Cord blood transplantation (CBT) is limited by low number of TNC and CD34+ cells, influencing the incidence and rate of hematopoietic recovery and risk of early transplant-related mortality. Ex vivo expansion is a strategy to increase the number of progenitor cells and improve clinical outcomes. Several early stage studies are evaluating the clinical benefit of expansion, mainly in a double CBT configuration. The StemEx® trial evaluates the potential contribution of expansion in a single CBT configuration. StemEx is manufactured from a portion of a single CBU, originally frozen in 2 separate fractions. CD133+ progenitor cells, purified from the smaller or equal fraction, are cultured for 21-23 days with cytokines and a copper chelator, TEPA, which delays differentiation and promotes expansion of progenitor cells with engraftment capabilities. A global pivotal registration study evaluating the safety and efficacy of StemEx in patients with hematological malignancies following myeloablative treatment is currently completing recruitment. Safety and efficacy outcomes of the study will be available in 2012. To date, 82 of 88 StemEx batches manufactured in 3 centralized GMP facilities, have been successfully expanded: median fold expansion of TNC, CD34+ cells and CFU over culture input were 399 (range 52-764), 75 (6-280) and 107 (43-662), respectively. Expansion of only a portion of the CBU resulted in a median 8.4 fold increase (0.8-90.3) in the number of CD34+ cells infused over the number that would be infused from the entire CBU without expansion. The CFU potential of culture seeded CD133+ cells measured at day-0 of production indicates the expansion potential of the cryopreserved CB cells. In all six batches which failed to expand, day-0 CFU was low, while day-0 CFU of all successfully expanded batches was within specification ranges. This information, available before patient myeloablation, strengthens the clinical applicability of StemEx. All StemEx batches were successfully delivered and infused. We are currently in the last stages of development of a frozen StemEx product. The added flexibility in the timing of transplantation allows for changes resulting from patient disease progression or complications. With the challenges of an ex vivo expanded product being successfully met in the current registration trial, and the development of a frozen product, StemEx demonstrates its feasibility as a quality alternative stem cell source for allogeneic HSCT.

Use of UCB as an alternative SC source is limited due to inadequate TNC & CD34+ cells, in that it is often difficult to find a single unit for successful engraftment in adolescents & adults. One strategy to overcome this limitation is to increase the number of cells by expansion of HPC. StemEx® was developed as a cell graft based on use of a copper chelator (TEPA) that delays differentiation & promotes expansion of HPC with engraftment capabilities. StemEx is manufactured from a fraction of a single CBU originally frozen in 2 separate fractions. CD133+ progenitor cells purified from the smaller or equal fraction are cultured for 21 days with cytokines & TEPA. The product is transplanted to the patient 24h after infusion of the unmanipulated fraction. A global pivotal Phase II/III registration study is underway to evaluate the safety & efficacy of StemEx in patients with advanced hematological malignancies. Taking into consideration the variability in the intrinsic potential & the cryopreservation processes of the CBUs, successful & robust manufacturing of clinical batches is one of the major challenges of this study. 61 StemEx batches have been manufactured so far in 3 centralized GMP facilities in US, EU & Israel. All the fresh hand-carried batches were successfully delivered to the centers within the StemEx stability period. Of the 61 batches, 58 successfully passed the in-process & final process quality control (IPQC & FPQC) criteria: median fold expansion of TNC, CD34+ cells & CFU over culture input values were 377 (52-743, n = 58), 74 (6-206, n = 57) & 111 (43-662, n = 56), respectively. Expansion of only a portion of the CBU resulted in a median 9.6 fold increase (0.8-90.3 n = 58) in the total number of CD34+ cells infused, over the theoretical number of CD34+ cells that would be infused from the entire CBU without expansion. Interestingly, all three batches that failed to expand also did not pass the IPQC criteria: ≥ 10 CFU/1000 freshly purified CD133+ cells at day 0 of production. This information, available before initiation of patient myeloablation, provides further confidence regarding the quality of the CBU & the StemEx product. With the significant production challenges of an ex vivo expanded product being successfully met in the current registration trial, StemEx study demonstrates the feasibility of this approach in satisfying an urgent unmet clinical need in the UCB transplantation setting.

