Concise Review: Advanced Cell Culture Models for Diamond Blackfan Anemia and Other Erythroid Disorders

Abstract

In vitro surrogate models of human erythropoiesis made many contributions to our understanding of the extrinsic and intrinsic regulation of this process in vivo and how they are altered in erythroid disorders. In the past, variability among the levels of hemoglobin F produced by adult erythroblasts generated in vitro by different laboratories identified stage of maturation, fetal bovine serum, and accessory cells as "confounding factors," that is, parameters intrinsically wired in the experimental approach that bias the results observed. The discovery of these factors facilitated the identification of drugs that accelerate terminal maturation or activate specific signaling pathways for the treatment of hemoglobinopathies. It also inspired studies to understand how erythropoiesis is regulated by macrophages present in the erythroid islands. Recent cell culture advances have greatly increased the number of human erythroid cells that can be generated in vitro and are used as experimental models to study diseases, such as Diamond Blackfan Anemia, which were previously poorly amenable to investigation. However, in addition to the confounding factors already identified, improvement in the culture models has introduced novel confounding factors, such as possible interactions between signaling from cKIT, the receptor for stem cell factor, and from the glucocorticoid receptor, the cell proliferation potential and the clinical state of the patients. This review will illustrate these new confounding factors and discuss their clinical translation potential to improve our understanding of Diamond Blackfan Anemia and other erythroid disorders. STEM CELLS 2018;36:172-179 SIGNIFICANCE STATEMENTBy contrast with theoretical science, experimental science tests models that by definition are imperfect because they contain "confounding factors." In the past, confounding factors identified in the in vitro models developed to study human erythropoiesis have greatly contributed to advance the field. Recent technological advances have greatly improved the power of analyses provided by in vitro models of human erythropoiesis. However, these improved models contain novel "confounding factors" that have yet to be fully discussed. This review will illustrate three novel confounding factors wired in advanced in vitro models of human erythropoiesis and discuss how their identification may drive additional studies to improve our understanding of the pathobiology of Diamond Blackfan Anemia and possibly other erythroid disorders.

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors

“…Analysis of cKIT metabolism identified the critical role played by CD63 in favoring endocytosis of cKIT, and therefore in quenching its signaling, in adult cells. In spite of the well-described effects of SCF on erythropoiesis, clinical attempts to use this growth factor for the treatment of EPO-unresponsive anemias have not been encouraging so far and an international trial in Diamond Blackfan Anemia was prematurely interrupted by the National Institute of Health after the death of one patient (58). Our observations suggest that CD63 antibodies, by neutralizing CD63, may prolong the canonical signaling of cKIT at physiological SCF levels increasing the expansion of the adult erythroid pool and.…”

Single-cell profiling of human bone marrow progenitors reveals mechanisms of failing erythropoiesis in Diamond-Blackfan anemia

“…Impaired translation or transcription of the master erythroidmegakaryocyte (MK) transcription factor GATA1 (11) due to defective ribosome biogenesis (12) and excess heme toxicity (13,14) has been suggested as a unifying mechanism of erythroid failure across DBA genotypes. However, inconsistent findings from human model cellular systems and cell lines (4,(14)(15)(16)) are yet to be reconciled, and it remains unclear whether additional mechanisms may contribute to erythroid failure in DBA (17). Furthermore, although the diagnostic criteria of DBA include presentation in infancy with virtually no mature bone marrow erythroblasts (EBs) (3), atypical presentations in later life with milder hematological manifestations are not uncommon (18)(19)(20)(21)(22).…”

Modelling erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I)

“…While ex vivo culture from HSCs reproduces aspects of normal erythropoiesis and provides a source of erythroid cells to study, a number of confounding factors have been identified that make interpretation of the data difficult. 14,38 Therefore, in order to investigate differences between patient-derived erythroblasts and those from healthy controls we took extreme care to stage the cultures using an array of methods that included FACS and CyTOF, which both rely on immunophenotyping cell populations. While such methods showed healthy and diseased erythroblasts were immunologically similar during the expansion phase and into early differentiation, aspects of disordered erythropoiesis became evident after this time by several parameters.…”