Adenosine-5'-triphosphate suppresses proliferation and migration capacity of human endometrial stem cells.

Abstract Extracellular ATP through the activation of the P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many types of cells, including stem cells. High plasticity, low immunogenicity and immunomodulation ability of mesenchymal stem cells derived from human endometrium (eMSCs) allow them to be considered a prominent tool for regenerative medicine. Here, we examined the role of ATP in the proliferation and migration of human eMSCs. Using a wound healing assay, we showed that ATP‐induced activation of purinergic receptors suppressed the migration ability of eMSCs. We found the expression of one of the ATP receptors, the P2X7 receptor in eMSCs. In spite of this, cell activation with specific P2X7 receptor agonist, BzATP did not significantly affect the cell migration. The allosteric P2X7 receptor inhibitor, AZ10606120 also did not prevent ATP‐induced inhibition of cell migration, confirming that inhibition occurs without P2X7 receptor involvement. Flow cytometry analysis showed that high concentrations of ATP did not have a cytotoxic effect on eMSCs. At the same time, ATP induced the cell cycle arrest, suppressed the proliferative and migration capacity of eMSCs and therefore could affect the regenerative potential of these cells.


| INTRODUC TI ON
Mesenchymal stem cells (MSCs) are derived from a wide range of adult tissues, including adipose tissue, cartilage, tendons, tooth pulp and periodontal ligament. By now the most common sources are bone marrow 1 and adipose tissue. 2 In recent years, a significant number of publications have appeared on the isolation of mesenchymal cells from the human endometrium. The important role of stem cells in the cyclic regeneration of the endometrium was discovered many years ago. [3][4][5] However, the population of endometrial stem cells (eMSCs) was first isolated from endometrial tissue samples and characterized only in 2004. 6,7 Later, researchers isolated and characterized stem cells from menstrual blood samples. 8 has been shown that eMSCs can be differentiated into cells of the tissues of the mesodermal (myocytes, cardiomyocytes, osteocytes, adipocytes, etc) and ectodermal (neurons) rows. 11 Together with the high availability of eMSCs, their key properties (high proliferative activity, genome stability, high level of plasticity) make these cells a very valuable substrate of cell therapy for many diseases.
Adenosine-5′-triphosphate (ATP) is an important multifunctional nucleotide serving as intracellular energy source. However, once outside the cell, it acts as signalling molecule, binding to purinergic receptors P2X or P2Y on the cell membrane. 12 It is known that nucleotides are released from damaged cells in the pathogenesis of different diseases. In addition, ATP is secreted by exocytosis from many cells, including endothelial cells, red blood cells, platelets or autonomic nerves, and acts as a neurotransmitter. [13][14][15] By binding to ATP, purinergic receptors trigger a chain of signalling events that affect the vital functions of many types of cells and tissues. 16 Recent investigations have shown that extracellular ATP regulates the migrating capacity of healthy 17,18 and cancer cells, [19][20][21][22] suggesting the involvement of purine receptors in cancer invasion or metastasis. Moreover, ATP-induced regulation of migration was found in stem cells and progenitor cells. [23][24][25][26] The capacity of stem cells to migrate to the specific tissues or organs is essential not only for normal tissue homeostasis, morphogenesis and repair, but also for creation of stem cell-based regenerative medicines. 23,24,27,28 This investigation was designed to explore the impact of extracellular ATP on migration and proliferation capacity of endometrial stem cells. Our results show that ATP decreases the migration capacity of eMSCs. In addition, our results demonstrate that the cultivation of cells in the presence of ATP potently suppresses cells growth by inducing accumulation of eMSCs in G0/G1 phase.