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 CuCl2. However, measurement of the cellular pool of labile Cu showed similar reduction (50% from the control) by all the TEPA forms, while CuCl2 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. (c) 2005 International Society for Experimental Hematology.

Ex-vivo expansion strategies of cord blood (CB) derived human progenitor cells (HPC) have been developed to provide an answer to the delayed time to engraftment and to the extended periods of neutropenia and thrombocytopenia encountered. These problems occur in transplants of CB products performed in adults due to the low yield of HPC. Reports correlating the clinical outcome with the number of CD34+ cells suggest that the transplantation of ex vivo expanded CD34+ cells may shorten the time to engraftment. The use of copper chelators such as tetraethylenepentamine (TEPA) has been shown to prolong expansion of HPC by inhibiting cell differentiation and thus allowing self-renewal of primitive HPC (Exp Hematol. 2004; 32:547). The variability observed in the expansion results, caused by the intrinsic differences among the various sources of CB units and processing methodologies, complicates the interpretation of published results. In the present report we summarize our results of CD34+ cell ex-vivo expansion of over 100 units in the presence of IL-6, TPO, Flt-3 ligand and SCF with and without TEPA. After 3 weeks, the total nuclear cell (TNC), colony forming unit (CFU), and the total CD34+ cell fold expansion of TEPA-treated cultures were 424±10.5 (n=230), 104±7 (n=112) and 19±3.2 (n=113), respectively, with no significant differences compared to controls. However, the percentage of the primitive subset of HPC, CD34+/38− cells, significantly (p<0.0001) increased in the TEPA-treated cultures (3.2%±0.2, n=59) vs. controls (1.6%±0.27, n=147). In contrast, after 5 weeks in culture, the TNC fold expansion was significantly (p<0.05) higher in TEPA-treated cultures compared to the controls, 1471±63.5 (n=89) vs. 1270 ±240 (n=55), respectively. The increase in TNC in TEPA-treated cultures did not result in increased HPC differentiation, but was accompanied by an increased self-renew capacity of CD34+ cells as represented by a 57±5.9 fold (n=47) vs. a 32±3.5-fold (n=38) amplification in the controls (p<0.0009). The overall fold expansion in culture analyzed by a Kaplan-Meier survival curve function demonstrate that the TNC, CFU, CD34+ and CD34+/38− cells derived from TEPA-treated cultures have higher in vitro survival probabilities than controls (p<0.0014). Cumulative values of all parameters were calculated and a transformation performed using the rank procedure. The results underline that TEPA increases CFU potential and CD34+ and CD34+/38− content during the ex-vivo expansion (p<0.01). The TEPA supplemented expansion technology was further tested after up-scaling of the processing and culturing procedures. AC133+ cells isolated by the CliniMACS device from frozen CB units obtained from 6 different banks. The expansion results of TNC, CD34+ cells, CFU and %CD34/38- were 337±23 (n=57), 21±4.3 (n=19), 133±27.5 (n=19) and 2.7% ±0.7 (n=19) fold, respectively. A clinical trial with TEPA expanded cultures for treatment of leukemia patients is currently ongoing at MD Anderson Cancer Center, USA.

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. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc.

CD38, originally described as a differentiation marker, has emerged as an important multifunctional transmembrane protein. Its most intriguing and well-characterized function is its ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. Of particular interest is its presence on the inner membrane of the nucleus, suggesting that CD38/cADPR may play a direct role in mediating nuclear activation and gene expression. Our studies on ex vivo expansion of Hematopoietic Stem Cells (HSCs) have led us to test whether alteration of CD38 function carries the potential of affecting cell fate decisions of HSCs. Inhibition of CD38 enzymatic activity was achieved by treating CD34+ cell cultures with nicotinamide (NA), a well-known base-exchange inhibitor demonstrated to inhibit the synthesis of cADPR from NAD.