oa Generation of red blood cells from pluripotent stem cells as an alternative to donated blood

We are working to generate human red blood cells from human pluripotent stem cells at sufficient levels of quality and safety to warrant entry to pre-clinical and clinical studies. These in vitro generated red blood cells (RBCs) offer an alternative to donated blood and may be of particular use for patients receiving repeated red cell transfusions including those with sickle cell disease and thalassaemias. In order to generate RBCs suitable for clinical use we have derived 10 new clinical grade GMP human embryonic stem cell lines. Our erythroid differentiation protocol is suspension culture based with no co-culture. The protocol, which lasts 24 days, achieves ~20,000 fold expansion in cell numbers and results in ~95% of the cells in culture expressing erythroid markers (glycophorin A, CD71, CD36)and having the characteristic morphology of haemoglobinised normoblasts. HPLC and quantitative PCR analysis of globin chains show that fetal alpha and gamma chains predominate with embryonic epsilon and zeta chains only comprising >5% of total globins. Minimal spontaneous enucleation (≤5%) has observed, however, nucleated erythroid cells examined for their ability to bind and release oxygen (in a Hemox Analyser) demonstrated a similar profile to cord blood derived RBCs, consistent with the presence of fetal haemoglobin in these cells. We are currently testing our differentiation protocol with induced pluripotent stem cells (iPSC) and dissecting the molecular profile of the hESC-derived day 24 normoblast population in order to better understand why these cells do not enucleate as readily as adult haematopoietic stem cell derived erythrocytes. In addition to these remaining biological issues, we are addressing the processes needed for GMP-grade scale up of the protocol to facilitate the production of large numbers of clinically acceptable RBCs.

J. C. Mountford1,4, D. Anstee2, A. Courtney6, L. Forrester4, W. Murphy3, S. Parsons2, P. de Sousa6,5 and M.L Turner1.

Scottish National Blood Transfusion Service1, NHS Blood and Transplant2, Irish Blood Transfusion Service3, University of Glasgow4, University of Edinburgh5 and Roslin Cells Ltd6. United Kingdom.