| Cell cultures
Experiments have been performed on mesenchymal stem cell line (eMSCs) derived from the human endometrium. Line (2804) was obtained from Department of Intracellular Signaling and Transport of Institute of Cytology RAS (Russia). 29 The experiments were approved by the Bioethics Committee of the Institute of Cytology of Russian Academy of Sciences and the principles of the Declaration of Helsinki. Cells exhibited properties typical for MSCs: they were adherent to plastic surface, displayed fibroblast-like morphology, they were multipotent, showed expression of MSC-positive surface markers CD13, CD29, CD44, CD73, CD90 and CD105 and were negative for the haematopoietic markers CD19, CD34, CD45, CD117, CD130 and HLADR (class II). Multipotency of eMSCs was tested by their ability to differentiate in other cell types, such as adipocytes and osteocytes. 29 Additionally, the isolated eMSCs partly (over 50%) expressed the pluripotency marker SSEA-4. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM/F12) with 10% foetal bovine serum (HyClone), 1% L-glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). The cells were cultured in culture flasks at 37°C in a humidified chamber with 5% CO 2 and subcultured twice per week.

| The cell migration assay
The experimental system for long-term live cell monitoring was installed on Zeiss AxioObserver Z1 microscope equipped with ×10 objective, PlasDIC contrast and humidified microscope chamber with temperature and 5% CO 2 control. For wound healing assay, eMSCs were seeded in 4 well plates, with pre-installed silicone inserts, in the amount of 60 000 cells per insert. The cells were cultured for further 24 hours, then inserts were taken out, the cells were washed and cultured in full culture media containing the reagents. The wound images were taken every 1h for 48h using 'Multidimensional acquisition' module of Axiovision 4.8.2 software. The absolute value of the distance the cells passed was calculated as the change in the perpendicular distance between the edges of the gap for 48 hours. The value was then normalized to the 0 hour of starting measurement.
Areas were calculated using ImageJ software (NIH). The percentage of area reduction or wound closure was calculated as follows: wound closure % = [(A t=0h -A t=∆h )/A t=0h ] × 100%, where A t=0h is the area of the wound measured immediately after drug addition, while A t=∆h is the area of the wound measured h (hours) after adding the drug. 30

| Calcium imaging
One day prior to the experiments, cells were seeded in 3-cm 2 Petri dishes containing a cover slides. After 24-48 hours, the medium was removed, cells were washed with serum-free medium and
After staining, coverslips were placed with Vectashield mounting medium (Vector Laboratories) and examined using the confocal microscope Olympus FV3000 (Olympus Corporation) with ×60 oil objective.

| Statistical analysis
The data are presented as the mean values of at least three independent experiments. Statistical significance was evaluated by Student's t test, and one-way ANOVA with Tukey's post hoc tests for multiple comparisons, P < .05 were considered to be significant.
Data are presented as the mean ± standard deviation (SD).

| The effect of ATP on migration of human endometrial stem cells (eMSCs)
Now, there is growing evidence that purinergic signalling triggered by ATP regulates the migration or homing of stem cells to the specific tissues or injuries. Therefore, first of all, the wound healing assay together with time-lapse imaging was used to establish the role of ATP in eMSCs migration. In order to avoid a possible uncontrolled release of ATP and other undesirable effects caused by cell damage, the wound in eMSCs culture was created by removal of a silicone insert as described in Material and Methods.

| Expression of P2X 7 receptor in eMSC
Numerous studies show that ATP-induced alterations in stem cell migration could arise from the activation of purinergic P2X 7 receptors. 20,31,32 Therefore, next, we investigated the expression of the P2X 7 R in endometrial stem cells. PCR primers were designed as indicated in Material and Methods. RT-PCR analysis revealed the 108-bp product corresponding to P2X 7 R transcript in eMSCs (Figure 2A). P2X 7 R protein expression was determined using Western blot analysis and polyclonal anti-P2X 7 R antibodies. Figure 2B shows the band ~70 kD, corresponding to the P2X 7 R.
Immunofluorescence confocal microscopy was used to examine the protein expression and intracellular distribution of P2X 7 R. The immunofluorescence signal was detected in the plasma membrane and in the cytoplasm of the cells labelled with the antibody recognizing the P2X 7 R ( Figure 2C).

| Ca 2+ response in stem cells activated by purinergic stimulation
Extracellular ATP operates as an autocrine and/or paracrine sig-

