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Hematopoiesis

Research lines

Differentiation of erythroblasts to erythrocytes   

Differentiation of erythroblasts to erythrocytes involves the assembly of membrane proteins into crucial structural complexes (junctional and ankyrin complexes) and the sorting of proteins during enucleation. 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 and aberrant erythropoiesis. Good knowledge about the assembly of these large membrane complexes and their association with the underlying cytoskeleton is crucial for our understanding of hemolytic disease, blood group presentation and general erythrocyte functionality.

In addition, it becomes increasingly clear that macrophages play a central role in controlling the end-stages of erythroid differentiation. We are currently setting up an in vitro co-culture system to study the requirements and function consequences of these erythroid-macrophage associations. These studies will help to understand and optimize reticulocyte maturation to erythrocytes.

Regulating Globin expression during fetal and adult erythropoiesis.

Human Cord blood and fetal liver erythropoiesis differs from adult erythropoiesis in several aspects, among which are differences in globin subunit expression. Late stage 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 (as fetal erythrocytes may have shorter life spans).

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 could thus putatively interfere with the expression of crucial erythrocyte structural proteins. As such we are also interested and are studying the basis of human variations within hereditary persistence of hemoglobin (HPFH) to identify novel regulators of globin switching. Evidence is accumulating showing that erythroblast globin expression may be influenced by specific niche cells and thus is under control of specific signal transduction. We are currently addressing this by performing co-culture experimenst with various cells from different developmental stages to understand and characterize these signals. Consequently, research in this links to aim 1 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. We have calculated that it takes 8 IPS lines to get 95% of the blood transfusions matched against the most important blood group systems. 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. We have setup an iPSC facility in which we generate iPSC lines from as little as 5ml of blood from which we culture human pro-erythroblasts and other hematopoietic progenitors to re-program. The facility has generated 30+ control and patient lines using lentiviral and episomal reprogramming methods and we are open to reprogram for external parties on a collaborative manner. We have setup a robust monolayer differentiation protocol to culture hematopoietic cells starting from single cells iPSC colonies.

Currently, we are optimizing the specification to mesodermal, endothelial and hematopoietic lineages to increase differentiation yield, for instance by studying endothelial to hematopoietic transition. In addition, we are optimizing the enucleation rate of iPSC-derived erythroblast during differentiation, e.g. by co-culture with specific cells. We make use of iPSC disease models and CRISPR-based genome editing to investigate the mechanisms behind control of these specification. Eventually, we aim to use iPSC to produce transfusion products.
Transfusion of in vitro cultured erythrocytes; clinical translation
Recently, we have been granted an NWO-TAS which allows us to begin translating our research to clinical application of in vitro cultured transfusion products. Within this grant we have made a completely defined good manufacturing practice grade media and culture conditions to generate erythrocytes from various sources that can be used for transfusion purposes.

We have collaborations with various companies and academics on the development of bioreactors, costs effectiveness and media definitions. Currently, we are utilizing a bioreactor that enables us to culture significantly more cells with lower media requirements (10-50 times) compared to static culture dishes. Within the NWO-TAS program, we aim to perform clinical trials with PBMC-derived erythrocytes within this decade followed by a trail with iPSC-derived erythrocytes within the first part of the next decade. This translational research is done in collaboration with the Amsterdam Medical Center and sanquins in house GMP-facility, the Laboratory for Cell Therapies.
Resources

Key Publications

  • Heideveld E, Masiello F, Marra M, Esteghamat F, Yağcı N, von Lindern M, Migliaccio AR, van den Akker E. CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield. Haematologica. 2015 Nov;100(11):1396-406.
  • Kostova EB, Beuger BM, Veldthuis M, van der Werff Ten Bosch J, Kühnle I, van den Akker E, van den Berg TK, van Zwieten R, van Bruggen R. Intrinsic defects in erythroid cells from familial hemophagocytic lymphohistiocytosis type 5 patients identify a role for STXBP2/Munc18-2 in erythropoiesis and phospholipid scrambling. Exp Hematol. 2015 Dec;43(12):1072-1076.e2.
  • 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.