Project leader: Emile van den Akker PhD
One of the main tasks of Sanquin Blood Supply is to supply hospitals with units of packed red cells. To guarantee future availability of erythrocytes for all patients that require transfusions we aim to generate erythrocytes in vitro to obtain a degree of donor independency and to eliminate donor-patient blood type variations and a more quality controlled product. This may be of particular interest for patients with rare blood group antigens that require recurrent transfusions. The ultimate goal is to culture erythrocytes from immortal induced pluripotent stem cells (IPS) or immortal pro-erythroblasts, providing a tool to generate specific low immunogenic erythrocytes of constant quality. As such we entertain three complementary research topics in the laboratory.
Differentiation of erythroblasts to erythrocytes
Differentiation of erythroblasts to erythrocytes involves the assembly of the plasma-membrane band 3 macro-complex and assembly of fetal/adult hemoglobins. These membrane complexes consists of 20+ proteins some of which are important blood group antigens, e.g. Rh, Aquaporin, band 3, GPA, LW, and Kell. Key functions of this protein complex include the regulation of deformability through protein 4.2 and ankyrin-dependent association to the underlying spectrin cytoskeleton, bicarbonate/chloride exchange as a function of erythrocyte CO2 transport and pH regulation, and RhAG is a putative ammonia transporter. Mutations in proteins comprising this macro-complex can lead to specific hemolytic anemias of variable severity. Using a human erythroblast culture, we found that the effects of these mutated proteins are already apparent during early erythropoiesis and have repercussions on macro-complex assembly, possibly affecting functionality. Hence it is important to study the assembly of these erythrocyte membrane protein complexes and the regulation of their expression during normal erythroblast. In 2011 we published the spatio-temporal assembly of the band 3 macro-complex by following the interaction, synthesis and intracellular routing of several proteins: 4.2/band 3 and RhAG/Rh. Good knowledge about the assembly of the band 3 complex and its association with the underlying cytoskeleton is crucial for our understanding of hemolytic disease, blood group presentation and general erythrocyte functionality. We are currently investigating the signals necessary to optimize reticulocyte maturation to erythrocytes and how post-translation modifications like phosphorylation can influence this process in health and disease.
Regulating Globin expression during fetal and adult erythropoiesis.
Cord blood erythropoiesis differs from adult erythropoiesis in several aspects, among which are differences in hemoglobins. Hemoglobin switching defined by replacing gamma-hemoglobin subunits (fetal (CB)) to beta hemoglobin subunits (adults) after birth is one of our key interests. It is important not only in a clinical setting aiming to re-express fetal globins in beta-globin-linked-pathologies but also in the production of erythrocytes from fetal tissues (IPS, CB) in which adult hemoglobin expressing erythrocytes need to be cultured. One of the key regulators of globin expression is the erythroid Kruppel like factor 1 (KLF1). We are studying the effects of various mutations of KLF1, clinically relevant or not, on specific globin expression to understand the role of this transcription factor in globin switching. Importantly, numerous proteins that comprise the band 3 macro-complex as defined above are also under transcriptional control of KLF1 (e.g. band 3, protein 4.2, ankyrin, CD44 etc.). Besides the effect of the KLF1 mutants on specific globin expression, these mutants thus could putatively interfere with the expression of crucial erythrocyte structural proteins. Consequently, both aims are linked to ultimately obtain an optimal erythroid differentiation protocol that yields specific fetal or adult program erythrocytes.
From IPS to erythroblasts to erythrocytes
Culturing erythrocytes from immortal induced pluripotent stem cells (IPS) potentially solves the donor dependency problem and provides a tool to generate specific low immunogenic erythrocytes. Lapillone et al. has calculated that it takes 24 IPS lines to get 99% of the patients blood transfusions matched against the most important blood group systems (ABO, Kell, Rh, MNS, Fy, Jk and P1) and 10 IPS lines are sufficient to provide 95% coverage. Although very much in its infancy, several groups have cultured erythrocytes from human IPS lines albeit with low enucleation efficiencies (4-10%) compared to human ES cell derived erythrocytes >50%. The low enucleation and differentiation rates may be due i) to endodermal and somatic epigenetic bias introduced by the choice of starting material used to generate the specific IPS lines and ii) by inefficient differentiation of IPS lines to hematopoietic lineages. Indeed, several groups have recently shown that epigenetics, in part, dictates the efficiency of IPS lines to differentiate into the different germline layers and/or downstream lineage stem cells such as the hematopoietic stem cell. Therefore we have generated IPS lines from human pro-erythroblasts. Presently, directed differentiation of IPS lines to embryonic bodies and mesodermal/hematopoietic lineages involves four growth factors BMP4, VEGF, bFGF and Wnt3. Preliminary results using feeder free IPS embryonic body differentiation shows between 10-15% CD34+ cells after 8 days of culture. To enhance differentiation of IPS cells to erythroblasts we currently test the effect of additional growth factors, co-culture protocols and over-expression of various transcription factors.
- Esteghamat F, Gillemans N, Bilic I, van den Akker E, Cantù I, van Gent T, Klingmüller U, van Lom K, von Lindern M, Grosveld F, Bryn van Dijk T, Busslinger M, Philipsen S. Erythropoiesis and globin switching in compound Klf1::Bcl11a mutant mice. Blood 2013; 121(13):2553-62.
- Pellegrin S, Heesom KJ, Satchwell TJ, Hawley BR, Daniels G, van den Akker E, Toye AM. Differential proteomic analysis of human erythroblasts undergoing apoptosis induced by epo-withdrawal. PLoS One 2012;7(6):e38356.
- Franco M, Collec E, Connes P, van den Akker E, Billette de Villemeur T, Belmatoug N, von Lindern M, Ameziane N, Hermine O, Colin Y, Le Van Kim C, Mignot C. Abnormal properties of red blood cells suggest a role in the pathophysiology of Gaucher disease. Blood 2013; 121(3):546-55.
- Satchwell TJ, Bell AJ, Pellegrin S, Kupzig S, Ridgwell K, Daniels G, Anstee DJ, van den Akker E*, Toye AM*. Critical band 3 multiprotein complex interactions establish early during human erythropoiesis. Blood 2011; 118(1):182-91. * contributed equally to this work.