1.5 cm laterally to the vertebrae. Uterus horns were pulled out very carefully to avoid any traumatization and intrauterine injections were performed. Three estrous cycles after AS induction the females were mated with Wistar males for a period of 3 months, and the number of pregnant rats and litter size were recorded. The control AS model rats were sacrificed before mating, and the uterine horns were taken for histological examination.


Frozen 10-μm sections of uterine horns were made. Slides were fixed in ethanol/methanol mixture for 2 min at −20 C° and stained with hematoxylin and eosin (H&E) or Trichrome. Structural alteration in uterus, fibrosis, and presence of inflammation and necrosis areas were assessed by the light microscopy.

Statistical analysis

The data are presented as the mean ± standard deviation (SD), when indicated. Difference in pregnancy rate was analyzed by Fisher’s exact test. The difference in the number of pups was tested by Kruskal-Wallis H-test (non-parametric ANOVA) followed by post hoc pairwise comparison using Mann-Whitney U test with Bonferroni correction when appropriate. All other data were treated using Student’s t test (two-tailed). Data were analyzed with IBM SPSS Statistics v.22 software (IBM Corp., Armonk, NY, USA). Differences were considered significant at p < 0.05.