The role of glucocorticoids in hematopoiesis in health and disease.
Hematopoiesis requires tight control of survival, proliferation, and differentiation of progenitors, regulated by lineage-specific and general growth factors and cytokines (or hormones). Steady-state erythropoiesis is controlled by EPO, while stress erythropoiesis is co- regulated by EPO, SCF and activation of the glucocorticoid receptor (GR). Diamond-Blackfan anemia syndrome (DBAS) is a bone marrow failure syndrome, characterized by impaired erythroid progenitor expansion, leading to severe anemia in most patients. Many patients with DBAS respond to treatment with glucocorticoids (GC), however this effect is often not long-lasting. The effects of GC in DBAS have been contributed to various mechanisms, including general p53 hyperactivation, which can also be relevant other bone marrow failure syndromes. Interestingly, in patients with Cushing syndrome, characterized by continuous “GC exposure”, it was recently demonstrated that GC-effects on erythropoiesis can be directly related to the nuclear/cytoplasmic transport of the GRalpha, reflected by increased cytoplasmic retention of GRalpha in patients that were “resistant” to GC stimulation. To increase our insights in the effects of GC on hematopoiesis, we have studied GC-effects in different models of healthy hematopoiesis and aberrant hematopoiesis in DBAS.
We have generated iPSC-lines from healthy controls and DBAS patients with distinct functional subtypes. iPSC-lines were used to generate hematopoietic organoids (HeOs) from which RBC precursors were obtained. iPSC-derived erythroblasts were harvested and the response to the GC analogue dexamethasone was followed during culture or upon acute stimulation. Assays included FACS analysis, qPCR for GC-target genes, total RNA-sequencing, and proliferation kinetics. iPSC-derived erythroblasts from healthy controls and DBAS patients (+/- GC-stimulation) were MACS-sorted and analyzed by RNA-sequencing. Data will be compared with previously acquired data in erythroblasts from different origins (e.g. cord blood) and publicly available data.
From our healthy and DBAS patient-derived iPSC-lines we successfully generated RBC-precursors that represented normal and impaired erythropoiesis (in DBAS), clinical severity and the in vivo response to GC-treatment. We identified GILZ as GC-responsive gene to determine the GC-receptor signaling kinetics. Together with proliferation kinetics and RNA-sequencing experiments, we used this to assess the response to GCs in HeO-derived proerythroblasts. Currently, we are investigating molecular mechanisms underlying the GC-response in our iPSC-models using RNA-sequencing data and comparative analyses.
Glucocorticoids play an important role in normal and stress hematopoiesis. Whereas the mechanisms underlying GC-induced erythropoiesis are not fully understood, iPSC-lines of healthy controls and patients with DBAS can be used as models to study these effects in more detail. In addition, iPSC-derived erythroblasts can be used to study “old and new” therapeutic interventions, targeting the GC-receptor or downstream effectors in patients with DBAS or other hypo-regenerative anemias as well as secondary erythrocytosis in Cushing syndrome.