Chelatable cellular copper modulates differentiation and self-renewal of cord blood-derived hematopoietic progenitor cells.

OBJECTIVES: We have demonstrated epigenetic modulation of CD34(+) cell differentiation by the high-affinity copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA slowed down the rate of CD34(+) cell differentiation and increased their engraftability in SCID mice. TEPA biological activity was attributed to its effect on cellular Cu levels as (a) treatment with TEPA resulted in reduction of cellular Cu, and (b) excess of Cu reversed TEPA's activity and accelerated differentiation. In the present study we further evaluated the role of cellular Cu in TEPA's biological activity. METHODS: The effects of Cu-chloride, TEPA, TEPA/Cu mixtures at various ratios, and a synthesized, stable, TEPA-Cu complex on short- and long-term cord blood-derived CD34(+) cell cultures as well as on the overall and chelatable cellular Cu were investigated. RESULTS: Addition of TEPA, TEPA/Cu mixtures at up to equimolar concentrations, and the TEPA-Cu complex to CD34(+) cell cultures resulted in inhibition of differentiation and enhancement of long-term self-renewal. Measurement of the overall cellular Cu by atomic absorption spectrophotometry showed 20 to 40% decrease by TEPA while the TEPA-Cu mixture and the TEPA-Cu complex increased cellular Cu by 10- to 20-fold, as did CuCl(2). However, measurement of the cellular pool of labile Cu showed similar reduction (50% from the control) by all the TEPA forms, while CuCl(2) increased it. Thus, inhibition of differentiation and enhancement of self-renewal of CD34(+) cells was correlated with reduction in the cellular chelatable Cu content. CONCLUSION: The results suggest that decreasing of the chelatable Cu pool, rather than overall Cu, is the mechanism that stands behind TEPA's biological activity.

Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice.

OBJECTIVE: We previously demonstrated that cellular copper is involved in the regulation of proliferation and differentiation of hematopoietic progenitor cells. Modulation of cellular copper was achieved by supplementing the culture with a copper chelator that reduces cell copper content, or copper salts, which elevate the level of cellular copper. In the present study, we evaluated the effect of short-term (3-week) treatment with the copper chelator tetraethylenepentamine (TEPA) on short- and long-term (up to 11 weeks) ex vivo expansion of hematopoietic progenitors, as well as on their SCID engraftment potential. MATERIALS AND METHODS: Cord blood-derived purified CD34+ cells were grown in liquid medium supplemented with the cytokines stem cell factor, thrombopoietin, Flt3 ligand, and IL-6, and the chelator TEPA for the first 3 weeks and then for up to 11 weeks with cytokines alone. Control cultures were supplemented with cytokines alone for the entire culture duration. Cultured cells were characterized by immunophenotyping and cloning (CFUc). Transplantability was assayed by injection of repurified CD34+ cells into NOD/SCID mice. RESULTS: In the short term, TEPA supported increased percentages of early progenitors over control cultures incubated with cytokines alone (CD34(+)CD38-, p=0.001 and CD34(+)Lin-, p=0.016). In the long term, TEPA pretreated cultures showed prolonged expansion of CD34+ cells (p=0.01) and CFUc (p=0.002) compared with that of untreated cultures. The SCID engraftment potential of CD34+ cells repurified from the TEPA-treated cultures was higher compared with that of the control, i.e., only cytokine-treated cultures (p=0.03). CONCLUSION: TEPA enabled preferential proliferation of early progenitor cells with the phenotype CD34(+)CD38- and CD34(+)CD38- Lin- during the first weeks of culture, resulting in the observed increased long-term ex vivo expansion and engraftment capabilities.