A Murine Stromal Cell Line Promotes the Expansion of CD34^hlgh+-Primitive Progenitor Cells Isolated from Human Umbilical Cord Blood in Combination with Human Cytokines

Abstract

The in vitro expansion of CD34⁻ cells is important for clinical applications such as transplantation and gene therapy with CD34⁺ cells isolated from human umbilical cord blood. In the present study, we developed a xenogenic coculture system involving HUCB-CD34⁺ cells and a murine stromal cell line, HESS-5 cells, in the presence of recombinant human (rh) cytokines. We examined the effects of combinations of cytokines, such as rh-IL-3, rh-SCF, rh-granulocyte colony-stimulating factor (G-CSF), rh-granulocyte-macrophage-CSF and h-erythropoietin (EPO), on the expansion of CD34^hlgh- cells and colony-forming progenitor cells (CFCs). The proliferation of CD34^high+ cells and CFCs was dramatically promoted on coculture with HESS-5 cells, and the expansion ratio of the CD34^hlgh+ cells showed good correlation with that of high-proliferative potential colony-forming cells (HPP-CFCs). The most potent combination of cytokines in this xenogenic coculture system for the expansion of CD34^high+ cells and HPP-CFCs was rh-IL-3 and rh-SCF. The proliferation of CD34^high+ cells was supported in the presence of HESS-5 cells with direct cell contact, but not observed in the indirect coculture involving a microporous membrane. Furthermore, we developed a unique coculture method, designated as the bilayer coculture method, involving CD34⁺ cells and HESS-5 cells using a microporous membrane. This expansion system will be applicable to the expansion of the primitive progenitor cells of HUCB-CD34⁻ cells and is worthy of consideration for the clinical application of HUCB-CD34⁻ cells.

Key Words:

• In vitro expansion
• transplantation
• CD34′ cells
• hematopoietic primitive progenitor cells
• hematopoiesis