Expansion and transduction of nonenriched human cord blood cells using HS-5 conditioned medium and FLT3-L

Cord blood (CB) stem cell transplantations have been associated with delaye d hematopoietic engraftment. This has most likely been due to the limited n umbers of hematopoietic short-term repopulating cells in CB. Ex vivo expans ion of CB has been attempted, and expansion of CD34-enriched CB has been su ccessful; however, CD34 enrichment procedures are in general associated wit h substantial cell loss. Thus, we have studied culture conditions for expan sion of nonenriched CB. Nonenriched CB cells were cultured for 21 days in t he presence of conditioned medium from the HS-5 stromal cell line and FLT3- L or alternatively in the presence of FLT3-L, stem cell factor (SCF), megak aryocite growth and development factor (MGDF), and granulocyte colony-stimu lating factor (G-CSF) (FSMG), either on fibronectin fragment CH-296-coated dishes or on uncoated dishes. With all four culture conditions, the number of mononuclear cells initially decreased until day 7 and then increased unt il the end of the expansion cultures. Overall expansion using HS-5 and FLT3 -L resulted in superior expansion of MNC and CFU-C (44-/34-fold) for both c ultures with and without CH-296 compared to FSMG (18-/17-fold). Expansion o n CH-296 was less efficient than expansion on tissue culture-treated wells without CH-296 for both conditions. We then studied the best time for trans duction on nonenriched CB. In contrast to enriched CD34 cells, we found for both conditions, HS-5/FLT3-L and growth factor cocktail, higher transducti on efficiencies when cells were transduced on day 7 as compared to day 2. G ene transfer rates up to 45% were achieved with both conditions, which corr esponded with the increased number of cells in S phase on day 7 compared to day 2. We conclude that HS-5 and FLT-3L allow efficient expansion and tran sduction of nonenriched CB.