TY - JOUR
T1 - Ex vivo expansion of megakaryocyte precursors from umbilical cord blood CD34 cells in a closed liquid culture system.
AU - Shaw, Peter H.
AU - Gilligan, Devin
AU - Wang, Xue Mei
AU - Thall, Peter F.
AU - Corey, Seth J.
N1 - Funding Information:
This work was supported by a Children's Oncology Group Chair's Development Award (National Childhood Cancer Foundation, Washington, DC) and a Research Advisory Committee Grant (Children's Hospital of Pittsburgh, Pittsburgh, PA).
PY - 2003/3
Y1 - 2003/3
N2 - Umbilical cord blood (UCB) provides a rich source of stem cells for transplantation after myeloablative therapy. One major disadvantage of UCB transplantation is delayed platelet engraftment. We propose to hasten platelet engraftment by expanding the number of megakaryocyte (MK) precursors (CD34/CD41 cells) through cytokine stimulation within a closed, pre-clinical liquid culture system. Clinical engraftment data suggest a 5- to 10-fold increase in MK precursors in a UCB unit can accelerate platelet engraftment, so this was our goal. Thirteen UCB samples from full-term births were Ficoll-separated and frozen for subsequent use. On thawing, the mononuclear cell population was positively selected for CD34(+) expression. The cells were cultured in gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: recombinant human interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin. MK lineage cell expansion was assessed using mononuclear cell count and flow cytometry (CD34/41, CD41, CD34/61, and CD61 expression) on days 7, 11, and 14. Optimal expansion of CD34/41 and CD41 cells was observed at day 11, with a median 6-fold and 33-fold increase in the starting cell doses, respectively. CD34/61 and CD61 cell expansion at day 11 was 7-fold and 14-fold, respectively. MK precursors can be successfully expanded from CD34(+) UCB cells in a closed liquid culture system using interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin to a level that should have a clinical impact in the transplantation setting. Our ex vivo expansion technique needs to be further optimized before it can be used in a pilot UCB transplantation trial.
AB - Umbilical cord blood (UCB) provides a rich source of stem cells for transplantation after myeloablative therapy. One major disadvantage of UCB transplantation is delayed platelet engraftment. We propose to hasten platelet engraftment by expanding the number of megakaryocyte (MK) precursors (CD34/CD41 cells) through cytokine stimulation within a closed, pre-clinical liquid culture system. Clinical engraftment data suggest a 5- to 10-fold increase in MK precursors in a UCB unit can accelerate platelet engraftment, so this was our goal. Thirteen UCB samples from full-term births were Ficoll-separated and frozen for subsequent use. On thawing, the mononuclear cell population was positively selected for CD34(+) expression. The cells were cultured in gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: recombinant human interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin. MK lineage cell expansion was assessed using mononuclear cell count and flow cytometry (CD34/41, CD41, CD34/61, and CD61 expression) on days 7, 11, and 14. Optimal expansion of CD34/41 and CD41 cells was observed at day 11, with a median 6-fold and 33-fold increase in the starting cell doses, respectively. CD34/61 and CD61 cell expansion at day 11 was 7-fold and 14-fold, respectively. MK precursors can be successfully expanded from CD34(+) UCB cells in a closed liquid culture system using interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin to a level that should have a clinical impact in the transplantation setting. Our ex vivo expansion technique needs to be further optimized before it can be used in a pilot UCB transplantation trial.
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U2 - 10.1016/S1083-8791(03)70003-2
DO - 10.1016/S1083-8791(03)70003-2
M3 - Article
C2 - 12652464
AN - SCOPUS:0642339113
SN - 1083-8791
VL - 9
SP - 151
EP - 156
JO - Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
JF - Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
IS - 3
ER -