normal dermal wounds, and this healing in turn is associated with increased angiogenesis in wound tissues [25]. Mobilization of MSCs into wound bed and their subsequent active participation in wound tissue neovascularization are critical steps towards successful wound healing [6–8,10,13,17–19]. Therefore, in the present investigation to explore the regulatory role of DA D2 receptors, if any, on mobilization of MSCs into wound site, the status of circulating MSCs in peripheral blood of both control and eticlopride treated back skin-injured mice had been compared at different time intervals by flow cytometry to determine the effect of inhibitory action of DA D2 receptors on the profile of circulat- ing MSCs. The results showed that treatment with DA D2 receptor antagonist eticlopride significantly increased the numbers of circulating MSCs (immunophenotypically CD342 CD452 CD105+ cells) [26] in peripheral blood of wound bearing mice in comparison to vehicle treated controls at different time intervals (3, 6, 12, 24, 36 and 48 hours after wounding) (Fig. 1A and 1B). In both control and treated animals, the number of circulating MSCs showed a sharp increase that reached peak at 6 hour after creation of wounds (Fig. 1A). However, this increase in the numbers of circulating MSCs were significantly higher in DA D2 receptor antagonist treated mice in comparison to vehicle treated controls at all time points (Fig. 1B). Similar results were also observed following treatment with another DA D2 receptor specific anta- gonist domperidone (results not shown). It is to be noted here that treatment with other DA receptor antagonists (D1, D3, D4 and D5) had no significant effects on the mobilization of MSCs (results not shown). This data confirmed that the action of DA was specific and was mediated through its D2 receptors present in MSCs. CD342 CD452 CD105+ Cells showed osteogenic and adipogenic differentiation potential in vitro Mesenchymal stem cells are multipotent in nature and have the unique ability to differentiate into various cell types among which osteogenic and adipogenic differentiation are considered as hallmarks of these adult stem cells [26]. Therefore, to confirm the identity of CD342 CD452 CD105+ cells as MSCs, their osteogenic and adipogenic differentiation potential were evaluated. Using lineage cell depletion kit (Miltenyi Biotec, Germany), mesenchymal lineage cells were isolated from total bone marrow cells by depletion of cells of haematopoietic lineage, cultured in MSC media and expanded in vitro for three passages [27]. Flow cytometry demonstrated that over 95% of the total cells were phenotypically CD342 CD452 CD105+ in nature, characteristics of MSCs (Fig. 1C). These CD342 CD452 CD105+ cells also expressed SSEA-4, a stage-specific embryonic antigen that identifies adult mesenchymal stem cell population from the bone marrow [28]. These cells were then cultured in appropriate induction media to induce osteocytes and adipocytes [29]. In the initial osteogenic culture, no mineralized cell was detected. However, after five weeks, mineralized cells positive for von Kossa staining showing calcium deposition were observed (Fig. 1D) thus indicating osteogenic differentiation of these cells. Similarly one week after adipogenic induction, formation of intracellular lipid vacuoles were detected in these cells. Thereafter, lipid accumulation increased along with the inductive periods, which was chemically stained by Oil Red O, a specific staining, which identifies lipid droplets in adipocytes, thereby, confirming adipocytic nature of these cells (Fig. 1E). Murine MSCs express DA D2 receptors To confirm the role of DA D2 receptors in the migration of MSCs into wound tissue, it was necessary to know whether these cells expressed DA D2 receptors. Therefore, the presence of DA D2 receptor was investigated by western blot and flow cytometry. Western blot analysis of in vitro expanded murine MSCs showed Dopamine and Mesenchymal Stem Cells presence of DA D2 receptors in these cells, which was further confirmed by flow cytometry analysis (Fig. 2A and 2B). Flow cytometry of CD452 CD342 CD105+ SSEA-4+ cells confirmed that almost 86% cells of the total MSC population expressed DA D2 receptors on their surfaces (Fig. 2A). Treatment with DA D2 receptor antagonist significantly accelerated mobilization of exogenously transplanted MSCs towards wound bed As it was evident that treatment with DA D2 receptor antagonist increased the number of circulating MSCs in wound bearing mice (Fig. 1A and 1B), therefore experiment was undertaken to determine mobilization of these circulating MSCs cells into the wound bed, the site where these cells are required to migrate in order to mediate wound angiogenesis and tissue repair. To investigate the effect of DA D2 receptor antagonist on the homing of MSCs into wound site, bone marrow derived and expanded MSCs (CD342 CD452 CD105+ cells) were labeled with BrdU in culture and were then injected i.v. via the tail veins into both control and eticlopride treated back skin-injured mice on day 1 after wounding. These cells were injected 2 hrs after DA D2 receptor antagonist treatment. The transplanted MSCs in the wound bed were identified by BrdU immunoreactivity. Our study revealed significantly higher number of BrdU labeled MSC transplants in DA D2 receptor antagonist treated wound tissues than vehicle treated controls on day 6 after creation of wound and completion of 4 day treatment schedule (10 mg/kg/day) of eticlopride (Fig. 2C and 2D). Increased integration of fluorophore-labeled (CM-Dil) transplanted MSCs into blood vessels in wound bed following DA D2 receptor antagonist treatment Our previous results had shown that treatment with DA D2 receptor antagonist eticlopride significantly increased the wound healing process by stimulating neovascularization [25]. Interest- ingly, our present results also revealed that this treatment was associated with increased number of migrated MSCs into wound sites (Fig. 2C and 2D). Therefore, we investigated whether this increased number of mobilized MSCs in wound tissue following DA D2 receptor antagonist treatment was also associated with increased incorporation of these cells into the neovessels of heal- ing wounds. Co-localization studies showed that on day 6 after wounding and completion of 4 day of eticlopride treatment schedule, exogenously transplanted MSCs, labeled with fluores- cent dye CM-Dil (red in colour), had incorporated into the newly formed blood vessels in wound site (CD31 positive cells, green in colour) in considerably higher numbers than vehicle treated controls (Fig. 3A). Furthermore our results also demonstrated that treatment with eticlopride in combination with transplanted MSCs resulted in significantly more angiogenesis in wound tissues in comparisons to the vehicle treated controls or animals treated with eticlopride or MSCs alone (Fig. 3B and 3C). DA through its D2 receptors regulate VEGF induced migration of MSCs in vitro In vivo results from the present investigation had shown that exogenously transplanted BrdU labeled MSCs migrated to the wound bed in significantly higher numbers in DA D2 receptor antagonist eticlopride treated wound bearing mice than vehicle treated controls (Fig. 2C). It was also evident from co-localization study that greater numbers of these cells had integrated into the neovessels of the wound bed (Fig. 3A). PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31682