The effect of Kisspeptin-10 on mesenchymal stem cells migration in vitro and in vivo

Abstract

Background: Kisspeptins (kp) activate a receptor coupled to a Gαq subunit (GPR54 or KiSS-1R) receptor to perform a variety of functions, including inhibition of cell motility, chemotaxis, and metastasis. In this study we have investigated whether kp-10, the most potent member of the kisspeptin family, can modulate CXCR4 (C-X-C chemokine receptor type 4) expression and mesenchymal stem cells (MSCs) migration that may influence the development of tumors.

Materials and methods: We compared the directional migration of MSCs treated with 10-100 or 500 nM kp-10 for 24 hours and no treated cells using an in vitro transmembrane migration assay. In addition, Chloromethylbenzamido Dialkylacarbocyanine (CM-Dil) labeled adipose-derived mesenchymal stem cells treated with 10-100 or 500 nM kp-10 and no treated cells were transfused via the tail vein to the melanoma tumor bearing C57BL/6 mice. After 24 hours, the mice were scarified, the tumors were dissected, and the tumor cell suspensions were analyzed by flow cytometry for detection of CM-Dil(+) MSCs.

Results: We have found that kp-10 increased the MSCs migration at 100 nM, while it decreased the MSCs migration at 500 nM, both in vitro and in vivo, with a significant increase of CXCR4 expression at 100 nM kp-10 compared to the no treated cells, but it had no significant difference between the various concentrations of kp-10.

Conclusion: Thus, our data showed that kp-10 can differently affect MSCs migration in various concentrations, probably through different effects on CXCR4 expression in various concentrations.

Keywords: CXCR4; Kisspeptin-10; mesenchymal stem cell; migration.

Figure 1

MSC Migration Assay. (A and B) Treated MSCs with 10-100 or 500 nM kp-10 for 24 hours and the no-treated cells were seeded into the top chamber of the Transwell (8 μm) in serum-free medium and 30% FCS or melanoma cells was added to the bottom camber. After incubation for 24 hours, at 37°C, the number of migrated cells across the membrane was counted by flow cytometry. All the results are indicative of three experiments. (c) Number of CM-Dil⁺ MSCs, 24 hours after injection, was measured in cell suspension of tumor by flow cytometry (n = 6). kp-10 significantly decreased migration of MSCs toward 30% FCS at the highest concentration compared to all groups and toward melanoma cells and tumor compared to 10 and 100 nM kp-10, but migration was increased at lower concentrations, especially at 100 nM kp-10, compared to other groups in all experiments. Results were compared between groups by the Kruskal-Wallis test (* P < 0.05)

Figure 2

Expression of CXCR4 in MSCs. Dot Plot shows the distribution pattern of sample cells, FL2 shows CXCR4⁺ cells. For each samples, 5 × 10⁴ cells were analyzed by flow cytometry. After 24 hours, the MSCs were treated with 10-100 or 500 nM kp-10 and the no-treated cells diluted in PBS and incubated with anti-mouse CXCR4 PE (dilution 1:200) or Rat IgG isotype control antibody for 45 minutes, in the dark, at room temperature. The fluorescence intensity was determined using flow cytometry. The expression of CXCR4 was increased at 100 nM compared to the no-treated cells, but it had no significant difference between various concentrations. The results are indicative of three experiments and are compared between groups with the Kruskal-Wallis test (*P < 0.05)