Akt regulates mobilization of MSCs by influencing polymerization dynamics of actin cytoskeleton, leading to actin polymerization and subsequently MSCs migration [43–44], we also determined the effect of DA on actin polymerization dynamics of MSCs. Our results indicated that DA mediated inhibition of MSC mobiliza- tion was associated with the inhibition of actin polymerization in these cells, and treatment with specific DA D2 receptor antagonist abrogated these effects. This further confirmed that the inhibitory effect of DA on MSC migration was specifically mediated through its D2 receptors. In summary, activation of DA D2 receptors in MSCs resulted in the inhibition of VEGF induced phosphoryla- tion of Akt, which in turn suppressed actin polymerization and subsequent migration of these stem cells. Recent studies by other groups have also shown that activation of the Akt increases the migratory activity of human MSCs, whereas treatment with PI3K inhibitor blocks activation of Akt and thereby, significantly inhibits migration of these cells [44]. Our results also demonstrated that like human MSCs, Akt also played a critical role in regulating migration of murine MSCs. This information generated from our present study indicate for the first time that physiological concentration of DA can negatively regulate mobilization of MSCs into wound sites and consequently influence angiogenesis in wound tissues. This novel finding is of clinical significance as DA D2 receptor specific antagonists being presently used for the treatment of other disorders may be utilized for accelerating mobilization of MSCs to expedite angiogenesis and hence tissue regeneration. Materials and Methods Experimental wound model and treatment All animal experiments were performed after approval by the Institutional Animal Care and Use Committees. The experiments were carried out in full thickness dermal wound bearing normal Swiss mice (4–6 weeks and weighing 22–25 g). The animals were anesthetized with intraperitoneal (i.p.) injection of 100 ml solution containing ketamine and xylazine mixture (2.215 and 0.175 mg, respectively; Sigma). The dorsal hair of the mouse was shaved and disinfected with an alcohol (70% ethanol) swab and two excisional wounds were created at the same cranial-caudal level on the dorso-medial back of each animal using an 8 mm dermal punch biopsy. At the end of the surgical procedure, cages were placed on a heating pad until mice fully recovered from anesthesia [25]. Following creation of wounds, the wound bearing mice were divided into two groups. Immediately after wounding, mice of the treatment group were injected i.p. with 10 mg/kg of eticlopride, specific DA D2 receptor antagonist (Sigma) in 300`ıl normal saline and continued for four consecutive days at an interval of 24 hours. The control group received similar volume of normal saline only [25]. The experiments were also repeated with another specific DA D2 receptor antagonist domperidone (Sigma). Isolation, purification and expansion of MSCs from mouse bone marrow MSCs were separated from bone marrow of normal Swiss mice by using MACS cell sorting system of Miltenyi Biotec (Germany). Bone marrow cells were collected by flushing the femurs and tibias from healthy female mice (6 weeks old). Cells obtained from bone marrow were incubated with a cocktail of biotin-conjugated monoclonal antibodies to remove the hema- topoetic cells and then separated by anti-biotin microbeads- conjugated secondary antibody. The Linneg fraction (containing Dopamine and Mesenchymal Stem Cells pure Mesenchymal lineage cells) was cultured in murine mesenchymal stem cell expansion medium (Millipore, USA). After 48 hours, the non-adherent cells were removed and fresh medium was added to the cells. Medium was changed every 2 or 3 days. The adherent spindle-shaped cells were further propagated for three passages [27]. Osteogenic and adipogenic differentiation of in vitro expanded MSCs Osteogenic differentiation was induced by culturing confluent mouse MSCs in a-minimum essential medium (a-MEM) supple- mented with 10% FCS, 10 mM b-glycerophosphate, 50 mg/ml ascorbic acid 2-phosphate and 10 nM dexamethasone for 14–28 days. The media was changed every 3–4 days. Osteogenic dif- ferentiation of murine MSCs was confirmed by staining min- eralized deposits in the extracellular matrix with von Kossa staining which stains calcium deposited in ECM [29]. Adipogenic differentiation was induced by culturing confluent mouse MSCs in a-minimum essential medium (a-MEM) sup- plemented with 10% FCS, 0.5 mM IBMX (3-isobutyl-1-methyl- xanthine), 10 mg/ml insulin and 1 mM dexamethasone (all from Sigma) for 21 days. The media was changed in every 3–4 days. Adipogenic differentiation of murine MSCs was confirmed by Oil-red O staining which stains lipid droplets within adipocytes [29]. Flow cytometry Antibodies used for flow cytometry analysis were anti-CD45 (FITC-conjugated conjugated; eBioscience), anti-CD34 (APC- conjugated; eBioscience), anti-CD105 (Phycoerythrin-conjugated; eBioscience), anti-SSEA-4 (Phycoerythrin-conjugated; R & D Systems), mouse anti-D2 DA receptor antibody (Santa Cruz), rat anti-mouse IgG-FITC (eBioscience) and anti-VEGFR-2 antibody (FITC-conjugated; BD biosciences). In order to determine the numbers of circulating MSCs in peripheral blood after injury, blood was collected from both control and DA D2 receptor antagonist treated wound bearing mice at different time intervals (3, 6, 12, 24, 36 and 48 hours after injury) by nicking the tip of the tail. Each time 400 ml blood was taken and mixed with anti- coagulant to prevent blood clotting. Then the collected blood samples were treated with RBC lysis buffer to remove RBCs and then incubated with different antibodies to evaluate the number of MSCs in circulation. Cells were initially gated to exclude dead cells and debris, and then the CD342 CD452 cell population was selected and evaluated for the presence of CD105, a surface marker of MSCs. According to the International Society for Cellular Therapy (ISCT), MSCs are positive for CD105 and negative for CD34 and CD45 [26]. Thus, the in vitro expanded spindle shaped Linneg cells (containing pure mesenchymal lineage cells) were examined for the presence or absence of specific surface markers to confirm their identity. In addition, the presence of DA D2 receptors and VEGFR-2 receptors on their surfaces were also determined in these cells by flow cytometry. Analyses were considered informative when adequate numbers of events (after acquisition of 10,000 cells per sample) were collected in the gated cells. Percentage of positive cells was finally determined after comparing them with matched isotype controls [25]. Detection of DA D2 receptors in MSCs by western blot In vitro expanded MSCs were lysed and the protein extracts from these cells were subjected to SDS-PAGE and then blotted onto PVDF membranes (Millipore). Mouse anti-D2 DA receptor IgG PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31682