Effects Of HMGB1 On Human Cord Blood CD34~+ Hematopoietic Progenitor Cells Homing, Proliferation And Differentiation And Their Mechanisms

Background and objective High mobility group box 1(HMGB1)is an abundant chromatin-binding protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells,can play a role in tissue regeneration. Presently, we build over this hypothesis. The fore treatment of haematopoietic stem cell transplantation(HSCT)will lead the bone marrow tissue damage, and release of HMGB1,which is expected to promote the migration of HSCs to the damaged tissue, and to play a role in reconstruction of hematopoiesis , but little is known about the mechanisms regulating these processes. Here, we elected to study both the release of HMGB1 from irradiation -treated mesenchymal stem cells (MSCs) and the effects of HMGB1 on human cord blood CD34+ hematopoietic progenitor cells homing,proliferation and differentiation and their mechanisms, in order to find new target in reconstruction of hematopoiesis.Methods MSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12Gy X-Gy irradiated was determined using a enzymelinked immunosorbent assay (ELISA). CD34+ cells were positively selected with a MACS CD34 isolation kit. Subsequently, freshly isolated CD34+ cells were cultured in the presence of HMGB1 for 6 days. Phenotype of cultured cells surface molecules (CD13,CD14,CD11c,CD41,CD71) were analyzed by flow cytometry. Also, the proliferation and differentiation ability of cord blood HSCs were characterized by colony forming cell assay. In addition, the receptors of HMGB1(RAGE,TLR2,TLR4) on cord blood CD34+ cells were detected by flow cytometry. The effects of HMGB1 on HSCs homing were evaluated by chemotaxis assay using Boyden chambers. To analyze the underlying mechanism of HMGB1-induced HSCs migration, we engaged inhibitory antibodies against its receptors, RAGE, TLR2, and TLR4. Results 1. HMGB1 is released from MSC treated with X-Gy irradiated. Level of HMGB1in the supernatant(4.3±0.9)ng/ml for MSC treated with 12Gy X-Gy irradiated was significantly higher when compared to control group(0.4±0.2)(p<0.01). 2. Expression of HMGB1 receptors in human cord blood CD34+ cells. Human cord blood CD34+ cells expressed the HMGB1 receptors, RAGE (43.1%±7.2%), TLR2(36.1%±6.6%) and TLR4 (23.1%±5.2%) . 3. HMGB1 induces the proliferation and differentiation of human cord blood CD34+ cells in vitro. The HMGB1-treated CD34+ cells possessed the higher proportion of CD13(32.6±5.9% vs 18.4±3.8%),CD14(25.4±4.4% vs 12.6±2.7%),CD11c(20.3±3.9% vs 9.8±2.1%),CD71(47.1±7.4% vs 26.6±4.6%) compared to control group. But, HMGB1 did not induce the generation of CD41+ cells (1.3±0.5% vs 1.1±0.4%).Colony forming cell assay indicate the growth of BFU-E,CFU-GM and total CFU upregulated. 4. HMGB1 stimulate migration of cord blood CD34+ cells HMGB1 stimulated migration of CD34+ cells in a concentration-dependent manner, when the concentration was 100ng/ml, the strong chemotaxis was shown(p<0.01). A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of CD34+, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated CD34+ migration.Conclusion Treatment of human MSC with X-Gy irradiation can cause release of HMGB1. Human cord blood CD34+ progenitor cells constitutively express functional HMGB1 receptors, RAGE, TLR2 and TLR4. HMGB1 can induce the differentiation of human cord CD34+ cells along the granulo-monocytic and erythroid system. A certain concentration of HMGB1 was able to induce the chemotaxis in vitro of CD34+ cells, and the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE.