Characterization of human erythroblasts produced in vitro using HEMA culture

In the context of the constant difficulty of obtaining supplies of blood products, the interest of disposing of complementary sources of red blood cells (RBCs) for transfusion is evident. Thus, an attempt to generate erythroid cells in vitro makes good sense. We explored in this work the characteristic of in vitro culture of erythroblasts (EBs) expanded from adult peripheral blood (AB) or cord blood (CB) mononuclear cells (MNCs), with the aim to define the best source for EBs amplification and to optimize the culture conditions to reach a clinical grade production of RBCs. With the aim to define the best source for EBs amplification, we studied the progenitor cell content and the amplification potential of AB- and CB-MNCs in the HEMA culture system. First, five distinct cell populations have been identified by CD34/CD36 staining in AB- and CB-MNCs at the beginning of the culture. They were morphologically and functionally (CFC, liquid culture) characterized. As expected, results showed that in CB the CD34pos content was higher than in AB (1.1±0.66 vs 0.14±0.05, p ≤0.05), while the frequency of erythroid colonies recovered in AB was not statistically different from that of CB. These observations inspired us to compare the expansion potential of these two sources. Results demonstrated that both CD34pos and CD34neg were able to generate EBs in culture. The CD34pos population had a greater proliferation potential, but MNCs generated greater number of EBs in culture both for AB and CB. In addition, we found that AB- and CB-MNCs have similar potential to generate erythroid cells in vitro: MNCs from discarded buffy coat of regular blood donations generate number of EBs comparable to those obtained by low volume CB. In order to reach the goal of optimization of EBs expansion, we performed a functional characterization of EBs obtained from AB- or CB-MNCs. First, we identified in AB-EBs the population that sustains the culture: the immature EBs (iEBs, CD36posCD235anegCD117posCD63low) that retain high self-renewal ability and able to generate greater number of EBs when repeatedly sorted from the bulk of culture. The comparison with CB-EBs revealed the presence of a higher percentage of iEBs than AB-EBs, but despite they showed the same antigenic profile, they highlighted distinct biological properties: they did not retain the ability to self-renew and failed to mature when repeatedly sorted. In fact, the phosphoproteomic profiles revealed differential signaling pathway active in AB- and CB-EBs. In conclusion, these results indicate that in HEMA culture, MNCs are better than CD34pos cells because they generate greater number of EBs. Furthermore, buffy coats from regular blood donations are a rich source of stem/progenitor cells which may have clinical potential and are not inferior to CB in generation of EBs. Moreover a molecular characterization of the signaling pathways activated during erythroid differentiation could be a novel approach to promote EBs expansion or the sequential purification of iEBs could be considered as an additional strategy to increase the expansion of these cells useful as transfusion products.