Ex vivo expansion of umbilical cord blood CD34+ subset hematopoietic stem cells on the covalently immobilized glycosaminoglycan/chitosan membrane surfaces

Stem cell transplantation (SCT) using umbilical cord blood (UCB) cell has been limited because of the low cell number from a single cord blood unit. Many groups are doing their researches through various methods to overcome the limitations. Glycosaminoglycans (GAGs) play a significant role in the regulation of hematopoiesis by the binding and activating of growth factors. However, it was difficult to evaluate whether proliferation of hematopoietic stem cell (HSC) was generated by soluble GAG detached from immobilized surface or by temporarily sustained ionically bound GAGs. Therefore, we evaluated biological effects of GAGs to cell cultures by forming ionically or covalently immobilized GAG onto chitosan surfaces and compared them with regard to GAG density and release kinetics. From the surface immobilization study we confirmed that a well-defined and evenly distributed membrane surfaces were constituted by both covalent and ionic binding of GAGs onto chitosan substrates when sufficient amount of GAGs were added. GAG desorption kinetic results indicated that surfaces with covalently immobilized membrane had quite constant desorption rate compared with ionic membrane surfaces. CD34+, CD34+38 -, or CD34+38+ cells isolated from cord blood were seeded onto ionically or covalently immobilized membrane surfaces in the IMDM medium with two growth factor combinations, IS (IL-3 and SCF) and STF (SCF, TPO, and FL) for 3 weeks. The total, CD34+ and CD34+38- populations were determined by flow cytometry. Cell cycle status was conducted by using 5'-bromo-2'-deoxyuridine (BrdU) with 7-amino-actinomycin D (7-AAD). CFU assays were evaluated for clonogenic efficiency of isolated or cultivated stem cells. Ex vivo expansion of CD34 +38- cells on surfaces with covalently immobilized GAGs showed better anti-apoptotic property, cell cycle progression to "S" phase, and expansion of immature cell population than on surfaces with ionically immobilized GAG. The results demonstrated that CD34+38 - cells expanded onto surfaces with covalently immobilized GAG for rapid proliferation of immature progenitor cells reduced growth factor amount, lowered apoptosis rate, and accelerated cell cycle status. CD44 antigen expression was a function of CD34+ cell proliferation and augmented proliferation caused by HA was blocked by anti-CD44 antibody to the surface CD44 ligand on progenitor cell populations. Therefore, the specific interaction between CD44 antigen and HA stimulated enhanced production of HSCs ex vivo.