Research On Microencapsulated Osteoblasts Support Cord Blood Hematopoietic Stem/Progenitor Cells Expansion

Human cord blood (CB) is rich in invaluable hematopoietic stem/progenitor cells (HSPC), having significant clinical application value. However, there is merely 50-100ml blood in single portion of CB, so it is just avalible to children with their weight less than 30kg. This drawback limits the efficacy of cord blood in transplantation. But researchers found that, HSPC would differentiated seriously in ex vivo expansion since large dose of cytokines were added, the HSPC would lose their stemness, and therefore transplantation effect is unsatisfied.Osteoblasts, as the essential component of the born marrow hematopoietic stem cell niche, can support HSPC expanding, but the mechanism of the action is still uncertain. Furthermore, the current culture modes, no matter direct contact culture or transwell culture, are all constraint by limited adherent area that stroma cell need to survive.In order to raise HSPC's expansion fold and investigate the mechanism of which osteoblasts support HSPC expansion, osteoblasts were embeded into gelatin-alginate-chitosan (GAC) microbeads in this thesis, co-culturing with HSPC in serum free, supplemented with relative low doses of purified recombinant human cytokines IMDM culture meida. The expansion of HSPC was evaluated by counting the total nucleated cell number, colony-forming assay and CD34~+ flow cytometric analysis. Meanwhile the pH value, glucose and lactic acid concentrations were detected everyday. Three key culture parameters' effects in supporting and maintaining HSPC were tested, including partial pressure of oxygen, diameter of microbeads, and ratio of cells. Then the concentrations of IL-6 and LIF that human osteoblasts secrete were measured and compared with the result of HSPC's expansion to determine their relationship.The results show that the condition of 5% oxygen partial pressure, 0.5mm bead diameter, and 2:1 ratio between OB and HSPC, is the optimum and there are 49.0±4.6-fold in hematopoietic total nucleated cells expansion, 87.6±8.3-fold in CD34~+ cells expansion, and 9.8±0.8-fold in CFU-Cs expansion. The research on cytokines shows that hematopoietic cell expansion is concerned with the concentration of IL-6; CD34~+ cell expansion and the sustaining of hematopoietic stemness are related with the concertration of LIF. These results indicate that the main support method of osteoblasts is by secreting cytokines.The utilizition of osteoblasts microbeads and hypoxic incubator to construct a novel ex vivo culture system can successfully simulate the hematopoietic environment in vivo. This system can support cord blood hemapotoietic stem/projenitor cells' expansion efficentlly. The mechanism through which osteoblasts support hemapotoietic cells expansion is also revealed. This work provides experimental base for further research on both elevating hemapotoietic cells' expansion and thoroughly explorating the interactive relationship between these two kinds of cells.