Objective: Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types. cells. The expression levels of the granulocyte subset-specific genes in the HL-60 cells were assayed by real-time polymerase chain reaction. Results: Our results revealed that BM-MSCs support the granulocytic differentiation of the human promyelocytic leukemia cell line HL-60. Conclusion: Based on the results Dynorphin A (1-13) Acetate of this study, we concluded that BM-MSCs may be an effective resource in reducing or even preventing ATRAs side effects and may promote differentiation for short medication periods. Though BM-MSCs are effective resources, more complementary studies are necessary to improve this differentiation mechanism in clinical cases. gene was used as an internal control (Table 1). Table 1 Primers for real-time polymerase chain reaction. Open in a separate window Statistical Analysis Data were reported as mean standard deviation and were analyzed using Graph Pad Prism v 5.00 (Graph Pad Software, Inc., La Jolla, CA, USA). Students t-test for single comparisons and two-way ANOVA for multigroup comparisons were used for analysis and p 0.01 was regarded as denoting statistical significance. Results Flow Cytometry Confirmation of the Nature of the BM-MSCs To WIN 55,212-2 mesylate biological activity verify the mesenchymal nature of the BM-MSCs, the surface antigens were assessed by flow cytometry, including CD14, CD19, CD34, CD45 CD90, CD105, and CD73. The characterization experiments performed in our study demonstrated that the BM-MSCs were negative in the expression of the hematopoietic markers for CD14, CD19, CD34, and CD45, and WIN 55,212-2 mesylate biological activity they had positive expression for CD90, CD105, and CD73 markers (Figure 1). Open in a separate window Figure 1 Flow cytometry analysis confirmed the mesenchymal nature of the bone marrow mesenchymal stem cells. The markers assessed by flow cytometry included CD14, CD19, CD34, CD45 CD90, CD105, and CD73. The experiments were done in triplicate. Morphological Changes of the Treated Cells To assess the morphological changes in the treated HL-60 cells, Wright-Giemsa staining was performed (Figures 2A-2D). The comparative study of the morphological changes in the HL-60 cells stained by Wright-Giemsa indicated that, in comparison to the control, the cells treated with ATRA and BM-MSCs individually had induced granulocytic differentiation of the HL-60 cells (Figures 2B and ?and2C)2C) and showed an additive effect when used with BM-MSCs in combination with ATRA (Figure 2D). While the control cells (Figure 2A) demonstrated typical morphology in the promyelocytic cells (a circular nucleus), the treated HL-60 cells exhibited a kidney-shaped nucleus and segmented nucleus and also had a reduced nuclear/cytoplasmic ratio. Open in a separate window Figure 2 BM-MSCs induced the granulocytic differentiation of HL-60 cells after 48 h of incubation and showed an additive effect with all-trans-retinoic acid (ATRA). The differentiation of the HL-60 cells was assessed by Wright-Giemsa staining: a) untreated HL-60 cells, b) HL-60 cells treated with ATRA, c) HL-60 cells treated with bone marrow mesenchymal stem cells, d) HL-60 cells treated with ATRA and BM-MSCs. Magnitude: 100x. CD11b WIN 55,212-2 mesylate biological activity Expression Increased in Treated HL-60 Cells In the treated HL-60 cells, an increase was observed in the percentage of CD11b marker expression, one of the main granulocytic differentiation markers measured by flow cytometry, after 48 h. Flow cytometry results displayed that the expression of the CD11b marker was 17.12%, 76.69%, 23.96%, and 96.4% in the untreated HL-60 cells, in the HL-60 cells treated with ATRA, in the HL-60 cells treated with BM-MSCs, and in the HL-60 cells treated with a combination of BM-MSCs and ATRA, respectively (Figure 3). The expression of CD11b significantly increased in the HL-60 cells treated with the combination of BM-MSCs and ATRA compared to the HL-60 cells treated with ATRA alone or with BM-MSCs alone. Open in a separate window Figure 3 The flow cytometric analysis of CD11b, a granulocytic differentiation marker, after 48 h: a) untreated HL-60 cells, b) HL-60 cells treated with BM-MSCs, c) HL-60 cells co-cultured with all-trans-retinoic acid (ATRA), d) HL-60 cells treated with BM-MSCs and ATRA. BM-MSCs and ATRA synergistically upregulated CD11b expression in cells treated with the combination of the two. The experiments were done in triplicate. Effects of BM-MSCs and ATRA on Gene Expression in HLA-60 Cells In the ATRA-treated HL-60 cells, there was a marked increase (p 0.05) in the gene expressions of CD11b, lysozyme, WIN 55,212-2 mesylate biological activity GCSFR, CD64, PU.1, and C/EBP-ALPHA from 1.00 to 8.33 (0.07), 5.53 (0.16), 3.36 (0.12), 1.94 (0.02), 1.26 (0.04), and 1.11 (0.02), respectively. There was no gene expression for C/EBP-BETA, C/EBP E, or CD16 (Figure 4). On the other hand, as revealed in Figure 4, in the HL-60 cells co-cultured with the BM-MSCs, there was significant increase (p 0.05) in CD11b, lysozyme, PU.1, CD64,.