Cord Blood Derived CD34+ and AC133+ Progenitor Cells Ex-Vivo Expanded in the Presence of Tetraethylenepentamine: Reproducibility among Cord Blood Units.
Authors: Grynspan F. , Peled T. , Rosenheimer-Goudsmid N. , Hana L. , Hasson N. , Mandel J. , Landau E. , Glukhman E. , Harel A. , Yudin D. , Adi S. , Olesinski E. , Daniely Y. , Hasson A. , Harati D. , Nagler A. , Fibach E. , Shpall E. 
Published: April 2020

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&lt;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&lt;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&lt;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&lt;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&lt;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.