Ex Vivo Culture and Amifostine Effects in Human Cord Blood.

Background

Human cord blood(CB) is the third most promising source of hematopoietic stem cells to the bone marrow and peripheral blood for replacing depressed bone marrow function in high dose chemotherapy in patient with cancer. The possibility of cord blood transplantation in adults was limited by the amount of cord blood that could be collected. Cord blood transplantation after ex vivo expansion with cytokines have already been tried in adults. Amifostine is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastic agents.

Objectives

The purposes of this study were to investigate expansion of progenitor and myeloid cells after ex vivo culture of mononuclear cells(MNCs) in umbilical cord blood with growth factor and characterize hematopoietic activities of amifostine.

Methods

MNCs were cultured and ex vivo expanded into myeloid progenitors by using hematopoietic growth factors(IL-1β, IL-3, IL-6, G-CSF, GM-CSF, SCF, EPO) which are known to stimulate differentiation and proliferation of myeloid progenitors. MNCs exposed to the appropriate amount of amifostine for 15 min were cultured in semisolid media and harvested at 24h intervals, and then apoptosis was assessed by flow cytometry.

Results

Myeloid colonies were successfully produced from MNCs. Maximal expansion was obtained with the combination of IL-3+SCF+G-CSF+GM-CSF. SCF was thought to be the most important growth factor for expansion of myeloid progenitor. Pretreatment with amifostine for 15 min stimulated formation of hematopoietic colonies at clinically relevant concentrations ranging from 1 to 100μM. Increase in colony number compare to control were comparable after pretreatment with amifostine (10μM), and CFU-GEMM and BFU-E were highly responsive. Further enhancement of colony was not observed after prolonging the duration of pre-incubation exposure to 1, 8 and 24 hours. Amifostine enhanced IL-1 and IL-3 induced formation of CFU-GEMM and BFU-E. Incubation of MNCs with amifostine in suspension culture increased recovery of secondary colonies. Treatment with amifostine retarded cell loss and apoptosis, and promoted cell survival at 24, 48 and 72 hours in cytokine-deficient medium.

Conclusions

In conclusion, cord blood MNCs can be successfully expanded into myeloid progenitors by using hematopoietic growth factors. This investigation extend the previously recognized hematologic effects of amifostine, and indicate that in addition to its cytoprotective properties, amifostine is a potent stimulant of hematopoietic progenitor growth.