Cellular copper content modulates differentiation and self‐renewal in cultures of cord blood‐derived CD34⁺ cells

Abstract

Summary. Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood-derived CD34 + cells were cultured in liquid medium supplemented with Kitligand, FLt3, interleukin 6 (IL-6), thrombopoietin and IL-3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/10 7 cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/10 7 cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/10 7 cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony-forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34 + cells in low Cu was extended (12-16 weeks), and in high Cu was shortened (2-4 weeks), compared with control cultures (6-8 weeks). These effects required modulation of intracellular Cu only during the first 1-3 weeks of the culture; the long-term effects persisted thereafter, suggesting that the decision process for either self-renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.

Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial

“…The molecule used was the copper chelator tetraethylenepentamine (TEPA), based on findings that cellular copper concentrations modulate cellular differentiation. 10,11 Adding the polyamine copper chelator TEPA to an early acting cytokines cocktail (thrombopoietin (TPO), interleukin-6 (IL-6), FLT-3 ligand (FLT3) and SCF) resulted in both proliferation of mature, committed CB cells (CD34 þ Lin þ ) and colony-forming units cells (CFUc) as well as robust expansion of the CD34 þ CD38À and CD34 þ LinÀ subpopulations. 11 Most importantly, the percentage of engrafted human progenitor cells as well as that of myeloid and lymphoid cells was reproducibly and significantly superior in SCID mice transplanted with TEPA-mediated ex vivo expanded cells than mice transplanted with similar numbers of cells expanded with the cytokine cocktail without TEPA, or with the corresponding cell fraction before expansion.…”

Umbilical cord blood graft enhancement strategies: has the time come to move these into the clinic?

“…NAM-mediated expansion resulted to a median 82-fold increase in the CD34 þ cell dose. Neutrophil engraftment was robust at a median time of 10 days (range [7][8][9][10][11][12][13][14] and durable with no increased toxicity. There were no graft failures and long-term engraftment was obtained from the expanded unit (authors' personal communications).…”

“…Cellular copper reduction in the presence of the copper chelator tetraethylenepentamine (TEPA) results in delays in progenitor differentiation and promotes expansion of cord blood-derived CD34 þ cells in cytokinesupplemented liquid medium. 13 Exposure of UCB progenitors to TEPA in cultures containing stimulatory cytokines (SCF, TPO, IL-6 and FLT-3 ligand) produced an 89-fold increase in CD34 þ cells, 14 which provided a scientific basis for a phase 1-2 clinical trial to test the feasibility and safety of TEPA-mediated HPC expansion. 15 In that study, a single UCB unit was divided, with one aliquot cultured in cytokine-enriched media containing TEPA for 21 days, which resulted in a 219-and 6-fold expansion of total nucleated cells and CD34 þ cells, respectively.…”

Clinical application of hematopoietic progenitor cell expansion: current status and future prospects

“…[108][109][110] Another interesting approach involves copper chelation, as this element promotes hematopoietic differentiation in vitro. 111 Clinical trials designed to combine chelation in combination with ex vivo cytokine exposure are underway.…”