Ex Vivo Expansion of Cryopreserved Cord Blood Cells

Kim, Samyong; Kim, Chul Hee; Bae, Gwang Bong; Kim, Hyun Soo; Park, Sang Jun; Kim, Jong Suk; Yun, Hwan Jung; Jo, Deog Yeon

Abstract

BACKGROUND: Cord blood (CB), which has no HLA restriction, is an alternative to bone marrow for hematopoietic stem cell transplantation. The use of cord blood, however, is limited by the number of progenitor/stem cells necessary to reconstitute the older child or adult. Therefore, ex vivo expansion of CB could have tremendous impact on diverse clinical settings. We studied the ex vivo expansion of isolated population of CD34+ cells from cryopreserved CB cells.
METHODS
CD34+ cells were isolated from cryopreserved CB mononuclear cells. Purified cells were cultured with various combinations of hematopoietic growth factors including erythropoietin (EPO), stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), granulocyte, macrophage-colony-stimulating factor (GM-CSF), interleukin-1beta (IL-1beta), IL-3, and IL-6. After 7, 10 or 14 days of culture, the fold increases of colony-forming unit- granulocyte, macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), colony-forming unit-mix (CFU-Mix), and high proliferative potential colony-forming cell (HPP-CFC) were evaluated.
RESULTS
Ten-day culture with the combination of EPO, SCF, G-CSF, IL-1beta, and IL-3 resulted in a median of 60-fold increase of CFU-GM, which was greater than those with the combinations of less than 5 growth factors. The addition of IL-6 or GM-CSF to this combination did not enhance CFU-GM expansion. Ten-day culture was significantly superior to 7-day culture for CFU-GM expansion. Prolongation of culture to 14 days, however, revealed decreased expansion of CFU-GM compared to 10 days. BFU-E and CFU-Mix were expanded to 2~5 folds in 7-day culture with the combination of EPO, SCF, and G-CSF. Further expansion was not achieved in 10-day culture and colonies disappeared in 14-day culture. HPP-CFC was expanded to a median of 7.5 folds in 7-day culture with the combination of EPO, SCF, G-CSF, IL-1beta, IL-3, and IL-6. Neither 10-day or 14 day-culture enhanced expansion of HPP-CFU.
CONCLUSION
Cryopreserved cord blood cells maintain ex vivo expansion potential. In our system, 10-day culture with the combination consisting of EPO, SCF, G-CSF, IL-1beta, and IL-3 seems to be adequate for hematopoietic progenitor/stem cell expansion from cryopreserved cord blood cells.