Cells loaded with dye Fura-2AM (as described in Material and
Methods) were set into the registration chamber with the flow system and analysed under a Zeiss Axiovert S100 epifluorescence inverted microscope. Images of the cells were taken every 2 seconds for 10 to 20 minutes with a Zeiss AxioCam Hrc camera. As shown in Figure 3A

| Role of P2X 7 receptors in eMSC migration
To assess whether P2X 7 R affects the eMSCs migration, the cell movement was monitored for 48 hours in the presence of receptor agonist, BzATP (0.1 mmol/L) ( Figure 4A,B). In addition, experiments were conducted using the selective P2X 7

| ATP affects the cell cycle in eMSCs
Next, we assumed that ATP-mediated inhibition of cell migration might occur because of changes in the proliferation rate of eMSC In general, the data show that ATP, applied to eMSCs does not affect the cell survival but causes a cell cycle delay at G1 phase and a decrease in cell growth rate.

| D ISCUSS I ON
Stem cells exist in all organs, providing the replacement of damaged and sick tissues. Damaged tissues and organs release numerous compounds, including intracellular ATP, which are triggers many cellular processes. ATP can be emitted by exocytosis, by traumatic cell lysis, or by passive leakage from damaged cells. 33 In healthy tissues, the extracellular ATP concentration is adjusted by ecto-nucleotidases, such as ecto-ATP/ADPase (CD39). 34 In damaged tissues, the impaired activity of CD39 and the release of ATP can result in accumulation of extracellular ATP. 35 Extracellular ATP through the activation of P2X and P2Y purinergic receptors affects the migration, proliferation and differentiation of many cell types, including stem cells. According to numerous reports, ATP can affect the proliferation and viability of some cell types, including stem cells. 27,[36][37][38] Here, we show that ATP in a dose-related manner suppressed the eMSCs proliferation, while the viability of cells was not altered throughout the period of their cultivation with ATP. Our data are consistent with the results reported for human bone marrow mesenchymal stem cells, 37 human endometrial stromal cells 39 and also neonatal rat cardiac fibroblasts, 40 which show that ATP regulates MSCs proliferation activity and probably is one of the factors determining the cell fate.
Many reproductive dysfunctions are connected with disturbed endometrial proliferation. The eMSCs are necessary for endometrial recovery during each menstrual cycle. Accordingly, decrease in the number of cells or disturbance of their functions can lead to the formation of thin endometrium that is unable to maintain gestation. 41 The potential of endometrial MSCs offers a broad perspective in the treatment of such diseases. Besides the eMSCs may play an important role in the pathophysiology of such diseases as human adenomyosis or endometriosis that are cause of infertility. 42,43 In conclusion, our results indicate that ATP suppresses the proliferation of eMSCs that accompanied by cells arrest in G1 phase of cell F I G U R E 5 ATP treatment affects the eMSC proliferation (FASC analysis). A, Cell cycle distribution of eMSC cells after 24 and 48 h of cultivation in the presence and in the absence of different concentrations of ATP. ATP treatment delays G0/G1-S transition. The data are presented as mean ± SD (n = 3), *P < .05. B, Dose-dependent effect of ATP on cells growth. The number of PI-negative ('live') cells is significantly reduced after the 48-h incubation with ATP (see 'Materials and Methods'). Growth curves of control (1, filled circles) and treated with 0.1, 1.0 and 5.0 mmol/L ATP eMSCs (2, filled rhombus; 3, square and 4, triangles, respectively). The data are presented as mean ± SD (n = 3), *P < .05. C, Representative histograms of eMSC distribution after 5 mmol/L ATP treatment; viability assay with PI staining. The percentage of dead cells after 24 (upper panel) and 48 h (lower panel) of cells incubation with ATP cycle. We believe that ATP-induced suppression in eMSC proliferation led to decrease in the migration capacity of these cells; however, the exact mechanism by which ATP acts on eMSCs needs further investigation. We are convinced that monitoring of processes associated with ATP-dependent regulation of cell migration and proliferation will allow to significantly improve the regenerative potential of eMSCs for use in tissue engineering and regenerative medicine.

ACK N OWLED G EM ENTS
This research was supported by grant of the Russian Science Foundation (RSF No 18-15-00106).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the results of this investigation are accessible from the corresponding author upon request.