Increased expansion and differentiation of cord blood products using a two-step expansion culture

[corrected] The use of allogeneic cord blood (CB) products as a source of cellular support for patients receiving high-dose chemotherapy has been limited primarily to smaller children due to the low numbers of cells in a CB unit. Ex vivo expansion of CB cells has been proposed as a method to increase the number of cells available for transplantation. Following high-dose chemotherapy administration, we transplanted adult patients with CB expanded in static culture for 10 days, in DM containing stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF). Patients achieved neutrophil engraftment in a median of 26 days (range 15 to 45). In an attempt to hasten the time to neutrophil engraftment, we developed a two-step culture system that results in increased expansion of total nucleated cells and further maturation of neutrophil precursors.

CD34(+) cells isolated from CB products were cultured for 7 days at 37 degrees C in 100-mL Teflon culture bags containing 50 mL of DM containing SCF, G-CSF, and MGDF (100 ng/mL). The cells were harvested from these bags after 7 days of incubation at 37 degrees C and transferred to 1-L Teflon bags containing 1 L of DM plus SCF, G-CSF, and MGDF. After a second culture period of 7 days, the cells were harvested, washed, and assayed for mature (granulocyte-macrophage colony-forming cells [GM-CFC]) and primitive progenitor cells (high proliferative potential colony-forming cells [HPP-CFC]).

The two-step cultures resulted in a median total nucleated cell expansion of 438-fold (range 286 to 952, N = 11); the original one-step cultures resulted in a median expansion of 98-fold (range 59 to 350, N = 5). Equivalent expansion of committed progenitor cells (GM-CFC) and primitive progenitor cells (HPP-CFC) was obtained. CD34(+) cells were expanded a median of 29-fold in the two-step cultures (N = 11). The two-step culture contained more mature neutrophil cells, by morphologic examination, than the one-step cultures, similar to ex vivo expanded peripheral blood progenitor cells (PBPC).

The two-step ex vivo expansion conditions described for CB resulted in increased numbers of total nucleated cells, GM-CFC, HPP-CFC, and CD34(+) cells and morphologically resembled ex vivo expanded PBPC, which have been shown to provide more rapid neutrophil engraftment than unexpanded PBPC. We propose that the availability of increased numbers of expanded CB cells may result in more rapid engraftment of neutrophils following infusion to transplant recipients.