Hyaluronic Acid Enhances To Differentiate into Myelo-Megakaryocytic Lineages during Ex-Vivo Expansion of Human Cord Blood(HCB) Using TPO, SCF and FL.

Hyaluronic acid(HA) is the major glycosaminoglycan component of extracellular matrices. CD44 is a ligand for HA and expresses on a wide variety of cell types. CD44 has been implicated in many cell-cell and cell-matrix interactions. In the present study, we studied the effects of HA on proliferation, differentiation and apoptosis of CD34+ cells during HCB ex vivo expansion using TPO, SCF and FL. CD34+ cells from cord blood MNC were cultured for 4 weeks in the combination of TPO, SCF and FL on HA-coated well and CD34+cells were cultured in identical conditions except HA-coated well. At day 4, 7, 14, 21, 18, we counted the number of expanded cells and analyzed the cell surface markers (CD3, CD14, CD19, CD34, CD44, CD61, CD66b) and apoptosis using 7-AAD staining. To analyze the effects of HA on CD34+ cell differentiation, anti-CD44 antibodies (clone A3D8 and 515) were used. The morphology on the expanded cells was examined using wright’s staining. In results, cell counts were increased in HA-coated well compared with the control group until D7, but proliferation rate was decreased after D7. Apoptosis portion of CD34+ using 7-AAD staining was increased in HA-coated well and % of CD34+ cells was decreased in HA-coated wells compared with that of the control groups. The expanded cells in HA-coated well were negative for CD3 (pan T cell), CD19 (B cell) but CD14 (monocyte), CD61 (megakaryocyte) and CD66b (neutrophil) were highly expressed than those of control groups. Apoptotic portion was also increased in the HA-coated group. CD66b (neutrophil) expression in HA-coated well was significantly higher than those of control groups at D14 and above findings were confirmed by wright’s stain. CD66 expression was reduced by the anti-CD44 antibody clone, A3D8 and however CD66 expression was increased by the clone, 515. Therefore, it might be possible that A3D8 and clone 515 recognize each different epitopes of CD44. In conclusion, CD34+ cell differentiation from HCB into CD66, CD14 and CD61 positive cells was enhanced by the contact of HA during ex vivo expansion using TPO, SCF and FL. This result suggests that the expanded human cord blood graft with HA might be useful as the “post-progenitor” to shorten the severe cytopenia period after myeloablative conditioning for HCB transplantation.