Erythropoiesis & Regenerative Hematology

The research in my group centers on the biology of erythropoiesis, with a dual focus on the generation of RBCs for transfusion purposes and the regulation of globin gene expression. These two themes are tightly interwoven, reflecting both the translational and fundamental aspects of erythropoiesis.

Generation of hematopoietic effector cells like red blood cells for transfusion purposes.

Classical cellular products like platelets and red blood cells are the most used cellular therapies worldwide. The research in my group has provided novel approaches and cellular systems to produce these classical products in vitro and donor independent, to allow optimal and individualized treatments. This has led to an in depth understanding of e.g. erythropoiesis and megakaryopoiesis, but also on general development of hematopoiesis placing our research firmly into the scientific community. The obtained knowledge is used not only to produce the classical cellular products but also to develop novel therapeutic products and diagnostic tools. RBCs with their unmatched biocompatibility and a safety record spanning over a century of clinical use, are now poised to become next-generation therapeutic vehicles. By genetically enhancing cultured RBCs (cRBCs) to carry specific cargo, we combine the proven safety of a well-characterized cell type with cutting-edge precision medicine, unlocking novel (immunological) applications that systemic therapies or other cell types cannot achieve. Our research lead upscaling efforts developing methodology to utilize turbulent 3D printed bioreactors (3l) to produce large amounts of cultured red blood cells, perform gene editing strategies to repair and/or amend the final product to the need of the patient. In additionan, also novel high throughput ways to detect allo-antibodies in plasma or to analyse erythrocyte morphologies that allow drug screening or patient monitoring were developed.
 

iPSC facility

In addition, I have setup an iPSC facility at Sanquin. In the facility we conduct reprogramming of somatic cells to iPSC lines using both integrative (lentiviral) and non-integrative approaches (Sendai, episomal). We have numerous control and patient lines that include for instance iPSC lines derived from patients with sickle cell disease, diamond blackfan anemia, grey platelet syndrome or specific leukemic translocations. Of note, specifically for DBAS we have a unique dataset of iPSC lines with various genetic origins. The iPSC facility conducts contract research and reprogramming on demand. With respect to their developmental stage, iPSC lines are comparable to embryonic stem cell and able to differentiate into all embryonic/fetal tissues depending on the cues given. Hematopoiesis and by extension erythropoiesis, occurs in three different independent spatially and temporally confined waves. In order of appearance these are the primitive wave followed by two definitive waves: the erythroid-myeloid progenitor (EMP) wave, and the Aorta-Gonad-Mesonephros (AGM)-wave. Adult hematopoiesis is a resultant of this last AGM wave. The last two waves are characterized by a process of trans-differentiation where specialized endothelial cells differentiate into hematopoietic stem and progenitor cells. Within my group we investigate the molecular cues that allow differentiation of iPSC into the different hematopoietic waves and lineages (including hematopoietic stem cells). We have models that recapitulate primitive and definitive wave hematopoiesis. The 3D protocols allow differentiation of iPSC to 3D hematopoietic organoids in turbulent (bioreactor) environments and yield all major hematopoietic lineages (erythroid, megakaryoid, myeloid, T-cell and hematopoietic progenitor cells) providing hematopoietic effector cell production starting points and cellular model systems for diseases.

We have collaborations with various companies and academic partners within Europe concerning our research and are embedded in many international and national consortia. For instance, we collaborate with PAN biotech with whom we have developed a completely novel culture media to grow hematopoietic cells, and we work together with the technical University and Delft and Gettinge to define the criteria to produce 3D printed stir-tank bioreactors that we are currently employing to upscale our red blood cell productions. Translational research is done in collaboration with the Laboratory for Cell Therapies at Sanquin.

Fetal and adult erythropoiesis, differences and similarities.

Human Cord blood and fetal liver erythropoiesis differs from adult erythropoiesis in several aspects. The regulation of developmentally different red blood cell population is of interest from several points of view.  One of our objectives is to develop and optimize a protocol for producing erythrocytes from iPSCs that closely resemble fetal red blood cells (RBCs) in structure and function. This will provide a safer alternative to adult RBCs for treating fetal anemia, reducing the risk of complications associated with current IUT and preterm transfusions. We therefor aim to characterize these different developmental waves of erythropoiesis and establish their viability for clinical application through rigorous functional testing and upscaling of production processes. In addition, these development waves of erythropoiesis are also defined by their different expression of globin genes: embryonic (zeta-epsilon), fetal (alpha-gamma) and adult (alpha-beta). The molecular pathways that control the so-called globin switch through replacing gamma-globin subunits with beta-globin subunits after birth is one of our key interests as it marks the clinical onset of beta-hemoglobinopathies where mutations in the beta-globin gene are causative, like sickle cell disease. Re-activation of gamma globin expression, and as a consequence fetal hemoglobin, at the expense of beta globin expression is known to be curative. 

We have generated several strategies (patented) to re-activate fetal hemoglobin expression. These strategies are currently evaluated and further developed into clinical applications. We use CRISPR  gene editing to force the re-expression of gamma-globin and fetal hemoglobin. We collaborate with several academic partners to evaluate different gene editing tools moving away from spyCas9, while also investing into in vivo systemic delivery of the gene editing machinery moving away from ex-vivo editing of hematopoietic stem cells followed by transplantation.