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Human adult pluripotent stem cells, stem cells of embryonic origin and induced pluripotent stem cells (iPS) provide cellular sources for new promising regenerative medicine approaches. Because these cells can be patient-specific, they allow considering a personalized medicine appropriate to the diagnosis of each. The generation of cultured red blood cells (cRBC) derived from stem cells is emblematic of personalized medicine. Indeed, these cells have the advantage of being selected according to a blood phenotype of interest and they may provide treatments to patients in situation of impossible transfusion (alloimmunized patients, rare phenotypes). Essential progresses have established proof of concept for this approach, still a concept some years ago. From adult stem cells, all steps of upstream research were successfully achieved, including the demonstration of the feasibility of injection into human. This leads us to believe that Red Blood Cells generated in vitro from stem cells will be the future players of blood transfusion. However, although theoretically ideal, these stem cells raise many biological challenges to overcome, although some tracks are identified.

We describe a technical approach permitting massive expansion of CD34+ stem cells (up to 1.95 x 10(6)-fold) and their full ex vivo conversion into mature red blood cells (RBCs). This three-step protocol can be adapted to hematopoietic stem cells (HSC) of various origins. First, cell proliferation and erythroid differentiation are induced in serum-free media supplemented with stem cell factor, interleukin-3 and erythropoietin (Epo) for 8 days. The cells are then co-cultured with either the murine stromal cell line MS-5 or human mesenchymal cells for 3 days in the presence of Epo alone. Finally, all exogenous factors are withdrawn and the cells are incubated on a simple stroma for up to 10 days. The ex vivo microenvironment strongly influences both the terminal maturation of erythroid cells and hemoglobin (Hb) synthesis. Critically, in vitro-generated RBCs have all the characteristics of functional native adult RBCs in terms of their enzyme content, membrane deformability, and capacity to fix and release oxygen. In addition, their behavior in the murine NOD/SCID model mirrors that of native RBCs. This new concept of "cultured RBCs" (cRBC) has major implications for basic research on terminal erythropoiesis and for patient management. Currently, the potential yield of functional red cells is compatible with clinical requirements, as several units of packed RBCs can be produced from a single donation. Importantly, infused cRBC would all have a life-span of about 120 days, whereas the mean half-life of normal donor RBCs is only 28 days. This would help to minimize the transfusion exposure of patients requiring regular treatment, thereby reducing the risk of iron overload and allo-immunization. The use of autologous CD34+ cells isolated from leukapheresis samples could be beneficial for patients who no longer tolerate allogeneic RBCs. This new method should also prove useful for analyzing the mechanisms of terminal erythropoiesis, including hemoglobin synthesis. Finally, it could provide a tool for investigating the lifecycle of blood parasites such as Plasmodium, the agent of malaria.